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  • 1.
    Ahlford, Katrin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Adolfsson, Hans
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Rhodium-catalyzed asymmetric transfer hydrogenation of alkyl and aryl ketones in aqueous media2008Ingår i: Green Chemistry, ISSN 1463-9262, E-ISSN 1463-9270, Vol. 10, nr 8, s. 832-835Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A novel lipophilic rhodium catalyst was evaluated in the enantioselective transfer hydrogenation of ketones in water using sodium formate as the hydride donor, and in the presence of sodium docecylsulfonate. Alkyl alkyl ketones were reduced in good yields and in moderate to good enantioselectivities, and the reduction of aryl alkyl ketones proceeded with excellent enantioselectivity (up to 97% ee).

  • 2.
    Andersson, August
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    A kinetic model for peptide-induced leakage from vesicles and cells2007Konferensbidrag (Övrig (populärvetenskap, debatt, mm))
  • 3.
    Ariöz, Candan
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ye, Weihua
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    al Bakali, Amin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ge, Changrong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Liebau, Jobst
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Götzke, Hansjörg
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Barth, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli2013Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 33, s. 5533-5544Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.

  • 4.
    Berglund, Anna-Karin
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Spånning, Erika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biverståhl, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Maddalo, Gianluca
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
    Tellgren-Roth, Christian
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dual targeting to Mitochondria and Chloroplasts: Characterization of Thr-tRNA Synthetase Targeting Peptide2009Ingår i: Molecular Plant, ISSN 1674-2052, Vol. 6, nr 2, s. 1298-1309Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins of Arabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides (dTPs). The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr–tRNA synthetase (ThrRS–dTP) studying organellar import of N- and C-terminal deletion constructs of ThrRS–dTP coupled to GFP. These results show that the 23 amino acid long N-terminal portion of ThrRS–dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS–dTP(2–60) to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS–dTP(2–60) inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS–dTP(2–60) indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

  • 5.
    Biverståhl, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bodor, Andrea
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K(+) channels2009Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1788, nr 9, s. 1976-86Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The membrane location of two fragments in two different K(+)-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative "paddle" domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T(1) and (13)C-(1)H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. (2)H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.

  • 6.
    Biverståhl, Henrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biophysical studies of the membrane location of the voltage-gated sensors in HsapBK and KvAPManuskript (Övrigt vetenskapligt)
  • 7. Björklund, Jörgen
    et al.
    Biverståhl, Henrik
    Gräslund, Astrid
    Mäler, Lena
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Real-time transmembrane translocation of penetratin driven by light-generated proton pumping.2006Ingår i: Biophys J, ISSN 0006-3495, Vol. 91, nr 4, s. L29-31Artikel i tidskrift (Refereegranskat)
  • 8.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane Interaction of Disease-Related Dynorphin A Variants2013Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 24, s. 4157-4167Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The membrane interaction properties of two single-residue variants, R6W and L5S, of the 17-amino acid neuropeptide dynorphin A (DynA) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Corresponding gene mutations have recently been discovered in humans and causatively linked to a neurodegenerative disorder. The peptides were investigated in buffer and in isotropic solutions of q = 0.3 bicelles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or DMPC (0.8) and 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DMPG) (0.2). The CD results and the NMR secondary chemical shifts show that R6W-DynA has a small a-helical fraction in buffer, which increases in the presence of bicelles, while L5S-DynA is mainly unstructured under all conditions studied here. R6W-DynA has an almost complete association with zwitterionic bicelles (similar to 90%, as probed by NMR diffusion experiments), similar to the behavior of wtDynA, while L5S-DynA has a weaker association (similar to 50%). For all peptides, the level of bicelle association is increased in negatively charged bicelles. The L5A-DynA peptide adopts a very shallow position in the headgroup region of the bicelle bilayer, as studied by paramagnetic spin relaxation enhancement experiments using paramagnetic probes. Similarly, the results show that R6W-DynA is more deeply buried in the bilayer, with only the C-terminal residues exposed to solvent, again more similar to the case of wild-type DynA. We suggest that the results presented here may explain the differences in cell toxicity of these disease-related neuropeptide variants.

  • 9.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The membrane interaction of dynorphin A depends on lipid head-group chargeManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    The influence of lipid bicelles on the dynamics of the opioid peptide DynA has been investigated by Nuclear Magnetic Resonance. DynA exerts its opioid effects mainly through interactions with the κ subtype of the opioid receptors, but has also been demonstrated to have direct interactions with membranes. Among other properties, it has been shown that the peptide causes membrane disruption and may penetrate bilayers. Despite the fact that DynA appears to bind tightly to model lipid bilayers, no structure induction has been observed. To further study the effect of membrane interactions we have here therefore measured the fast local dynamics of DynA specifically labeled with 15N in three backbone amide sites (Gly2, Leu5 and Leu12) in fast-tumbling bicelles, both with and without the incorporation of the negatively charged dimyristoylglycerol. We also examined the amide exchange in the two bicelles. We find that despite the fact that DynA is largely unstructured in both types of bicelles, the peptide has restricted backbone dynamics, which depends on the presence of negatively charged lipids. Moreover we see that the lipid dependence is not uniform throughout the sequence, but is most noticeable for Leu5, which precedes an unusually basic stretch of amino acid residues. The findings indicate that this basic sequence may be of significance for bilayer recognition. Finally, we note that the dynamical behavior of the peptide is much more influenced by the lipid surroundings than what the structural properties are.

  • 10.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kurnik, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oliveberg, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Danielsson, Jens
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the kappa opioid receptor using a soluble protein scaffold2014Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, nr 3, s. 814-824Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the kappa opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble beta-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.

  • 11.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, Mathias
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Analysing DHPC/DMPC bicelles by diffusion NMR and multivariate decomposition2015Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1848, nr 11, s. 2910-2917Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Mixtures of lipids and detergents are known to form bicelles at certain parameter ranges, but many uncertainties remain concerning the details of the phase behaviour of these mixtures and the morphology of the formed lipid assemblies. Here we used nuclear magnetic resonance (NMR) diffusion data in combination with the multivariate processing method speedy component resolution (SCORE) to analyse mixtures of 1,2-dihexanoyl-snglycero-3-phosphocholine (DHPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the relative concentration q = [DMPC]/[DHPC] = 0.5 at total lipid concentrations ranging from 15 to 300 mM. With this approach we were able to resolve the heavily overlapping mixture spectra into component spectra and obtained reliable diffusion coefficients for lipid concentrations in the range 15 to 300 mM, although at high concentrations (250-300 mM), non-negativity constraints or overfactoring was required to successfully decompose the data. At 50-300 mM total lipid concentration, the radii estimated from the diffusion coefficient of DMPC indicate assemblies of the appropriate bicelle size, although small size variations exist, while at lower concentrations the morphology appears to change to larger assemblies. Taken together, the results suggest that for q = 0.5 DMPC/DHPC mixtures there is a relatively broad concentration range above 50 mM where bicelles may reliably be assumed to adopt the 'classical' bicelle morphology. The study clearly demonstrates the usefulness of our approach for accurately determining physical properties of complex mixtures such as bicelles. Both reliable diffusion coefficients and chemical shifts can be derived from overlapping data. This should prove useful for analysing the behaviour of other, more complex, lipid mixtures.

  • 12.
    Björnerås, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, Mathias
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Analysing the morphology of DHPC/DMPC complexes by diffusion NMRManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Mixtures of lipids and detergents are known to form bicelles at certain parameter ranges, but many un-certainties remain concerning the details of the phase be-haviour of these mixtures and the morphology of the formed lipid assemblies. Here we used NMR diffusion data in com-bination with the multivariate processing method SCORE to analyze mixtures of DHPC and DMPC with the relative concentration q=[DMPC]/[DHPC]=0.5 at total lipid con-centrations from 15 to 300 mM. With this approach we were able to resolve the heavily overlapping mixture spectra into component spectra and obtained reliable diffusion coeffi-cients for lipid concentrations in the range 15 to 200 mM. Between 200 and 300 mM, the similar diffusion coefficients in combination with substantial signal overlap makes it difficult to get very reliable spectra and diffusion coeffi-cients with standard processing parameters, but overfactoring provided useful diffusion coefficient estimates also at these concentrations. At 50–300 mM total lipid concentration, the radii estimated from the diffusion coeffi-cient of DMPC indicate assemblies of the appropriate bicelle size, although small size variations exist, while at lower concentrations the morphology appears to change to larger assemblies. Taken together, the results suggest that for q=0.5 DMPC/DHPC mixtures there is a relatively broad concentration range above 50 mM where bicelles may relia-bly be assumed to adopt the 'classical' bicelle morphology. At lower concentrations there is evidence for a more com-plex morphology with more than one type of lipid assembly in the sample.

  • 13. Bodor, Andrea
    et al.
    Kover, Katalin E.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane interactions in small fast-tumbling bicelles as studied by P-31 NMR2015Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1848, nr 3, s. 760-766Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Small fast-tumbling bicelles are ideal for studies of membrane interactions at molecular level; they allow analysis of lipid properties using solution-state NMR. In the present study we used P-31 NMR relaxation to obtain detailed information on lipid head-group dynamics. We explored the effect of two topologically different membrane-interacting peptides on bicelles containing either dimyristoylphosphocholine (DMPC), or a mixture of DMPC and dimyristoylphosphoglycerol (DMPG), and dihexanoylphosphocholine (DHPC). KALP21 is a model transmembrane peptide, designed to span a DMPC bilayer and dynorphin B is a membrane surface active neuropeptide. KALP21 causes significant increase in bicelle size, as evidenced by both dynamic light scattering and P-31 T-2 relaxation measurements. The effect of dynorphin B on bicelle size is more modest, although significant effects on T-2 relaxation are observed at higher temperatures. A comparison of P-31 T-1 values for the lipids with and without the peptides showed that dynorphin B has a greater effect on lipid head-group dynamics than KALP21, especially at elevated temperatures. From the field-dependence of T-1 relaxation data, a correlation time describing the overall lipid motion was derived. Results indicate that the positively charged dynorphin B decreases the mobility of the lipid molecules - in particular for the negatively charged DMPG - while KALP21 has a more modest influence. Our results demonstrate that while a transmembrane peptide has severe effects on overall bilayer properties, the surface bound peptide has a more dramatic effect in reducing lipid head-group mobility. These observations may be of general importance for understanding peptide-membrane interactions.

  • 14. Brown, Christian
    et al.
    Szpryngiel, Scarlett
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kuang, Guanglin
    Srivastava, Vaibhav
    Ye, Weihua
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    McKee, Lauren S.
    Yaoquan, Tu
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bulone, Vincent
    Structural and functional characterization of the microtubule interacting and trafficking domains of two oomycete chitin synthases2016Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 283, nr 16, s. 3072-3088Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a Microtubule Interacting and Trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed and characterized. Both were shown to interact strongly with the plasma membrane component phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modelling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes. 

  • 15.
    Bárány-Wallje, Elsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Andersson, August
    Gräslund, Astrid
    Mäler, Lena
    Dynamics of transportan in bicelles is surface charge dependent.2006Ingår i: J Biomol NMR, ISSN 0925-2738, Vol. 35, nr 2, s. 137-47Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In this study we investigated the dynamic behavior of the chimeric cell-penetrating peptide transportan in membrane-like environments using NMR. Backbone amide 15N spin relaxation was used to investigate the dynamics in two bicelles: neutral DMPC bicelles and partly negatively charged DMPG-containing bicelles.

    The structure of the peptide as judged from CD and chemical shifts is similar in the two cases. Both the overall motion as well as the local dynamics is, however, different in the two types of bicelles. The overall dynamics of the peptide is significantly slower in the partly negatively charged bicelle environment, as evidenced by longer global correlation times for all measured sites.

    The local motion, as judged from generalized order parameters, is for all sites in the peptide more restricted when bound to negatively charged bicelles than when bound to neutral bicelles (increase in S2 is on average 0.11±0.07). The slower dynamics of transportan in charged membrane model systems cause significant line broadening in the proton NMR spectrum, which in certain cases limits the observation of 1H signals for transportan when bound to the membrane. The effect of transportan on DMPC and DHPC motion in zwitterionic bicelles was also investigated, and the motion of both components in the bicelle was found to be affected.

  • 16.
    Bárány-Wallje, Elsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. biofysik.
    Andersson, August
    Gräslund, Astrid
    Mäler, Lena
    NMR solution structure and position of transportan in neutral phospholipid bicelles.2004Ingår i: FEBS Lett, ISSN 0014-5793, Vol. 567, nr 2-3, s. 265-9Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transportan is a chimeric cell-penetrating peptide constructed from the peptides galanin and mastoparan, which has the ability to internalize living cells carrying a hydrophilic load. In this study, we have determined the NMR solution structure and investigated the position of transportan in neutral bicelles. The structure revealed a well-defined -helix in the C-terminal mastoparan part of the peptide and a weaker tendency to form an -helix in the N-terminal domain. The position of the peptide in relation to the membrane, as studied by adding paramagnetic probes, shows that the peptide lies parallel to, and in the head-group region of the membrane surface. This result is supported by amide proton secondary chemical shifts.

  • 17.
    Bárány-Wallje, Elsa
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structure and dynamics of galanin in phospholipid bicelles2007Konferensbidrag (Övrig (populärvetenskap, debatt, mm))
  • 18.
    Dinic, Jelena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Biverståhl, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Parmryd, Ingela
    Stockholms universitet, Naturvetenskapliga fakulteten, Wenner-Grens institut.
    Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing2011Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1808, nr 1, s. 298-306Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.

  • 19.
    Gräslund, Astrid
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Testing membrane interactions of CPPs2011Ingår i: Methods in molecular biology (Clifton, N.J.), ISSN 1940-6029, Vol. 683, s. 33-40Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The chapter deals with some biophysical methods used for investigating CPP-induced changes in membrane properties by spectroscopy methods such as fluorescence or NMR and methods used for probing CPP-induced leakage in membranes. Some useful model systems for biomembranes are described. These include large unilamellar phospholipid vesicles (LUVs) of well-defined size (diameter typically 100 nm). A protocol for the preparation of such vesicles is included. The leakage studies make use of LUVs with entrapped dye molecules. The NMR studies make use of mixed micelles (bicelles) as a membrane mimetic system, which can be oriented in the magnetic field of the spectrometer.

  • 20.
    Haglund, Ellinor
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öman, Tommy
    Department of chemistry, Umeå university.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öhman, Anders
    Department of chemistry, Umeå university.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oliveberg, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The HD-exchange motions of ribosomal protein S6 are insensitive to reversal of the protein-folding pathway.2009Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 1091-6490, Vol. 106, nr 51, s. 21619-21624Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (sigma1 and sigma2) defining two competing pathways in the folding energy landscape: sigma1 --> sigma2 and sigma2 --> sigma1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.

  • 21. Jornvall, Hans
    et al.
    Lindahl, Emma
    Astorga-Wells, Juan
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Holmlund, Anna
    Melles, Ermias
    Alvelius, Gunvor
    Nerelius, Charlotte
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Johansson, Jan
    Oligomerization and insulin interactions of proinsulin C-peptide: Threefold relationships to properties of insulin2010Ingår i: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 391, nr 3, s. 1561-1566Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Three principally different sites of action have been reported for proinsulin C-peptide, at surface-mediated, intracellular, and extracellular locations. Following up on the latter, we now find that (i) mass spectrometric analyses reveal the presence of the C-peptide monomer in apparent equilibrium with a low-yield set of oligomers in weakly acidic or basic aqueous solutions, even at low peptide concentrations (sub-mu M). It further shows not only C-peptide to interact with insulin oligomers (known before), but also the other way around. (ii) Polyacrylamide gel electrophoresis of C-peptide shows detectable oligomers upon Western blotting. Formation of thioflavin T positive material was also detected. (iii) Cleavage patterns of analogues are compatible with C-peptide as a substrate of insulin degrading enzyme. Combined, the results demonstrate three links with insulin properties, in a manner reminiscent of amyloidogenic peptides and their chaperons in other systems. If so, peripheral C-peptide/insulin interactions, absolute amounts of both peptides and their ratios may be relevant to consider in diabetic and associated diseases.

  • 22.
    Liebau, Jobst
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Fu, Biao
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brown, Christian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    New insights into the membrane association mechanism of the glycosyltransferase WaaG from Escherichia coli2018Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1860, nr 3, s. 683-690Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Monotopic glycosyltransferases (GTs) interact with membranes via electrostatic interactions. The N-terminal domain is permanently anchored to the membrane while the membrane interaction of the C-terminal domain is believed to be weaker so that it undergoes a functionally relevant conformational change upon donor or acceptor binding. Here, we studied the applicability of this model to the glycosyltransferase WaaG. WaaG is involved in the synthesis of lipopolysaccharides (LPS) in Gram-negative bacteria and was previously categorized as a monotopic GT. We analyzed the binding of WaaG to membranes by stopped-flow fluorescence and NMR diffusion experiments. We find that electrostatic interactions are required to bind WaaG to membranes while mere hydrophobic interactions are not sufficient. WaaG senses the membrane's surface charge density but there is no preferential binding to specific anionic lipids. However, the binding is weaker than expected for monotopic GTs but similar to peripheral GTs. Therefore, WaaG may be a peripheral GT and this could be of functional relevance in vivo since LPS synthesis occurs only when WaaG is membrane-bound. We could not observe a C-terminal domain movement under our experimental conditions.

  • 23.
    Liebau, Jobst
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Fu, Biao
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brown, Christian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The glycosyltransferase WaaG: a peripheral membrane protein?Manuskript (preprint) (Övrigt vetenskapligt)
  • 24.
    Liebau, Jobst
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Immersion Depths of Lipid Carbons in Bicelles Measured by Paramagnetic Relaxation Enhancement2017Ingår i: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 121, nr 32, s. 7660-7670Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Myriads of biological processes occur in or at cellular lipid membranes. Knowledge about the localization of proteins, lipids, and other molecules within biological membranes is thus crucial for the understanding of such processes. Here, we present a method to determine the immersion depths of lipid carbon atoms in membranes by paramagnetic relaxation enhancement (PRE) caused by the presence of doxylated lipids. As membrane mimetics, we employ small isotropic bicelles made of synthetic lipids and of natural Escherichia coli phospholipid extract. Bicelles are particularly suitable for solution state NMR since they maintain a lipid bilayer while they are at the same time amenable to solution state NMR experiments. PREs were measured in the presence of different doxylated lipids with the nitroxide radical located in the headgroup and at various positions in the acyl chain. Theoretical PREs were calculated assuming a simple bicelle model using the Solomon–Bloembergen equations. Immersion depths of the lipid carbon atoms were obtained by a least-squares fit of the theoretical to the experimental PREs. The carbon immersion depths correspond well to results obtained by other methods and differences do not exceed 3–5 Å. This means that the method presented here provides sufficient resolution to distinguish the localization of carbons in different regions of the lipid bilayer, despite considerable simplifications of the underlying theory. These simplifications include a simple form of the spectral density function, which we find is sufficient to reliably determine immersion depths. A more complicated spectral density function that includes bicelle, lipid, and local motions may only improve the results if its parametrization is good enough. The approach presented here may be extended to the determination of protein localization in membranes employing realistic membrane mimetics like the bicelles made of E. coli phospholipid extract used here.

  • 25.
    Liebau, Jobst
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pettersson, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Szpryngiel, Scarlett
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane Interaction of the Glycosyltransferase WaaG2015Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 109, nr 3, s. 552-563Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The glycosyltransferase WaaG is involved in the synthesis of lipopolysaccharides that constitute the outer leaflet of the outer membrane in Gram-negative bacteria such as Escherichia coli. WaaG has been identified as a potential antibiotic target, and inhibitor scaffolds have previously been investigated. WaaG is located at the cytosolic side of the inner membrane, where the enzyme catalyzes the transfer of the first outer-core glucose to the inner core of nascent lipopolysaccharides. Here, we characterized the binding of WaaG to membrane models designed to mimic the inner membrane of E. coli. Based on the crystal structure, we identified an exposed and largely a-helical 30-residue sequence, with a net positive charge and several aromatic amino acids, as a putative membrane-interacting region of WaaG (MIR-WaaG). We studied the peptide corresponding to this sequence, along with its bilayer interactions, using circular dichroism, fluorescence quenching, fluorescence anisotropy, and NMR. In the presence of dodecylphosphocholine, MIR-WaaG was observed to adopt a three-dimensional structure remarkably similar to the segment in the crystal structure. We found that the membrane interaction of WaaG is conferred at least in part by MIR-WaaG and that electrostatic interactions play a key role in binding. Moreover, we propose a mechanism of anchoring WaaG to the inner membrane of E. coli, where the central part of MIR-WaaG inserts into one leaflet of the bilayer. In this model, electrostatic interactions as well as surface-exposed Tyr residues bind WaaG to the membrane.

  • 26.
    Liebau, Jobst
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Pettersson, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Zuber, Philipp
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ariöz, Candan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Chalmers University of Technology, Sweden.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Fast-tumbling bicelles constructed from native Escherichia coli lipids2016Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, nr 9, s. 2097-2105Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Solution-state NMR requires small membrane mimetic systems to allow for acquiring high-resolution data. At the same time these mimetics should faithfully mimic biological membranes. Here we characterized two novel fast-tumbling bicelle systems with lipids from two Escherichia coli strains. While strain 1 (AD93WT) contains a characteristic E. coli lipid composition, strain 2 (AD93-PE) is not capable of synthesizing the most abundant lipid in E. coli, phosphatidylethanolamine. The lipid and acyl chain compositions were characterized by P-31 and C-13 NMR. Depending on growth temperature and phase, the lipid composition varies substantially, which means that the bicelle composition can be tuned by using lipids from cells grown at different temperatures and growth phases. The hydrodynamic radii of the bicelles were determined from translational diffusion coefficients and NMR spin relaxation was measured to investigate lipid properties in the bicelles. We find that the lipid dynamics are unaffected by variations in lipid composition, suggesting that the bilayer is in a fluid phase under all conditions investigated here. Backbone glycerol carbons are the most rigid positions in all lipids, while head-group carbons and the first carbons of the acyl chain are somewhat more flexible. The flexibility increases down the acyl chain to almost unrestricted motion at its end. Carbons in double bonds and cyclopropane moieties are substantially restricted in their motional freedom. The bicelle systems characterized here are thus found to faithfully mimic E. coli inner membranes and are therefore useful for membrane interaction studies of proteins with E. coli inner membranes by solution-state NMR.

  • 27.
    Liebau, Jobst
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ye, Weihua
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Characterization of fast-tumbling isotropic bicelles by PFG diffusion NMR2017Ingår i: Magnetic Resonance in Chemistry, ISSN 0749-1581, E-ISSN 1097-458X, Vol. 55, nr 5, s. 395-404Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Small isotropic bicelles are versatile membrane mimetics, which, in contrast tomicelles, provide a lipid bilayer and are at the same time suitable for solution-state NMR studies. The lipid composition of the bilayer is flexible allowing for incorporation of various head groups and acyl chain types. In bicelles, lipids are solubilized by detergents, which are localized in the rimof the disk-shaped lipid bilayer. Bicelles have been characterized by a broad array of biophysical methods, pulsed-field gradient NMR (PFG NMR) being one of them. PFG NMR can readily be used to measure diffusion coefficients of macromolecules. It is thus employed to characterize bicelle size and morphology. Even more importantly, PFG NMR can be used to study the degree of protein association to membranes. Here, we present the advances that have been made in producing small, fast-tumbling isotropic bicelles from a variety of lipids and detergents, together with insights on the morphology of such mixtures gained from PFG NMR. Furthermore, we review approaches to study protein-membrane interaction by PFG NMR.

  • 28.
    Lind, Jesper
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane Interactions of Dynorphins2006Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, s. 15931-15940Artikel i tidskrift (Refereegranskat)
  • 29.
    Lind, Jesper
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindahl, Emma
    Perálvarez-Marín, Alex
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Holmlund, Anna
    Jörnvall, Hans
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural features of proinsulin C-peptide oligomeric and amyloid states2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr 18, s. 3759-68Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The formation and structure of proinsulin C-peptide oligomers has been investigated by PAGE, NMR spectroscopy and dynamic light scattering. The results obtained show that C-peptide forms oligomers of different sizes, and that their formation and size distribution is altered by salt and divalent metal ions, which indicates that the aggregation process is mediated by electrostatic interactions. It is further demonstrated that the size distribution of the C-peptide oligomers, in agreement with previous studies, is altered by insulin, which supports a physiologically relevant interaction between these two peptides. A small fraction of oligomers has previously been suggested to be in equilibrium with a dominant fraction of soluble monomers, and this pattern also is observed in the present study. The addition of modest amounts of sodium dodecyl sulphate at low pH increases the relative amount of oligomers, and this effect was used to investigate the details of both oligomer formation and structure by a combination of biophysical techniques. The structural properties of the SDS-induced oligomers, as obtained by thioflavin T fluorescence, CD spectroscopy and IR spectroscopy, demonstrate that soluble aggregates are predominantly in β-sheet conformation, and that the oligomerization process shows characteristic features of amyloid formation. The formation of large, insoluble, β-sheet amyloid-like structures will alter the equilibrium between monomeric C-peptide and oligomers. This leads to the conclusion that the oligomerization of C-peptide may be relevant also at low concentrations.

  • 30.
    Lind, Jesper
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordin, Jon
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lipid dynamics in fast-tumbling bicelles with varying bilayer thickness: Effect of model transmembrane peptides2008Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1778, nr 11, s. 2526-2534Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The morphology of q=0.5 fast-tumbling bicelles prepared with three different acyl chain lengths has been investigated by NMR. It is shown that bicelles prepared with DLPC (12 C) and DHPC are on average larger than those containing DMPC or DPPC (14 and 16 C) and DHPC, which may be due to a higher degree of mixing between DLPC and DHPC. The fast internal mobility of the lipids was determined from natural abundance carbon-13 relaxation. A similar dynamical behaviour of the phospholipids in the three different bicelles was observed, although the DPPC lipid acyl chain displayed a somewhat lower degree of mobility, as evidenced by higher generalized order parameters throughout the acyl chain. Carbon-13 relaxation was also used to determine the effect of different model transmembrane peptides, with flanking Lys residues, on the lipid dynamics in the three different bicelles. All peptides had the effect of increasing the order parameters for the DLPC lipid, while no effect was observed on the longer lipid chains. This effect may be explained by a mismatch between the hydrophobic length of the peptides and the DLPC lipid acyl chain.

  • 31.
    Lind, Jesper
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rämö, Tuulia
    Rosén Klement, Maria L.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bárány-Wallje, Elsa
    Epand, Richard M.
    Epand, Raquel F.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    High cationic charge and bilayer interface-binding helices in a regulatory lipid glycosyltransferase2007Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, nr 19, s. 5664-5677Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In the prokaryote Acholeplasma laidlawii, membrane bilayer properties are sensed and regulated by two interface glycosyltransferases (GTs), synthesizing major nonbilayer- (alMGS GT) and bilayer-prone glucolipids. These enzymes are of similar structure, as many soluble GTs, but are sensitive to lipid charge and curvature stress properties. Multivariate and bioinformatic sequence analyses show that such interface enzymes, in relation to soluble ones of similar fold, are characterized by high cationic charge, certain distances between small and cationic amino acids, and by amphipathic helices. Varying surface contents of Lys/Arg pairs and Trp indicate different membrane-binding subclasses. A predicted potential (cationic) binding helix from alMGS was structurally verified by solution NMR and CD. The helix conformation was induced by a zwitterionic as well as anionic lipid environment, and the peptide was confined to the bilayer interface. Bilayer affinity of the peptide, analyzed by surface plasmon resonance, was higher than that for soluble membrane-seeking proteins/peptides and rose with anionic lipid content. Interface intercalation was supported by phase equilibria in membrane lipid mixtures, analyzed by 31P NMR and DSC. An analogous, potentially binding helix has a similar location in the structurally determined Escherichia coli cell wall precursor GT MurG. These two helices have little sequence conservation in alMGS and MurG homologues but maintain their amphipathic character. The evolutionary modification of the alMGS binding helix and its location close to the acceptor substrate site imply a functional importance in enzyme catalysis, potentially providing a mechanism by which glycolipid synthesis will be sensitive to membrane surface charge and intrinsic curvature strain.

  • 32.
    Lindholm, Ljubica
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ariöz, Candan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jawurek, Michael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Liebau, Jobst
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Ballmoos, Christoph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Barth, Andreas
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Effect of lipid bilayer properties on the photocycle of green proteorhodopsin2015Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1847, nr 8, s. 698-708Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocyde was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Eschetichia coil was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-beta-D-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurifled phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coil cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.

  • 33. Metola, Ane
    et al.
    Bouchet, Ana M.
    Alonso-Marino, Marian
    Diercks, Tammo
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Goni, Felix M.
    Viguera, Ana R.
    Purification and characterization of the colicin A immunity protein in detergent micelles2017Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1859, nr 11, s. 2181-2192Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the colicin A immunity protein, was characterized here in detergent micelles by circular dichroism (CD), size exclusion chromatography, chemical cross-linking, nuclear magnetic resonance (NMR) spectroscopy, cysteine accessibility, and colicin A binding in detergent micelles. Bile salt derivatives induced extensive protein polymerization that precluded further investigation. The physical characterization of detergent-solubilized protein indicates that phosphate-containing detergents are more efficient in extracting, solubilizing and maintaining Cai in a monomeric state. Yet, their capacity to ensure protein activity, reconstitution, helix packing, and high-quality NMR spectra was inferior to that of milder detergents. Solvent ionic strength and composition greatly modified the solubilizing capacity of milder detergents. Most importantly, binding to the colicin A pore-forming domain (pf-ColA) occurred almost exclusively in sugar-derived detergents. The relative performance of the different detergents in each experiment depends on their impact not only on Cai structure, solubility and oligomerization state, but also on other reaction components and technical aspects. Thus, proteoliposomes were best obtained from protein in LDAO micelles, possibly also due to indirect effects on the lipidic bilayer. The compatibility of a detergent with Cai/pf-ColA complex formation is influenced by its effect on the conformational landscape of each protein, where detergent-mediated pf-ColA denaturation could also lead to negative results. The NMR spectra were greatly affected by the solubility, monodispersity, fold and dynamics of the protein-detergent complexes, and none of those tested here provided NMR spectra of sufficient quality to allow for peak assignment. Cai function could be proven in alkyl glycosides and not in those detergents that afforded the best solubility, reconstitution efficiency or spectral quality indicating that these criteria cannot be taken as unambiguous proof of nativeness without the support of direct activity measurements.

  • 34.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Solution NMR studies of cell-penetrating peptides in model membrane systems2013Ingår i: Advanced Drug Delivery Reviews, ISSN 0169-409X, E-ISSN 1872-8294, Vol. 65, nr 8, s. 1002-1011Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are a class of short, often cationic peptides that have the capability to translocate across cellular membranes, and although the translocation most likely involves several pathways, they interact directly with membranes, as well as with model bilayers. Most CPPs attain a three-dimensional structure when interacting with bilayers, while they are more or less unstructured in aqueous solution. To understand the relationship between structure and the effect that CPPs have on membranes it is of great importance to investigate CPPs at atomic resolution in a suitable membrane model. Moreover, the location in bilayers is likely to be correlated with the translocation mechanism. Solution-state NMR offers a unique possibility to investigate structure, dynamics and location of proteins and peptides in bilayers. This review focuses on solution NMR as a tool for investigating CPP-lipid interactions. Structural propensities and cell-penetrating capabilities can be derived from a combination of CPP solution structures and studies of the effect that the peptides have on bilayers and the localization in a bilayer.

  • 35.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Solution NMR studies of peptide-lipid interactions in model membranes2012Ingår i: Molecular membrane biology, ISSN 0968-7688, E-ISSN 1464-5203, Vol. 29, nr 5 SI, s. 155-176Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Many important processes in life take place in or around the cell membranes. Lipids have different properties regarding their membrane-forming capacities, their mobility, shape, size and surface charge, and all of these factors influence the way that proteins and peptides interact with the membrane. In order for us to correctly understand these interactions, we need to be able to study all aspects of the interplay between lipids and peptides and proteins. Solution-state NMR offers a somewhat unique possibility to investigate structure, dynamics and location of proteins and peptides in bilayers. This review focuses on solution NMR as a tool for investigating peptide-lipid interaction, and special attention is given to the various membrane mimetics that are used to model the membrane. Examples from the field of cell-penetrating peptides and their lipid interactions will be given. The importance of studying lipid and peptide dynamics, which reflect on the effect that peptides have on bilayers, is highlighted, and in this respect, also the need for realistic membrane models.

  • 36.
    Mäler, Lena
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    NMR studies of three-dimensional structure and positioning of CPPs in membrane model systems2011Ingår i: Methods in molecular biology (Clifton, N.J.), ISSN 1940-6029, Vol. 683, s. 57-67Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    CPPs are generally short cationic peptides that have the capability to interact directly with membranes. Most CPPs attain a three-dimensional structure when interacting with bilayers, while they are more or less unstructured in aqueous solution. To understand the relationship between structure and the effect that CPPs have on membranes, it is of great importance to investigate CPPs with atomic resolution in a suitable membrane model. Nuclear magnetic resonance (NMR) is an excellent technique both for studying solution structures of peptides as well as for investigating their location within a model bilayer. This chapter outlines protocols for producing model membrane systems for NMR investigations as well as the basic NMR tools for determining the three-dimensional structure of CPPs and for investigating the details in lipid-peptide interactions, i.e., the localization of the CPP in the bilayer.

  • 37.
    Papadopoulos, E
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oglecka, K
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, L
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jarvet, J
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wright, PE
    Dyson, HJ
    Gräslund, A
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    NMR solution structure of the peptide fragment 1-30, derived from unprocessed mouse Doppel protein, in DHPC micelles.2006Ingår i: Biochemistry, ISSN 0006-2960, Vol. 45, nr 1, s. 159-66Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The downstream prion-like Doppel (Dpl) protein is a homologue related to the prion protein (PrP). Dpl is expressed in the brains of mice that do not express PrP, and Dpl is known to be toxic to neurons. One mode of toxicity has been suggested to involve direct membrane interactions. PrP under certain conditions of cell trafficking retains an uncleaved signal peptide, which may also hold for the much less studied Dpl. For a peptide with a sequence derived from the N-terminal part (1-30) of mouse Dpl (mDpl(1-30)) CD spectroscopy shows about 40% alpha-helical structure in DHPC and SDS micelles. In aqueous solution it is mostly a random coil. The three-dimensional solution structure was determined by NMR for mDpl(1-30) associated with DHPC micelles. 2D 1H NMR spectra of the peptide in q = 0.25 DMPC/DHPC bicelles only showed signals from the unstructured termini, indicating that the structured part of the peptide resides within the lipid bilayer. Together with 2H2O exchange data in the DHPC micelle solvent, these results show an alpha-helix protected from solvent exchange between residues 7 and 19, and suggest that the alpha-helical segment can adopt a transmembrane localization also in a membrane. Leakage studies with entrapped calcein in large unilamellar phospholipid vesicles showed that the peptide is almost as membrane perturbing as melittin, known to form pores in membranes. The results suggest a possible channel formation mechanism for the unprocessed Dpl protein, which may be related to toxicity through direct cell membrane interaction and damage.

  • 38.
    Pettersson, Pontus
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Patrick, Joan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jakob, Mario
    Klösgen, Ralf Bernd
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Soluble TatA forms oligomers that interact with membranes: structure and insertion studies of a versatile protein transporterManuskript (preprint) (Övrigt vetenskapligt)
  • 39.
    Pettersson, Pontus
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ye, Weihua
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Jakob, Mario
    Tannert, Franzisca
    Klösgen, Ralf Bernd
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structure and dynamics of plant TatA in micelles and lipid bilayers studied by solution NMR2018Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 285, nr 10, s. 1886-1906Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The twin-arginine translocase (Tat) transports folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. In Gram-negative bacteria and chloroplasts, the translocon consists of three subunits, TatA, TatB, and TatC, of which TatA is responsible for the actual membrane translocation of the substrate. Herein we report on the structure, dynamics, and lipid interactions of a fully functional C-terminally truncated core TatA' from Arabidopsisthaliana using solution-state NMR. Our results show that TatA consists of a short N-terminal transmembrane helix (TMH), a short connecting linker (hinge) and a long region with propensity to form an amphiphilic helix (APH). The dynamics of TatA were characterized using N-15 relaxation NMR in combination with model-free analysis. The TMH has order parameters characteristic of a well-structured helix, the hinge is somewhat less rigid, while the APH has lower order parameters indicating structural flexibility. The TMH is short with a surprisingly low protection from solvent, and only the first part of the APH is protected to some extent. In order to uncover possible differences in TatA's structure and dynamics in detergent compared to in a lipid bilayer, fast-tumbling bicelles and large unilamellar vesicles were used. Results indicate that the helicity of TatA increases in both the TMH and APH in the presence of lipids, and that the N-terminal part of the TMH is significantly more rigid. The results indicate that plant TatA has a significant structural plasticity and a capability to adapt to local environments.

  • 40. Stromqvist, Johan
    et al.
    Chmyrov, Andriy
    Johansson, Sofia
    Andersson, August
    Maler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Widengren, Jerker
    Quenching of Triplet State Fluorophores for Studying Diffusion-Mediated Reactions in Lipid Membranes2010Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 99, nr 11, s. 3821-3830Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    An approach to study bimolecular interactions in model lipid bilayers and biological membranes is introduced, exploiting the influence of membrane associated electron spin resonance labels on the triplet state kinetics of membrane bound fluorophores Singlet triplet state transitions within the dye Lissamine Rhodamine B (LRB) were studied when free in aqueous solutions, with LRB bound to a lipid in a liposome and in the presence of different local concentrations of the electron spin resonance label TEMPO By monitoring the triplet state kinetics via variations in the fluorescence signal, in this study using fluorescence correlation spectroscopy a strong fluorescence signal can be combined with the ability to monitor low frequency molecular interactions at timescales much longer than the fluorescence lifetimes Both in solution and in membranes the measured relative changes in the singlet triplet transitions rates were found to well reflect the expected collisional frequencies between the LRB and TEMPO molecules These collisional rates could also be monitored at local TEMPO concentrations where practically no quenching of the excited state of the fluorophores can be detected The proposed strategy is broadly applicable in terms of possible read out means types of molecular interactions that can be followed, and in what environments these interactions can be measured

  • 41.
    Ståhl, Annelie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nilsson, Stefan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lundberg, Pontus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi.
    Bhushan, Shashi
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biverståhl, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Moberg, Per
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Morisett, Magali
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vener, Alexander
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi och neurotoxikologi.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Two Novel Targeting Peptide Degrading Proteases, PrePs, in Mitochondria and Chloroplasts, so Similar and Still Different2005Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 349, nr 4, s. 847-860Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F1β pre-sequence, peptides derived from the F1β pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F1β pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P1 position and small, uncharged amino acids or serine residues in the P1P′1

    position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the α-helical elements of the F1β pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic α-helix and positively charged amino acid residues and degraded the F1β pre-sequence into 10–16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F1β pre-sequence into 10–23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.

  • 42.
    Szpryngiel, Scarlett
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ge, Changrong
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lakovleva, Irina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Georgiev, Alexander
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wieslander, Åke
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lipid Interacting Regions in Phosphate Stress Glycosyltransferase atDGD2 from Arabidopsis thaliana2011Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, nr 21, s. 4451-4466Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227–245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130–148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227–245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.

  • 43.
    Szpryngiel, Scarlett
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Insights into the membrane interacting properties of the C-terminal domain of the monotopic glycosyltransferase DGD2 in A. thalianaManuskript (preprint) (Övrigt vetenskapligt)
  • 44.
    Szpryngiel, Scarlett
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Insights into the Membrane Interacting Properties of the C-Terminal Domain of the Monotopic Glycosyltransferase DGD2 in Arabidopsis thaliana2016Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, nr 49, s. 6776-6786Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Glycosyltransferases (GTs) are responsible for regulating the membrane composition of plants. The synthesis of one of the main lipids in the membrane, the galactolipid digalactosyldiacylglycerol, is regulated by the enzyme digalactosyldiacylglycerol synthase 2 (atDGD2) under starving conditions, such as phosphate shortage. The enzyme belongs to the GT-B fold, characterized by two beta/alpha/beta Rossmann domains that are connected by a flexible linker. atDGD2 has previously been shown to attach to lipid membranes by the N-terminal domain via interactions with negatively charged lipids. The role of the C-terminal domain in the membrane interaction is, however, not known. Here we have used a combination of in silico prediction methods and biophysical experimental techniques to shed light on the membrane interacting properties of the C-terminal domain. Our results demonstrate that there is an amphipathic sequence, corresponding to residues V240-E258, that interacts with lipids in a charge-dependent way. A second sequence was identified as being potentially important, with a high charge density, but no amphipathic character. The features of the plant atDGD2 observed here are similar in prokaryotic glycosyltransferases. On the basis of our results, and by analogy to other glycosyltransferases, we propose that atDGD2 interacts with the membrane through the N-terminus and with parts of the C-terminus acting as a switch, allowing for a dynamic interaction with the membrane.

  • 45.
    Szpryngiel, Scarlett
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Oliveberg, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Diffuse binding of Zn2+ to the denatured ensemble of Cu/Zn superoxide dismutase 12015Ingår i: FEBS Open Bio, E-ISSN 2211-5463, Vol. 5, s. 56-63Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The stability and structural properties of the metalloprotein superoxide dismutase 1 (SOD1) are found to depend critically on metal ions. Native SOD1 monomers coordinate one structural Zn2+ and one redox-active Cu2+/1+ to the active site. To do this, the Zn2+ ions need to interact with the SOD1 protein on the denatured side of the folding barrier, prior to the formation of the folding nucleus. In this study, we have examined at residue level the nature of this early Zn2+ binding by NMR studies on the urea denatured-state of SOD1. Nearly complete backbone chemical shift assignments were obtained in 9 M urea at physiological pH, conditions at which NMR studies are scarce. Our results demonstrate that SOD1 is predominantly unstructured under these conditions. Chemical-shift changes upon Zn2+ titration show that denatured SOD1 retains a significant affinity to Zn2+ ions, even in 9 M urea. However, the Zn2+ interactions are not limited to the native metal-binding ligands in the two binding sites, but are seen for all His residues. Moreover, the native Cu2+/1+ ligand H46 seems not to bind as well as the other His residues, while the nearby non-native H43 does bind, indicating that the binding geometry is relaxed. The result suggests that the Zn2+-binding observed to catalyze folding of SOD1 in physiological buffer is initiated by diffuse, non-specific coordination to the coil, which subsequently funnels by ligand exchange into the native coordination geometry of the folded monomer. Altogether, this diffuse binding is a result with fundamental implications for folding of metalloproteins in general.

  • 46.
    Unnerståle, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lind, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Papadopoulos, Evangelos
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Solution structure of the HsapBK K+ channel voltage-sensor paddle sequence2009Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 25, s. 5813-5821Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Voltage-gated potassium channels open and close in response to changes in the membrane potential. In this study, we have determined the NMR solution structure of the putative S3b-S4 voltage-sensor paddle fragment, the part that moves to mediate voltage gating, of the HsapBK potassium channel in dodecylphosphocholine (DPC) micelles. This paper presents the first structure of the S3b-S4 fragment from a BK channel. Diffusion coefficients as determined from PFG NMR experiments showed that a well-defined complex between the peptide and DPC molecules was formed. The structure reveals a helix-turn-helix motif, which is in agreement with crystal structures of other voltage-gated potassium channels, thus indicating that it is feasible to study the isolated fragment. The paddle motifs generally contain several basic residues, implicated in the gating. The critical Arg residues in this structure all reside on the surface, which is in agreement with crystal structures of K(v) channels. Similarities in the structure of the S3b-S4 fragment in BK and K(v) channels as well as important differences are seen, which may be important for explaining the details in paddle movement within a bilayer.

  • 47.
    Unnerståle, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Madani, Fatemeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gräslund, Astrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Membrane-perturbing properties of two Arg-rich paddle domains from voltage-gated sensors in the KvAP and HsapBK K+ channels2012Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 19, s. 3982-3992Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Voltage-gated K+ channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a “paddle” domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the Kv channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic–hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.

  • 48.
    Unnerståle, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    pH-Dependent Interaction between C-Peptide and Phospholipid Bicelles2012Ingår i: Journal of Biophysics, ISSN 1687-8019, s. 185907-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    C-peptide is the connecting peptide between the A and B chains of insulin in proinsulin. In this paper, we investigate the interaction between C-peptide and phospholipid bicelles, by circular dichroism and nuclear magnetic resonance spectroscopy, and in particular the pH dependence of this interaction. The results demonstrate that C-peptide is largely unstructured independent of pH, but that a weak structural induction towards a short stretch of β-sheet is induced at low pH, corresponding to the isoelectric point of the peptide. Furthermore, it is demonstrated that C-peptide associates with neutral phospholipid bicelles as well as acidic phospholipid bicelles at this low pH. C-peptide does not undergo a large structural rearrangement as a consequence of lipid interaction, which indicates that the folding and binding are uncoupled. In vivo, local variations in environment, including pH, may cause C-peptide to associate with lipids, which may affect the aggregation state of the peptide.

  • 49.
    Unnerståle, Sofia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Draheim, Roger R.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural characterization of AS1-membrane interactions from a subset of HAMP domains2011Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1808, nr 10, s. 2403-2412Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    HAMP domains convert an extracellular sensory input into an intracellular signaling response in a wide variety of membrane-embedded bacterial proteins. These domains are almost invariably found adjacent to the inner leaflet of the cell membrane. We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of four different, well-characterized HAMP domains with several membrane model systems. The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli osmosensor EnvZ(Ec), the E. coli nitrate/nitrite sensor NarX(Ec), and the aspartate chemoreceptor of E. coli (Tar(Ec)). Far-UV CD and NMR spectroscopy were used to monitor the induction of secondary structure upon association with neutral or acidic large unilamellar vesicles (LUVs) and bicelles. We observed significant increases in alpha-helicity within AS1 from NarX(Ec) and Tar(Ec) but not in AS1 from the other proteins. To characterize these interactions further, we determined the solution structure of AS1 from Tar(Ec) associated with acidic bicelles. The bulk of AS1 formed an amphipathic alpha-helix, whereas the N-terminal control cable, the region between TM2 and AS1, remained unstructured. We observed that the conserved prolyl residue found in AS1 of many membrane-adjacent HAMP domains defined the boundary between the unstructured and helical regions. In addition, two positively charged residues that flank the hydrophobic surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We interpret these results within the context of the helix-interaction model for HAMP signaling and propose roles for AS1-membrane interactions during the membrane assembly and transmembrane communication of HAMP-containing receptors.

  • 50.
    Ye, Weihua
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Liebau, Jobst
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mäler, Lena
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    New Membrane Mimetics with Galactolipids: Lipid Properties in Fast-Tumbling Bicelles2013Ingår i: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, nr 4, s. 1044-1050Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Galactolipids are the main structural component of plant chloroplastic (thylakoid) membranes and of blue-green algae cell membranes. The predominant lipids in this class are monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG). We here present a method for the preparation of bicelles that contain these galactolipids together with a characterization of the bicelles, and the lipids within the bicelles. NMR diffusion data show that up to 3096 of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in a q = 0.5 DMPC/DHPC lipid matrix can be replaced with either monogalactosyl-diacylglycerol or digalactosyl-diacylglycerol and that these lipids incorporate into the bicelles. No evidence for phase separation is observed. Bicelles made with monogalactosyl-diacylglycerol are significantly larger than bicelles containing only DMPC, already with only 1096 of the DMPC replaced with the galactolipid. The effect of digalactosyl-diacylglycerol on bicelle size is much smaller. These observations are likely to be correlated with the different bilayer-forming properties of the lipids. Monogalactosyl-diacylglycerol is a non-bilayer-forming lipid, while digalactosyl-diacylglycerol is a bilayer-forming lipid. Both galactolipids display extensive local motion within the bilayer, as evidenced by natural abundance carbon-13 relaxation of the lipid molecules. The sugar headgroup regions are motionally restricted and cannot be described by a model that does not take into account anisotropic reorientation of the sugar units. No significant effect of the galactolipids on DMPC dynamics was observed. Our results indicate that these bicelles may become useful as model membrane mimetic media for studies of galactolipid-protein interactions.

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