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  • 1.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jarvet, Jüri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. The National Institute of Chemical Physics and Biophysics, Estonia.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ionic Strength Modulation of the Free Energy Landscape of A beta(40) Peptide Fibril Formation2016In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 21, 6893-6902 p.Article in journal (Refereed)
    Abstract [en]

    Protein misfolding and formation of cross-beta structured amyloid fibrils are linked to, many neurodegenerative disorders. Although recently developed,quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid beta peptide (A beta 40) self-association, implicated in Alzheimer's disease. We found that physiological ionic strength accelerates A beta 40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between Monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different beta-structured states with =similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (F-beta*) and a mature stable State (F-beta), where salt favors formation of the F-beta fibril morphology. Addition of salt to preformed F-beta* accelerates transition to F-beta, underlining the dynamic nature of A beta 40 fibrils in solution. On the basis of,these results we suggest a model where salt decreases the free-energy barrier for A beta 40 folding to the F-beta state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states.

  • 2.
    Andersson, Julia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hauser, Karin
    Karjalainen, Eeva-Liisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Protonation and hydrogen bonding of Ca2+ site residues in the E2P phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase studied by a combination of infrared spectroscopy and electrostatic calculations.2008In: Biophys J, ISSN 1542-0086, Vol. 94, no 2, 600-11 p.Article in journal (Refereed)
    Abstract [en]

    Protonation of the Ca(2+) ligands of the SR Ca(2+)-ATPase (SERCA1a) was studied by a combination of rapid scan FTIR spectroscopy and electrostatic calculations. With FTIR spectroscopy, we investigated the pH dependence of C=O bands of the Ca(2+)-free phosphoenzyme (E2P) and obtained direct experimental evidence for the protonation of carboxyl groups upon Ca(2+) release. At least three of the infrared signals from protonated carboxyl groups of E2P are pH dependent with pK(a) values near 8.3: a band at 1758 cm(-1) characteristic of nonhydrogen-bonded carbonyl groups, a shoulder at 1720 cm(-1), and part of a band at 1710 cm(-1), both characteristic of hydrogen-bonded carbonyl groups. The bands are thus assigned to H(+) binding residues, some of which are involved in H(+) countertransport. At pH 9, bands at 1743 and 1710 cm(-1) remain which we do not attribute to Ca(2+)/H(+) exchange. We also obtained evidence for a pH-dependent conformational change in beta-sheet or turn structures of the ATPase. With MCCE on the E2P analog E2(TG+MgF(4)(2-)), we assigned infrared bands to specific residues and analyzed whether or not the carbonyl groups of the acidic Ca(2+) ligands are hydrogen bonded. The carbonyl groups of Glu(771), Asp(800), and Glu(908) were found to be hydrogen bonded and will thus contribute to the lower wave number bands. The carbonyl group of some side-chain conformations of Asp(800) is left without a hydrogen-bonding partner; they will therefore contribute to the higher wave number band.

  • 3. Arbesu Valdivia, Alejandro
    et al.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Romero Batista, Yamilet
    Kumar, Saroj
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Characterization of recombinant antibodies for cancer therapy by infrared spectroscopy2013In: Biologicals (Print), ISSN 1045-1056, E-ISSN 1095-8320, Vol. 41, no 2, 104-110 p.Article in journal (Refereed)
    Abstract [en]

    Fourier transform infrared (FTIR) spectroscopy was used to study the structure of the recombinant antibodies 1E10, anti-CD20 and hR3, which are used as anti-cancer therapeutic drugs. We tested their sensitivity against different conditions and treatments such as pH, temperature, freeze-thaw cycles and drying, which are relevant for the practical usefulness of the drugs. All antibodies were stable against moderate temperature increases (up to 50 degrees C) and pH changes (range 5-9). 1E10 was sensitive to extreme pH values (pH 3 and 12), whereas hR3 was most sensitive to temperature (at and above 60 degrees C). We did not observe any significant changes upon freeze-thaw and drying treatments. The secondary structure content of all three antibodies was estimated to be similar to that of IgG with similar to 64% beta-sheet, 0% alpha-helix and similar to 36% other structure. (C) 2012 The International Alliance for Biological Standardization.

  • 4.
    Ariöz, Candan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Götzke, Hansjörg
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindholm, Ljubica
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, Jonny
    Edwards, Katarina
    Daley, Daniel O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Heterologous overexpression of a monotopic glucosyltransferase (MGS) induces fatty acid remodeling in Escherichia coli membranes:  2014In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 7, 1862-1870 p.Article in journal (Refereed)
    Abstract [en]

    The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22 h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~ 62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.

  • 5.
    Ariöz, Candan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ye, Weihua
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    al Bakali, Amin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ge, Changrong
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liebau, Jobst
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Götzke, Hansjörg
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 33, 5533-5544 p.Article in journal (Refereed)
    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.

  • 6.
    Baldassarre, Maurizio
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Baronio, Cesare M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla A.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Amyloid beta-peptides 1-40 and 1-42 form oligomers with mixed beta-sheets2017In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 8, no 12, 8247-8254 p.Article in journal (Refereed)
    Abstract [en]

    Two main amyloid-beta peptides of different length (A beta(40) and A beta(42)) are involved in Alzheimer's disease. Their relative abundance is decisive for the severity of the disease and mixed oligomers may contribute to the toxic species. However, little is know about the extent of mixing. To study whether A beta(40) and A beta(42) co-aggregate, we used Fourier transform infrared spectroscopy in combination with C-13-labeling and spectrum calculation and focused on the amide I vibration, which is sensitive to backbone structure. Mixtures of monomeric labeled A beta(40) and unlabeled A beta(42) (and vice versa) were co-incubated for similar to 20 min and their infrared spectrum recorded. The position of the main C-13-amide I' band shifted to higher wavenumbers with increasing admixture of C-12-peptide due to the presence of C-12-amides in the vicinity of C-13-amides. The results indicate that A beta(40) and A beta(42) form mixed oligomers with a largely random distribution of A beta(40) and A beta(42) strands in their beta-sheets. The structures of the mixed oligomers are intermediate between those of the pure oligomers. There is no indication that one of the peptides forces the backbone structure of its oligomers on the other peptide when they are mixed as monomers. We also demonstrate that isotope-edited infrared spectroscopy can distinguish aggregation modulators that integrate into the backbone structure of their interaction partner from those that do not. As an example for the latter case, the pro-inflammatory calcium binding protein S100A9 is shown not to incorporate into the b-sheets of A beta(42).

  • 7.
    Baldassarre, Maurizio
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pushing the detection limit of infrared spectroscopy for structural analysis of dilute protein samples.2014In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 139, no 21, 5393-5399 p.Article in journal (Refereed)
    Abstract [en]

    Fourier-transform infrared spectroscopy is a powerful and versatile tool to investigate the structure and dynamics of proteins in solution. The intrinsically low extinction coefficient of the amide I mode, the main structure-related oscillator, together with the high infrared absorptivity of aqueous media, requires that proteins are studied at high concentrations (>10 mg L(-1)). This may represent a challenge in the study of aggregation-prone proteins and peptides, and questions the significance of structural data obtained for proteins physiologically existing at much lower concentrations. Here we describe the development of a simple experimental approach that increases the detection limit of protein structure analysis by infrared spectroscopy. Our approach relies on custom-made filters to isolate the amide I region (1700-1600 cm(-1)) from irrelevant spectral regions. The sensitivity of the instrument is then increased by background attenuation, an approach consisting in the use of a neutral density filter, such as a non-scattering metal grid, to attentuate the intensity of the background spectrum. When the filters and grid are combined, a 2.4-fold improvement in the noise level can be obtained. We have successfully tested this approach using a highly diluted solution of pyruvate kinase in deuterated medium (0.2% w/v), and found that it provides spectra of a quality comparable to those recorded with a 10-fold higher protein concentration.

  • 8.
    Baldassarre, Maurizio
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The carbonate/bicarbonate system as a pH indicator for infrared spectroscopy2014In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 139, no 9, 2167-2176 p.Article in journal (Refereed)
    Abstract [en]

    Caged compounds capable of inducing large pH-jumps upon UV illumination have represented a breakthrough in time-resolved infrared spectroscopy of acidification-triggered phenomena, but their use is hampered by the inability to control the initial pH as well as to measure the final pH in mu L volumes. We have developed an experimental approach that accurately measures the initial and final pH values in pH-jump experiments. Our approach exploits the concomitant presence of two or more inorganic ions, such as carbonate and bicarbonate, that are added to the sample at a known concentration. The difference spectrum obtained in the infrared measurement is fitted to isolate the bands arising from the appearance or disappearance of either protonation state, and is then compared to a synthetic library of difference spectra generated using both qualitative (band position and width, extinction coefficient, pK) and quantitative (concentration, pathlength) parameters of the reporter ions. We have tested this approach in UV-photolysis experiments of 1-(2-nitrophenyl)ethyl sulfate in the presence of different concentrations of Na2CO3 and successfully used the infrared absorption of the carbonate and the bicarbonate ions to determine the initial and final pH values before and after the pH-jump, respectively.

  • 9.
    Baldassarre, Maurizio
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bennett, Matthew
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Simultaneous acquisition of infrared, fluorescence and light scattering spectra of proteins: direct evidence for pre-fibrillar species in amyloid fibril formation2016In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 141, no 3, 963-973 p.Article in journal (Refereed)
    Abstract [en]

    Different spectroscopic approaches are often used to probe specific aspects of amyloid fibril formation but are usually performed separately and under different conditions. This makes it problematic to relate different aspects of the aggregation process when these are monitored by different methods. We report on a multispectral approach for simultaneous acquisition of infrared, fluorescence and light scattering spectra of proteins undergoing aggregation. We have applied our approach to study beta-lactoglobulin, a milk protein known to form amyloid fibrils under well-established conditions. Our real-time multispectral measurements show that unfolding of this protein is followed by formation of early aggregates consisting of intermolecular beta-sheets with a typical infrared absorption at similar to 1619 cm(-1) in (H2O)-H-2. These aggregates, which lead to an increase in the light scattering signal, do not bind the amyloid-specific fluorophore ThT and therefore consist of oligomers or protofibrils. Fibril growth is then observed as a sigmoidal increase in ThT fluorescence. After similar to 25 h, a plateau is observed in the intensities of ThT emission and of the band at 1619 cm(-1), indicating that no new fibrils are forming. However, a second phase in the light scattering signal taking place after similar to 25 h suggests that the fibrils are assembling into larger structures, known as mature fibrils. This is associated with an upshift of the main beta-sheet band in the infrared spectrum. TEM analyses confirmed the existence of thick fibrils comprising 3-5 filaments.

  • 10.
    Baldassarre, Maurizio
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Li, Chenge
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eremina, Nadejda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Goormaghtigh, Erik
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Simultaneous Fitting of Absorption Spectra and Their Second Derivatives for an Improved Analysis of Protein Infrared Spectra2015In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 20, no 7, 12599-12622 p.Article in journal (Refereed)
    Abstract [en]

    Infrared spectroscopy is a powerful tool in protein science due to its sensitivity to changes in secondary structure or conformation. In order to take advantage of the full power of infrared spectroscopy in structural studies of proteins, complex band contours, such as the amide I band, have to be decomposed into their main component bands, a process referred to as curve fitting. In this paper, we report on an improved curve fitting approach in which absorption spectra and second derivative spectra are fitted simultaneously. Our approach, which we name co-fitting, leads to a more reliable modelling of the experimental data because it uses more spectral information than the standard approach of fitting only the absorption spectrum. It also avoids that the fitting routine becomes trapped in local minima. We have tested the proposed approach using infrared absorption spectra of three mixed α/β proteins with different degrees of spectral overlap in the amide I region: ribonuclease A, pyruvate kinase, and aconitase.

  • 11.
    Barth, A
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hauser, K
    Andersson, J
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karjalainen, E-L
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton countertransport by the Ca2+ pump2007Conference paper (Other (popular science, discussion, etc.))
  • 12.
    Barth, A
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hauser, K
    Andersson, J
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karjalainen, E-L
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton countertransport by the Ca2+ pump: Pathways and protonation sites studied by infrared spectroscopy and computation2007Conference paper (Other (popular science, discussion, etc.))
  • 13.
    Barth, A
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hauser, K
    Andersson, J
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karjalainen, E-L
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Protonation of acidic Ca2+ ligands of the Ca2+ pump studied by infrared spectroscopy and computation2007Conference paper (Other (popular science, discussion, etc.))
  • 14.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Infrared Spectroscopy - Past and Present2009In: Biological and Biomedical Infrared Spectroscopy / [ed] P.I. Haris, A. Barth, Amsterdam: IOS Press BV , 2009, 1-52 p.Chapter in book (Other (popular science, discussion, etc.))
    Abstract [en]

    History of infrared spectroscopy as well as current technology and applications are reviewed.

  • 15.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Infrared spectroscopy of proteins2007In: Biochim Biophys Acta: Bioenergetics, ISSN 0006-3002, Vol. 1767, no 9, 1073-101 p.Article, review/survey (Other (popular science, discussion, etc.))
    Abstract [en]

    This review discusses the application of infrared spectroscopy to the study of proteins. The focus is on the mid-infrared spectral region and the study of protein reactions by reaction-induced infrared difference spectroscopy.

  • 16.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Quantifying bond distortions in transient enzyme species by a combination of density functional theory calculations and time-resolved infrared difference spectroscopy. Implications for the mechanism of dephosphorylation of the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a)2015In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1847, no 10, 1036-1043 p.Article in journal (Refereed)
    Abstract [en]

    The sarcoplasmic Ca2+-ATPase (SERCA1a) forms two phosphoenzyme intermediates during Ca2+ pumping. The second intermediate E2P hydrolyzes rapidly, which is essential for the rapid removal of Ca2+ from the cytosol of muscle cells. The present work studies whether a weakening of the scissile P-O bond in the E2P ground state facilitates dephosphorylation. To this end, the experimentally known vibrational spectrum of the E2P phosphate group was calculated with density functional theory (DFT) using structural models at two levels of structural complexity: (i) Models of acetyl phosphate in simple environments and (ii) similar to 150 atom models of the catalytic site. It was found that the enzyme environment distorts the structure of the phosphate group: one of the terminal P-O bonds is shorter in the catalytic site indicating weaker interactions than in water. However, the bond that bridges phosphate and Asp351 is unaffected. This indicates that the scissile P-O bond is not weakened by the enzyme environment of E2P. A second finding was that the catalytic site of the E2P state in aqueous solution appears to adopt a structure as in the crystals with BeF3-, where the ATPase is in a non-reactive conformation. The reactant state of the dephosphorylation reaction differs from the E2P ground state: Glu183 faces Asp351 and positions the attacking water molecule. This state has a 0.04 angstrom longer, and thus weaker, bridging P-O bond. The reactant state is not detected in our experiments, indicating that its energy is at least 1 kcal/mol higher than that of the E2P ground state.

  • 17.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural dynamics of the Ca2+-ATPase studied by time-resolved infrared spectroscopy2008In: Spectroscopy, Vol. 22, 63-82 p.Article, review/survey (Other (popular science, discussion, etc.))
    Abstract [en]

    Protonation of acidic residues in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca(2+)-bound state Ca(2)E1, in the Ca(2+)-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with MgF(4)(2-) E2(TG+MgF(4)(2-)). Around physiological pH, all acidic Ca(2+) ligands (Glu(309), Glu(771), Asp(800), and Glu(908)) were unprotonated in Ca(2)E1; in E2(TG) and E2(TG+MgF(4)(2-)) Glu(771), Asp(800), and Glu(908) were protonated. Glu(771) and Glu(908) had calculated pK(a) values larger than 14 in E2(TG) and E2(TG+MgF(4)(2-)), whereas Asp(800) titrated with calculated pK(a) values near 7.5. Glu(309) had very different pK(a) values in the Ca(2+)-free states: 8.4 in E2(TG+MgF(4)(2-)) and 4.7 in E2(TG) because of a different local backbone conformation. This indicates that Glu(309) can switch between a high and a low pK(a) mode, depending on the local backbone conformation. Protonated Glu(309) occupied predominantly two main, very differently orientated side-chain conformations in E2(TG+MgF(4)(2-)): one oriented inward toward the other Ca(2+) ligands and one oriented outward toward a protein channel that seems to be in contact with the cytoplasm. Upon deprotonation, Glu(309) adopted completely the outwardly orientated side-chain conformation. The contact of Glu(309) with the cytoplasm in E2(TG+MgF(4)(2-)) makes this residue unlikely to bind lumenal protons. Instead it might serve as a proton shuttle between Ca(2+)-binding site I and the cytoplasm. Glu(771), Asp(800), and Glu(908) are proposed to take part in proton countertransport.

  • 18.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The study of protein reactions by reaction-induced infrared difference spectroscopy2009In: Biological and Biomedical Infrared Spectroscopy / [ed] P.I. Haris, A. Barth, Amsterdam: IOS Press BV , 2009, 53-78 p.Chapter in book (Other academic)
    Abstract [en]

    Reaction-induced infrared difference spectroscopy of proteins is reviewed. This technique enables detailed characterization of enzyme function on the level of single bonds of proteins, cofactors or substrates. Discussed are methods to initiate protein reactions in the infrared samples, general aspects of spectra interpretation, measurements of enzyme activity and studies of protein function at the example of the Ca2+ pump.

  • 19.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Two sides of the same coin: How enzymes distort substrates and vice versa. An infrared spectroscopic view on pyruvate kinase and Ca2+-ATPase2016In: Biomedical Spectroscopy and Imaging, ISSN 2212-8794, Vol. 5, no 2, 101-114 p.Article in journal (Refereed)
    Abstract [en]

    This review summarises our infrared spectroscopy and density functional theory studies on the mutual interactions between enzymes and their substrates. We investigated phosphoenolpyruvate bound to pyruvate kinase (EC 2.7.1.40, M1 isozyme), ATP bound to the Ca2+-ATPase (SERCA1a), and the aspartylphosphate moiety of the Ca2+-ATPase phosphoenzyme E2P. Conformational changes of the enzymes and distortions of substrate structure are discussed. In all cases, the infrared absorption of the substrate in the enzyme environment could be identified by a combination of reaction-induced difference spectroscopy and isotopic labelling. The experimentally-determined vibrational frequencies were interpreted in structural terms using experimental correlations or modelling of the active site in density functional theory calculations. For none of the three systems, a weakening of the bond that is cleaved in the following enzymatic reaction could be detected in the ground state of the enzyme-substrate complex. However, for the dephosphorylation reaction of the Ca2+-ATPase phosphoenzyme E2P, a high energy intermediate, not detected in experiments, is the reactant state according to density functional theory calculations.

  • 20.
    Barth, Andreas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haris, Parvez I.De Montfort University, Leicester, UK.
    Biological and Biomedical Infrared Spectroscopy2009Collection (editor) (Other academic)
  • 21. Corrie, J
    et al.
    Munasinghe, V
    Rudbeck, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Photochemistry and thermal decarboxylation of alpha-phosphoryloxy-p-nitrophenylacetates2009In: Photochemistry and Photobiology, ISSN 0031-8655, E-ISSN 1751-1097, Vol. 85, 1089-1096 p.Article in journal (Refereed)
    Abstract [en]

    α-Carboxy-4-nitrobenzyl phosphate 4 and its derived monomethyl phosphate ester 5 were synthesized and purified by anion-exchange chromatography. A gradient of LiCl was necessary for elution of the anion-exchange column to avoid unexpected thermal decarboxylation that occurred during vacuum evaporation when the volatile triethylammonium bicarbonate buffer was used. Photolysis of each compound was accompanied by decarboxylation, and 4 released inorganic phosphate with near-100% stoichiometry. Time-resolved infrared spectroscopy of the photolysis reaction, coupled with density functional theory calculations of vibrational frequencies, enabled us to infer a mechanism for the photolytic pathway, although there was some evidence for a second pathway also being operative. In contrast to the results for 4, photolysis of 5 appeared to release little or no monomethyl phosphate.

  • 22. Corrie, John E T
    et al.
    Munasinghe, V Ranjit N
    Trentham, David R
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Studies of decarboxylation in photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds.2008In: Photochem Photobiol Sci, ISSN 1474-905X, Vol. 7, no 1, 84-97 p.Article in journal (Refereed)
    Abstract [en]

    Photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds, studied here by time-resolved IR and UV spectroscopy, involves at least two pathways. In one, a conventional 2-nitrobenzyl type rearrangement takes place to release the photoprotected species via rapid decay of an aci-nitro intermediate. The alpha-carboxylate moiety of the CNB group is retained and the final by-product from this pathway is 2-nitrosophenylglyoxylate. Direct measurements of product formation confirmed that release via this pathway is faster for CNB-caged compounds than for related caged compounds without an alpha-carboxylate substituent and a rationale for the faster release rate is proposed. In a second pathway, photodecarboxylation of the starting material occurs: this pathway leads only to a slow, minor release of the photoprotected species. The extent to which the latter pathway contributes is affected by the nature of buffer salts in the irradiated solution. It was more prominent in an amine-based buffer (MOPS) than in phosphate buffer.

  • 23.
    Eremina, Nadejda
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Use of Creatine Kinase To Induce Multistep Reactions in Infrared Spectroscopic Experiments2013In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 48, 14967-14972 p.Article in journal (Refereed)
    Abstract [en]

    An extension of current approaches to trigger enzymatic reactions in reaction-induced infrared difference spectroscopy experiments is described. A common procedure is to add a compound that induces a reaction in the protein of interest. To be able to induce multistep reactions, we explored here the use of creatine kinase (CK) for the study of phosphate transfer mechanisms. The enzymatic reaction of CK could be followed using bands at 1614 and 979 cm(-1) for creatine phosphate consumption, at 944 cm(-1) for ADP consumption, and at 1243, 992, and 917 cm(-1) for ATP formation. The potential of CK to induce multistep reactions in infrared spectroscopic experiments was demonstrated using the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) as the protein of interest. ADP binding to the ATPase was triggered by photolytic release of ADP from P-3-1-(2-nitro)phenylethyl ADP (caged ADP). CK added in small amounts converted the released ADP to ATP on the time scale of minutes. This phosphorylated the ATPase and led to the formation of the first phosphoenzyme intermediate Ca(2)E1P. Thus a difference spectrum could be obtained that reflected the reaction from the ADP ATPase complex to the first phosphoenzyme intermediate. Comparison with a phosphorylation spectrum obtained when the initial state was the ATP ATPase complex revealed the contribution of ATP's gamma-phosphate to the conformational change of the ATPase upon nucleotide binding: gamma-phosphate binding modifies the structure of a beta-sheet, likely in the phosphorylation domain, and shifts its spectral position from similar to 1640 to similar to 1630 cm(-1). Upon phosphorylation of the ATPase, the beta-sheet relaxes back to a structure that is intermediate between that adopted in the ADP bound state and that in the ATP bound state.

  • 24.
    Eriksson, Sylvia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eremina, Nadejda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Harryson, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane-Induced Folding of the Plant Stress Dehydrin Lti302016In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 171, no 2, 932-943 p.Article in journal (Refereed)
    Abstract [en]

    Dehydrins are disordered proteins that are expressed in plants as a response to embryogenesis and water-related stress. The molecular function and structural action of the dehydrins are yet elusive, but increasing evidence points to a role in protecting the structure and functional dynamics of cell membranes. An intriguing example is the cold-induced dehydrin Lti30 that binds to membranes by its conserved K segments. Moreover, this binding can be regulated by pH and phosphorylation and shifts the membrane phase transition to lower temperatures, consistent with the protein's postulated function in cold stress. In this study, we reveal how the Lti30-membrane interplay works structurally at atomic level resolution in Arabidopsis (Arabidopsis thaliana). Nuclear magnetic resonance analysis suggests that negatively charged lipid head groups electrostatically capture the protein's disordered K segments, which locally fold up into a-helical segments on the membrane surface. Thus, Lti30 conforms to the general theme of structure-function relationships by folding upon binding, in spite of its disordered, atypically hydrophilic and repetitive sequence signatures. Moreover, the fixed and well-defined structure of the membrane-bound K segments suggests that dehydrins have the molecular prerequisites for higher level binding specificity and regulation, raising new questions about the complexity of their biological function.

  • 25.
    Gewert, Berit
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Ogonowski, Martin
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry. Aquabiota Water Research, Sweden.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    MacLeod, Matthew
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Abundance and composition of near surface microplastics and plastic debris in the Stockholm Archipelago, Baltic Sea2017In: Marine Pollution Bulletin, ISSN 0025-326X, E-ISSN 1879-3363, Vol. 120, no 1-2, 292-302 p.Article in journal (Refereed)
    Abstract [en]

    We collected plastic debris in the Stockholm Archipelago using a manta trawl, and additionally along a transect in the Baltic Sea from the island of Gotland to Stockholm in a citizen science study. The samples were concentrated by filtration and organic material was digested using hydrogen peroxide. Suspected plastic material was isolated by visual sorting and 59 of these were selected to be characterized with Fourier transform infrared spectroscopy. Polypropylene and polyethylene were the most abundant plastics identified among the samples (53% and 24% respectively). We found nearly ten times higher abundance of plastics near central Stockholm than in offshore areas (4.2 x 10(5) plastics km(-2) compared to 4.7 x 10(4) plastics km(-2)). The abundance of plastic debris near Stockholm was similar to urban areas in California, USA, and the overall abundance in the Stockholm Archipelago was similar to plastic abundance reported in the northwestern Mediterranean Sea.

  • 26. Hauser, K
    et al.
    Barth, A
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Side-Chain Protonation and Mobility in the Sarcoplasmic Reticulum Ca2+-ATPase: Implications for Proton Countertransport and Ca2+ Release.2007In: Biophys J, ISSN 0006-3495, Vol. 93, no 9, 3259-70 p.Article in journal (Refereed)
    Abstract [en]

    Protonation of acidic residues in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA 1a) was studied by multiconformation continuum electrostatic calculations in the Ca(2+)-bound state Ca(2)E1, in the Ca(2+)-free state E2(TG) with bound thapsigargin, and in the E2P (ADP-insensitive phosphoenzyme) analog state with MgF(4)(2-) E2(TG+MgF(4)(2-)). Around physiological pH, all acidic Ca(2+) ligands (Glu(309), Glu(771), Asp(800), and Glu(908)) were unprotonated in Ca(2)E1; in E2(TG) and E2(TG+MgF(4)(2-)) Glu(771), Asp(800), and Glu(908) were protonated. Glu(771) and Glu(908) had calculated pK(a) values larger than 14 in E2(TG) and E2(TG+MgF(4)(2-)), whereas Asp(800) titrated with calculated pK(a) values near 7.5. Glu(309) had very different pK(a) values in the Ca(2+)-free states: 8.4 in E2(TG+MgF(4)(2-)) and 4.7 in E2(TG) because of a different local backbone conformation. This indicates that Glu(309) can switch between a high and a low pK(a) mode, depending on the local backbone conformation. Protonated Glu(309) occupied predominantly two main, very differently orientated side-chain conformations in E2(TG+MgF(4)(2-)): one oriented inward toward the other Ca(2+) ligands and one oriented outward toward a protein channel that seems to be in contact with the cytoplasm. Upon deprotonation, Glu(309) adopted completely the outwardly orientated side-chain conformation. The contact of Glu(309) with the cytoplasm in E2(TG+MgF(4)(2-)) makes this residue unlikely to bind lumenal protons. Instead it might serve as a proton shuttle between Ca(2+)-binding site I and the cytoplasm. Glu(771), Asp(800), and Glu(908) are proposed to take part in proton countertransport.

  • 27.
    Hugonin, Loïc
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Perálvarez-Marín, Alex
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Secondary structure transitions and aggregation induced in dynorphin neuropeptides by the detergent sodium dodecyl sulfate.2008In: Biochim Biophys Acta, ISSN 0006-3002, Vol. 1778, no 11, 2580-7 p.Article in journal (Refereed)
    Abstract [en]

    Dynorphins, endogeneous opioid neuropeptides, function as ligands to the opioid kappa receptors and also induce non-opioid effects in neurons, probably related to direct membrane interactions. We have characterized the structure transitions of dynorphins (big dynorphin, dynorphin A and dynorphin B) induced by the detergent sodium dodecyl sulfate (SDS). In SDS titrations monitored by circular dichroism, we observed secondary structure conversions of the peptides from random coil to alpha-helix with a highly aggregated intermediate. As determined by Fourier transform infrared spectroscopy, this intermediate exhibited beta-sheet structure for dynorphin B and big dynorphin. In contrast, aggregated dynorphin A was alpha-helical without considerable beta-sheet content. Hydrophobicity analysis indicates that the YGGFLRR motif present in all dynorphins is prone to be inserted in the membrane. Comparing big dynorphin with dynorphin A and dynorphin B, we suggest that the potent neurotoxicity of big dynorphin could be related to the combination of amino acid sequences and secondary structure propensities of dynorphin A and dynorphin B, which may generate a synergistic effect for big dynorphin membrane perturbing properties. The induced aggregated alpha-helix of dynorphin A is also correlated with membrane perturbations, whereas the beta-sheet of dynorphin B does not correlate with membrane perturbations.

  • 28. Kaltofen, Sabine
    et al.
    Li, Chenge
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Huang, Po-Ssu
    Serpell, Louise C.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    André, Ingemar
    Computational De Novo Design of a Self-Assembling Peptide with Predefined Structure2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 2, 550-562 p.Article in journal (Refereed)
    Abstract [en]

    Protein and peptide self-assembly is a powerful design principle for engineering of new biomolecules. More sophisticated biomaterials could be built if both the structure of the overall assembly and that of the self-assembling building block could be controlled. To approach this problem, we developed a computational design protocol to enable de novo design of self-assembling peptides with predefined structure. The protocol was used to design a peptide building block with a beta alpha beta fold that self-assembles into fibrillar structures. The peptide associates into a double beta-sheet structure with tightly packed a-helices decorating the exterior of the fibrils. Using circular dichroism, Fourier transform infrared spectroscopy, electron microscopy and X-ray fiber diffraction, we demonstrate that the peptide adopts the designed conformation. The results demonstrate that computational protein design can be used to engineer protein and peptide assemblies with predefined three-dimensional structures, which can serve as scaffolds for the development of functional biomaterials. Rationally designed proteins and peptides could also be used to investigate the subtle energetic and entropic tradeoffs in natural self-assembly processes and the relation between assembly structure and assembly mechanism. We demonstrate that the de novo designed peptide self-assembles with a mechanism that is more complicated than expected, in a process where small changes in solution conditions can lead to significant differences in assembly properties and conformation. These results highlight that formation of structured protein/peptide assemblies is often dependent on the formation of weak but highly precise intermolecular interactions.

  • 29.
    Karjalainen, Eeva-Liisa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vibrational Coupling between Helices Influences the Amide I Infrared Absorption of Proteins: Application to Bacteriorhodopsin and Rhodopsin2012In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, no 15, 4448-4456 p.Article in journal (Refereed)
    Abstract [en]

    The amide 1 spectrum of multimers of helical protein segments was simulated using transition dipole coupling (TDC) for long-range interactions between individual amide oscillators and DFT data from dipeptides (la Cour Jansen et al. J. Chem. Phys. 2006, 125, 44312) for nearest neighbor interactions. Vibrational coupling between amide groups on different helices shift the helix absorption to higher wavenumbers. This effect is small for helix dimers (1 cm(-1)) at 10 angstrom distance and only moderately affected by changes in the relative orientation between the helices. However, the effect becomes considerable when several helices are bundled in membrane proteins. Particular examples are the 7-helix membrane proteins bacteriorhodopsin (BR) and rhodopsin, where the upshift is 4.3 and 5.3 cm(-1) respectively, due to interhelical coupling within a BR monomer. A further upshift of 4.0 cm(-1) occurs when BR monomers associate to trimers. We propose that interhelical vibrational coupling explains the experimentally observed unusually high wavenumber of the amide I band of BR

  • 30.
    Karjalainen, Eeva-Liisa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ersmark, Tore
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Optimization of Model Parameters for Describing the Amide I Spectrum of a Large Set of Proteins2012In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, no 16, 4831-4842 p.Article in journal (Refereed)
    Abstract [en]

    A new simulation protocol for the prediction of the infrared absorption of the amide I vibration of proteins was developed. The method incorporates known effects on the intrinsic frequencies (backbone conformation, interpeptide and peptide-solvent hydrogen bonding) and couplings (nearest neighbor coupling, transition dipole coupling) of amide I oscillators in a parametrized manner. Model parameters for the simulation of amide I spectra were determined through fitting and optimization of simulated spectra to experimentally measured infrared spectra of 44 proteins that represent maximum structural variation in terms of different folds and secondary structure contents. Prediction of protein spectra using the optimized parameters resulted in good agreement with experimental spectra and in a considerable improvement compared to a description involving only transition dipole coupling.

  • 31.
    Karjalainen, Eeva-Liisa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hardell, Amelie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Toward a general method to observe the phosphate groups of phosphoenzymes with infrared spectroscopy2006In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 91, no 6, 2282-2289 p.Article in journal (Refereed)
    Abstract [en]

    A general method to study the phosphate group of phosphoenzymes with infrared difference spectroscopy by helper enzyme-induced isotope exchange was developed. This allows the selective monitoring of the phosphate P-O vibrations in large proteins, which provides detailed information on several band parameters. Here, isotopic exchange was achieved at the oxygen atoms of the catalytically important phosphate group that transiently binds to the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). [γ-18O3]ATP phosphorylated the ATPase, which produced phosphoenzyme that was initially isotopically labeled. The helper enzyme adenylate kinase regenerated the substrate ATP from ADP (added or generated upon ATP hydrolysis) with different isotopic composition than used initially. With time this produced the unlabeled phosphoenzyme. The method was tested on the ADP-insensitive phosphoenzyme state of the Ca2+-ATPase for which the vibrational frequencies of the phosphate group are known, and it was established that the helper enzyme is effective in mediating the isotope exchange process.

  • 32.
    Karjalainen, Eeva-Liisa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hauser, Karin
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton paths in the sarcoplasmic reticulum Ca2+-ATPase 2007In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, no 11, 1310-1318 p.Article in journal (Refereed)
    Abstract [en]

    The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) pumps Ca(2+) and countertransport protons. Proton pathways in the Ca(2+) bound and Ca(2+)-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca(2+) bound state Ca(2)E1, one of the proposed Ca(2+) entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca(2+)-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca(2+) binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca(2+) dissociation pathways. We suggest that separate proton and Ca(2+) pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.

  • 33.
    Karjalainen, Eeva-Liisa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ravi, Harish Kumar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Simulation of the Amide I Absorption of Stacked β-Sheets2011In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 115, no 4, 749-757 p.Article in journal (Refereed)
    Abstract [en]

    Aggregated β-sheet structures are associated with amyloid and prion diseases. Techniques capable of revealing detailed structural and dynamical information on β-sheet structure are thus of great biomedical and biophysical interest. In this work, the infrared (IR) amide I spectral characteristics of stacked β-sheets were modeled using the transition dipole coupling model. For a test set of β-sheet stacks, the simulated amide I spectrum was analyzed with respect to the following parameters; intersheet distance, relative rotation of the sheets with respect to each other and the effect of number of sheets stacked. The amide I maximum shifts about 5 cm(-1) to higher wavenumbers when the intersheet distance between two identical β-sheets decreases from 20 to 5 Å. Rotation around the normal of one of the sheets relative to the other results in maximum intersheet coupling near 0° and 180°. Upon of rotation from 0° to 90° at an intersheet distance of 9 Å, the amide I maximum shifts about 3 cm(-1). Tilting of one of the sheets by 30° from the normal results in a shift of the amide I maximum by less than 1 cm(-1). When stacking several β-sheets along the normal, the amide I maximum shifts to higher wavenumbers with increasing stack size. The amide I maximum shifts about 6 cm(-1) when stacking four sheets with an intersheet distance of 9 Å. The study provides an aid in the interpretation of the IR amide I region for experiments involving β-sheets and creates awareness of the many effects that determine the spectrum of β-sheet structures.

  • 34.
    Krasteva, Maria
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structures of the Ca2+-ATPase complexes with ATP, AMPPCP and AMPPNP. An FTIR study.2007In: Biochim Biophys Acta: Bioenergetics, ISSN 0006-3002, Vol. 1767, no 1, 114-23 p.Article in journal (Refereed)
    Abstract [en]

    We studied binding of ATP and of the ATP analogs adenosine 5'-(beta,gamma-methylene)triphosphate (AMPCP) and beta,gamma-imidoadenosine 5'-triphosphate (AMPPNP) to the Ca(2+)-ATPase of the sarcoplasmic reticulum membrane (SERCA1a) with time-resolved infrared spectroscopy. In our experiments, ATP reacted with ATPase which had AMPPCP or AMPPNP bound. These experiments monitored exchange of ATP analog by ATP and phosphorylation to the first phosphoenzyme intermediate Ca(2)E1P. These reactions were triggered by the release of ATP from caged ATP. Only small differences in infrared absorption were observed between the ATP complex and the complexes with AMPPCP and AMPPNP indicating that overall the interactions between nucleotide and ATPase are similar and that all complexes adopt a closed conformation. The spectral differences between ATP and AMPPCP complex were more pronounced at high Ca(2+) concentration (10 mM). They are likely due to a different position of the gamma-phosphate which affects the beta-sheet in the P domain.

  • 35.
    Krasteva, Maria
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kumar, Saroj
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A dialysis accessory for attenuated total reflection infrared spectroscopy2006In: Spectroscopy, ISSN 0712-4813, Vol. 20, no 3, 89-94 p.Article in journal (Refereed)
    Abstract [en]

    A dialysis accessory for attenuated total reflection (ATR) infrared spectroscopy is described together with an evaluation based on known systems with well-studied infrared spectra, such as chemical oxidation and reduction of cytochrome c and substrate binding to the Ca2+-ATPase. Changes in the infrared spectra of the two proteins are successfully monitored with the dialysis accessory. The accessory was developed in our laboratory for the diamond 9-reflections SensIR ATR unit. It can be used to study absorbance changes of macromolecules which are induced by low molecular weight compounds, for example the binding of substrates, inhibitors or ions to macromolecules as well as effects of pH, ionic strength or denaturants on the structure of macromolecules. The dialysis accessory confines the macromolecule of interest to a sample compartment created between the ATR crystal and the dialysis membrane. On the other side of the dialysis membrane, a reservoir for the sample medium is created. In this way the low molecular weight compound of interest can exchange freely between the reservoir and the sample compartment via the dialysis membrane. This provides a flexible way to change sample conditions for the macromolecule of interest, allowing for example initiation of ligand binding.

  • 36.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Effects of ions on ligand binding to pyruvate kinase: Mapping the binding site by infrared spectroscopyIn: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207Article in journal (Refereed)
  • 37.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Effects of Ions on Ligand Binding to Pyruvate Kinase: Mapping the Binding Site with Infrared Spectroscopy2011In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 115, no 20, 6784-6789 p.Article in journal (Refereed)
    Abstract [en]

    The effects of mono- and divalent ions (Li(+), K(+), Na(+), Cs(+), Mg(2+), Ca(2+); Mn(2+), Zn(2+)) on the binding of phosphoenolpyruvate (PEP) to rabbit muscle pyruvate kinase (PK) were studied by attenuated total reflection infrared spectroscopy in combination with a dialysis accessory. The experiments assessed the structural change of the protein as well as the binding mode of PEP. They indicated that a signal at 1638 cm(-1) assigned to a beta sheet was perturbed differently with Na(+) as compared to the other monovalent ions. Otherwise, we obtained similar conformational changes in the presence of different monovalent cations, and therefore, it seems unlikely that the ion effects on activity are due to an ion effect on the structure of the PEP:PK complex. With different divalent cations, a particularly large conformational change was observed with Mn(2+) and attributed to a more closed conformation of the complex The absorption of bound PEP was also detected. The antisymmetric stretching vibration of the carboxylate group of bound PEP indicates a more homogeneous binding mode for Mn(2+) compared to the other divalent ions. The symmetric stretching vibration depends on both monovalent and divalent ions, indicating that the dihedral angle O-C(1)-C(2)-O is affected by the ions in the catalytic site. Little change in the bond strengths of PEP is observed, indicating that the PEP:PK complex does not adopt a reactive conformation.

  • 38.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Following Enzyme Activity with Infrared Spectroscopy2010In: Sensors, ISSN 1424-8220, E-ISSN 1424-8220, Vol. 10, no 4, 2626-2637 p.Article in journal (Refereed)
    Abstract [en]

    Fourier transform infrared (FTIR) spectroscopy provides a direct, ""on-line"" monitor of enzymatic reactions. Measurement of enzymatic activity is based on the fact that the infrared spectra of reactants and products of an enzymatic reaction are usually different. Several examples are given using the enzymes pyruvate kinase, fumarase and alcohol dehydrogenase. The main advantage of the infrared method is that it observes the reaction of interest directly, i.e., no activity assay is required to convert the progress of the reaction into an observable quantity.

  • 39.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ligand induced conformational change of pyruvate kinase studied by ATR-FTIR spectroscopy2007Conference paper (Refereed)
  • 40.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Phosphoenolpyruvate and Mg2+ binding to pyruvate kinase monitored by infrared spectroscopy2010In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 98, no 9, 1931-1940 p.Article in journal (Refereed)
    Abstract [en]

    Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg2+ binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg2+ binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-C-13(2)-labeled PEP. We obtained the following assignments: 1589 cm(-1) (antisymmetric carboxylate stretching vibration); 1415 cm(-1) (symmetric carboxylate stretching vibration); 1214 cm(-1) (C-O stretching vibration); 1124 and 1110 cm(-1) (asymmetric PO32- stretching vibrations); and 967 cm(-1) (symmetric PO32- stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm-1. The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na+ exhibited the same band positions as in the presence of K+, indicating very similar interaction strengths between ligand and protein in both cases.

  • 41.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Allosteric Effect of Fructose Bisphosphate on Muscle Pyruvate Kinase Studied by Infrared Spectroscopy2011In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 115, no 39, 11501-11505 p.Article in journal (Refereed)
    Abstract [en]

    Pyruvate kinase exhibits allosteric properties. The allosteric effect of fructose 1,6-bisphosphate (FBP) on phosphoenolpyruvate (PEP) binding to rabbit muscle pyruvate kinase (PK) in the presence of various ions (mg(2+), mn(2+), Na(+)) was studied by attenuated total reflection infrared spectroscopy in combination with a dialysis accessory. The experiments indicated that FBP binding causes conformational changes of PK that are of the same order of magnitude as those of PEP binding. The conformational change of PEP binding to PK/Mg(2+)/K(+) in the presence of FBP was about twice as large as in its absence, which is tentatively ascribed to a higher occupancy the closed state. The affinity for PEP increased in the presence of Mg(2+) and K(+). No such effects were observed with the other ion combinations Mn(2+)/K(+) and Mg(2+)/Na(+) or in D(2)O (with Mg(2+)/K(+)), and therefore we did not detect an allosteric effect on PEP binding under these conditions.

  • 42.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eremina, Nadejda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Detection of Ligand Binding to Proteins through Observation of Hydration Water2012In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, no 48, 13968-13974 p.Article in journal (Refereed)
    Abstract [en]

    Drug development is impeded by the need to design for each drug target a test that detects the binding of drug candidate molecules to the target protein. Therefore, a general method to detect ligand binding is highly desirable. Here, we present an observation toward developing such a method, which is based on monitoring a change in water absorption by infrared spectroscopy. Infrared spectroscopy has high sensitivity for water, and changes in its hydrogen bond pattern can be observed. We studied absorption changes of water upon the addition of phosphenolpyruvate or Mg2+ to pyruvate kinase. In each case, there is a decrease in the absorption of water in the 3000-3100 cm(-1) region on the low wavenumber side of the OH stretching vibration when a ligand binds to the protein. Our results suggest that the weaker water absorption is due to the release of protein-bound water into bulk water during ligand binding. This observation has high potential for drug development as well as for basic research because it can lead to a general method for detecting molecular association events that (i) is label-free, (ii) works with both binding partners being in aqueous solution, and (iii) is based on a universal process that takes place in all binding events.

  • 43.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eremina, Nadejda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ligand binding detected by change in water absorption by infrared spectroscopyManuscript (preprint) (Other academic)
  • 44.
    Kumar, Saroj
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Li, Chenge
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. chenge.li@dbb.su.se.
    Montigny, Cedric
    le Maire, Marc
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Conformational changes of recombinant Ca2+-ATPase studied by reaction-induced infrared difference spectroscopy2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 21, 5398-5407 p.Article in journal (Refereed)
    Abstract [en]

    Recombinant Ca2+-ATPase was expressed in Saccharomycescerevisiae with a biotin-acceptor domain linked to its C-terminus by a thrombin cleavage site. We obtained 200g of similar to 70% pure recombinant sarcoendoplasmic reticulum Ca2+-ATPase isoform1a (SERCA1a) from a 6-L yeast culture. The catalytic cycle of SERCA1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states (Ca(2)E1P and Ca(2)E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca(2)E1 had the same spectral features as those from the native rabbit SERCA1a. The enzyme-specific activity for the active enzyme fraction in both samples was also similar. The results show that the recombinant protein obtained from the yeast-based expression system has similar structural and dynamic properties as native rabbit SERCA1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.

  • 45.
    Li, Chenge
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kumar, Saroj
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Montigny, Cedric
    le Mairebcd, Marc
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca2+-ATPase2014In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 139, no 17, 4231-4240 p.Article in journal (Refereed)
    Abstract [en]

    Infrared spectroscopy was used to characterise recombinant sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). In the amide I region, its spectrum differed from that of Ca2+-ATPase prepared from rabbit fast twitch muscle below 1650 cm(-1). A band at 1642 cm(-1) is reduced in the spectrum of the recombinant protein and a band at 1631 cm(-1) is more prominent. By comparison of amide 1 band areas with the known secondary structure content of the protein, we assigned the 1642 cm(-1) band to beta-sheet structure. Further investigation revealed that the 1642 cm(-1) band decreased and the 1631 cm-1 band increased upon storage at room temperature and upon repeated washing of a protein film with water. Also protein aggregates obtained after solubilisation of the rabbit muscle enzyme showed a prominent band at 1631 cm(-1), whereas the spectrum of solubilised ATPase resembled that of the membrane bound protein. The spectral position of the 1631 cm(-1) band is similar to that of a band observed for inclusion bodies of other proteins. The findings show that the absence of the 1642 cm(-1) band and the presence of a prominent band at 1631 cm(-1) indicate protein aggregation and can be used as a quality marker for the optimisation of recombinant protein production. We conclude that recombinant production of SERCA1a, storage at room temperature, repeated washing and aggregation after solubilisation all modify existing beta-sheets in the cytosolic domains so that they become similar to those found in inclusion bodies of other proteins.

  • 46.
    Lindholm, Ljubica
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ariöz, Candan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jawurek, Michael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liebau, Jobst
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Effect of lipid bilayer properties on the photocycle of green proteorhodopsin2015In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1847, no 8, 698-708 p.Article in journal (Refereed)
    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.

  • 47.
    Liu, Man
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karjalainen, Eeva-Liisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Use of Helper Enzymes for ADP Removal in Infrared Spectroscopic Experiments: Application to Ca2+-ATPase2005In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 88, no 5, 3615-3624 p.Article in journal (Refereed)
    Abstract [en]

    Adenylate kinase (AdK) and apyrase were employed as helper enzymes to remove ADP in infrared spectroscopic experiments that study the sarcoplasmic reticulum Ca2+-ATPase. The infrared absorbance changes of their enzymatic reactions were characterized and used to monitor enzyme activity. AdK transforms ADP to ATP and AMP, whereas apyrase consumes ATP and ADP to generate AMP and inorganic phosphate. The benefits of using them as helper enzymes are severalfold: i), both remove ADP generated after ATP hydrolysis by ATPase, which enables repeat of ATP-release experiments several times with the same sample without interference by ADP; ii), AdK helps maintain the presence of ATP for a longer time by regenerating 50% of the initial ATP; iii), apyrase generates free Pi, which can help stabilize the ADP-insensitive phosphoenzyme (E2P); and iv), apyrase can be used to monitor ADP dissociation from transient enzyme intermediates with relatively high affinity to ADP, as shown here for ADP dissociation from the ADP-sensitive phosphoenzyme intermediate (Ca2E1P). The respective infrared spectra indicate that ADP dissociation relaxes the closed conformation immediately after phosphorylation partially back toward the open conformation of Ca2E1 but does not trigger the transition to E2P. The helper enzyme approach can be extended to study other nucleotide-dependent proteins.

  • 48.
    Mandal, Paulami
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eremina, Nadejda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Formation of Two Different Types of Oligomers in the Early Phase of pH-Induced Aggregation of the Alzheimer A beta(12-28) Peptide2012In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 116, no 41, 12389-12397 p.Article in journal (Refereed)
    Abstract [en]

    The early phase in the aggregation process of the Alzheimer's peptide A beta(12-28) with both protected and unprotected ends was studied by time-resolved infrared spectroscopy and circular dichroism spectroscopy. Aggregation in the time-resolved experiments was initiated by a rapid pH drop caused by the photolysis of 1-(2-nitrophenyl)ethyl sulfate (caged sulfate). The infrared spectra indicate two different types of aggregates from both versions of the A beta(12-28) peptide. One type has small and/or twisted beta sheets with a beta-sheet band at 1627 cm(-1), They form fast (within 60 ms), presumably from initial aggregates, and their spectral signature is consistent with a beta-barrel structure. The other type arises relatively slowly from unstructured monomers on the seconds-to-minutes time scale and forms at lower pH than the first type. These beta sheets are antiparallel, planar, and large and show an absorption band at 1622 cm(-1) that shifts to 1617 cm(-1) in 12 min with most of the shift occurring in 10 s.

  • 49. Marter, Kathrin
    et al.
    Wetzel, Janina
    Eichhorst, Jenny
    Eremina, Nadja
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Leboulle, Gerard
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wiesner, Burkhard
    Eisenhardt, Dorothea
    Inhibition of Protein Synthesis with Highly Soluble Caged Compounds2017In: ChemistrySelect, ISSN 2365-6549, Vol. 2, no 22, 6212-6217 p.Article in journal (Refereed)
    Abstract [en]

    To target protein synthesis in defined areas, e.g. neuropiles of small brains or subcellular structures, locally restricted inhibition of protein synthesis is needed and can be realized by caged compounds of protein synthesis inhibitors (PSI). Since organic solvents interfere with protein synthesis themselves, the use of water-soluble caged PSIs is a prerequisite in studies on protein synthesis. Such compounds are sparsely available. We developed and characterized efficient highly soluble caged compounds of the PSIs anisomycin and emetine masking their biological activity with a {8-[bis(carboxymethyl)aminomethyl]-6-bromo-7-hydroxycoumarin-4-yl}methoxycarbonyl (BBHCMOC) derivative. The absorption spectra of the resulting BBHCMOC-caged anisomycin and BBHCMOC-caged emetine show long-wavelength maxima and the extinction coefficients are high, allowing uncaging under non-damaging light conditions. When uncaged, these caged PSIs reliably inhibit protein synthesis in an in vitro translation system and in cell culture. Taken the whole spectrum of properties into account, our BBHCMOC-caged PSIs are highly qualified for in vivo studies.

  • 50.
    Peralvarez-Marin, Alex
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mateos, Laura
    Zhang, Ce
    Singh, Shalini
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cedazo-Minguez, Angel
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Morozova-Roche, Ludmilla
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Influence of Residue 22 on the Folding, Aggregation Profile, and Toxicity of the Alzheimer's Amyloid beta Peptide2009In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 97, no 1, 277-285 p.Article in journal (Refereed)
    Abstract [en]

    Several biophysical techniques have been used to determine differences in the aggregation profile (i.e., the secondary structure, aggregation propensity, dynamics, and morphology of amyloid structures) and the effects on cell viability of three variants of the amyloid beta peptide involved in Alzheimer's disease. We focused our study on the Glu(22) residue, comparing the effects of freshly prepared samples and samples aged for at least 20 days. In the aged samples, a high propensity for aggregation and beta-sheet secondary structure appears when residue 22 is capable of establishing polar (Glu(22) in wild-type) or hydrophobic (Val(22) in E22V) interactions. The Arctic variant (E22G) presents a mixture of mostly disordered and a-helix structures (with low beta-sheet contribution). Analysis of transmission electron micrographs and atomic force microscopy images of the peptide variants after aging showed significant quantitative and qualitative differences in the morphology of the formed aggregates. The effect on human neuroblastoma cells of these A beta(12-28) variants does not correlate with the amount of beta-sheet of the aggregates. In samples allowed to age, the native sequence was found to have an insignificant effect on cell viability, whereas the Arctic variant (E22G), the E22V variant, and the slightly-aggregating control (F19G-F20G) had more prominent effects.

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