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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, p. 6893-6902Article 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, p. 600-11Article 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, p. 104-110Article 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, p. 1862-1870Article 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.

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    Paper II
  • 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, p. 5533-5544Article 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. Arndt, Tina
    et al.
    Greco, Gabriele
    Schmuck, Benjamin
    Bunz, Jessica
    Shilkova, Olga
    Francis, Juanita
    Pugno, Nicola M.
    Jaudzems, Kristaps
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Jan
    Rising, Anna
    Engineered Spider Silk Proteins for Biomimetic Spinning of Fibers with Toughness Equal to Dragline Silks2022In: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 32, no 23, article id 2200986Article in journal (Refereed)
    Abstract [en]

    Spider silk is the toughest fiber found in nature, and bulk production of artificial spider silk that matches its mechanical properties remains elusive. Development of miniature spider silk proteins (mini-spidroins) has made large-scale fiber production economically feasible, but the fibers’ mechanical properties are inferior to native silk. The spider silk fiber's tensile strength is conferred by poly-alanine stretches that are zipped together by tight side chain packing in β-sheet crystals. Spidroins are secreted so they must be void of long stretches of hydrophobic residues, since such segments get inserted into the endoplasmic reticulum membrane. At the same time, hydrophobic residues have high β-strand propensity and can mediate tight inter-β-sheet interactions, features that are attractive for generation of strong artificial silks. Protein production in prokaryotes can circumvent biological laws that spiders, being eukaryotic organisms, must obey, and the authors thus design mini-spidroins that are predicted to more avidly form stronger β-sheets than the wildtype protein. Biomimetic spinning of the engineered mini-spidroins indeed results in fibers with increased tensile strength and two fiber types display toughness equal to native dragline silks. Bioreactor expression and purification result in a protein yield of ≈9 g L−1 which is in line with requirements for economically feasible bulk scale production.

  • 7. Arndt, Tina
    et al.
    Jaudzems, Kristaps
    Shilkova, Olga
    Francis, Juanita
    Johansson, Mathias
    Laity, Peter R.
    Sahin, Cagla
    Chatterjee, Urmimala
    Kronqvist, Nina
    Barajas-Ledesma, Edgar
    Kumar, Rakesh
    Chen, Gefei
    Strömberg, Roger
    Abelein, Axel
    Langton, Maud
    Landreh, Michael
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holland, Chris
    Johansson, Jan
    Rising, Anna
    Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform2022In: Nature Communications, E-ISSN 2041-1723, Vol. 13, article id 4695Article in journal (Refereed)
    Abstract [en]

    Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to β-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.

  • 8.
    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, p. 8247-8254Article 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).

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  • 9.
    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, p. 5393-5399Article 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.

  • 10.
    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, p. 2167-2176Article 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.

  • 11.
    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, p. 963-973Article 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.

  • 12.
    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 1431-5157, E-ISSN 1420-3049, Vol. 20, no 7, p. 12599-12622Article 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.

  • 13.
    Baronio, Cesare M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Baldassarre, Maurizio
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Insight into the internal structure of amyloid-β oligomers by isotope-edited Fourier transform infrared spectroscopy2019In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 21, no 16, p. 8587-8597Article in journal (Refereed)
    Abstract [en]

    The internal structure of amyloid-β (Aβ) oligomers was investigated with isotope-edited Fourier transform infrared spectroscopy. Homo-oligomers of Aβ(40) and Aβ(42) were prepared from unlabeled and C-13, N-15-labeled monomeric Aβ and from mixtures of these. For the unlabeled peptides, two main bands were observed in (H2O)-H-2 at 1685 and 1622 cm(-1) for Aβ(40) and at 1685 and 1626 cm(-1) for Aβ(42). These band positions indicate that the number of strands per sheet is at least four. The obtained experimental amide I spectra were simulated using a number of structural models (antiparallel β-sheets, β-barrels and a dodecamer structure). According to experiments and calculations, the main C-13-band shifts down at increasing molar ratio of labeled peptides. This shift occurs when vibrational coupling becomes possible between C-13-amide groups in close-by strands. It is small, when intervening C-12-strands increase the distance between C-13-strands; it is large, when many neighboring strands are labeled. The shift depends on the internal structure of the peptides within the oligomers, i.e. on the building block that each peptide molecule contributes to the β-sheets of the oligomers. The shift is largest, when individual peptides contribute just a single strand surrounded by strands from other peptide molecules. It is smaller when each molecule forms two or three adjacent strands. As indicated by a comparison between experiment and computation, the number of adjacent β-strands per peptide molecule is two for Aβ(40) oligomers and two or more for Aβ(42) oligomers. Our results are well explained by regular, antiparallel β-sheets or β-barrels.

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  • 14.
    Baronio, Cesare M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Refining protein amide I spectrum simulations with simple yet effective electrostatic models for local wavenumbers and dipole derivative magnitudes2024In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 26, no 2, p. 1166-1181Article in journal (Refereed)
    Abstract [en]

    Analysis of the amide I band of proteins is probably the most wide-spread application of bioanalytical infrared spectroscopy. Although highly desirable for a more detailed structural interpretation, a quantitative description of this absorption band is still difficult. This work optimized several electrostatic models with the aim to reproduce the effect of the protein environment on the intrinsic wavenumber of a local amide I oscillator. We considered the main secondary structures – α-helices, parallel and antiparallel β-sheets – with a maximum of 21 amide groups. The models were based on the electric potential and/or the electric field component along the CO bond at up to four atoms in an amide group. They were bench-marked by comparison to Hessian matrices reconstructed from density functional theory calculations at the BPW91, 6-31G** level. The performance of the electrostatic models depended on the charge set used to calculate the electric field and potential. Gromos and DSSP charge sets, used in common force fields, were not optimal for the better performing models. A good compromise between performance and the stability of model parameters was achieved by a model that considered the electric field at the positions of the oxygen, nitrogen, and hydrogen atoms of the considered amide group. The model describes also some aspects of the local conformation effect and performs similar on its own as in combination with an explicit implementation of the local conformation effect. It is better than a combination of a local hydrogen bonding model with the local conformation effect. Even though the short-range hydrogen bonding model performs worse, it captures important aspects of the local wavenumber sensitivity to the molecular surroundings. We improved also the description of the coupling between local amide I oscillators by developing an electrostatic model for the dependency of the dipole derivative magnitude on the protein environment.

  • 15.
    Baronio, Cesare M.
    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 Amide I Spectrum of Proteins—Optimization of Transition Dipole Coupling Parameters Using Density Functional Theory Calculations2020In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 124, no 9, p. 1703-1714Article in journal (Refereed)
    Abstract [en]

    The amide I region of the infrared spectrum is related to the protein backbone conformation and can provide important structural information. However, the interpretation of the experimental results is hampered because the theoretical description of the amide I spectrum is still under development. Quantum mechanical calculations, for example, using density functional theory (DFT), can be used to study the amide I spectrum of small systems, but the high computational cost makes them inapplicable to proteins. Other approaches that solve the eigenvalues of the coupled amide I oscillator system are used instead. An important interaction to be considered is transition dipole coupling (TDC). Its calculation depends on the parameters of the transition dipole moment. This work aims to find the optimal parameters for TDC in three major secondary structures: α-helices, antiparallel β-sheets, and parallel β-sheets. The parameters were suggested through a comparison between DFT and TDC calculations. The comparison showed a good agreement for the spectral shape and for the wavenumbers of the normal modes for all secondary structures. The matching between the two methods improved when hydrogen bonding to the amide oxygen was considered. Optimal parameters for individual secondary structures were also suggested.

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  • 16.
    Baronio, Cesare M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Li, Huimin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Song, Guangyuan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Annecke, Henry
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gustafsson, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Martinez-Carranza, Markel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The amide I spectrum of parallel β-sheet proteinsManuscript (preprint) (Other academic)
    Abstract [en]

    The amide I absorption of the polypeptide backbone has long been used to analyze the secondary structure of proteins. This approach has gained additional attention in the context of amyloid diseases where a particular focus is on the distinction between parallel and antiparallel β-sheets because these structures often discriminate between pre-fibrillar structures and fibrils. Some earlier infrared spectra with typical features of antiparallel β-sheets were interpreted as arising from the parallel β-sheets of fibrils. Therefore, the ability of infrared spectroscopy to distinguish between both types of β-sheets is debated. While it is established that regular, antiparallel β-sheets give rise to a high wavenumber band near 1690 cm-1, it is less clear whether or not this band may also occur for parallel β-sheets. Here we present and analyze the amide I spectra of two β-helix proteins, SV2 and Pent. The overall shape of the proteins is that of a cuboid which has parallel β-sheets on its four sides, which are connected by bends. The main features of their amide I spectrum are a band at 1665, and two bands between 1645 and 1628 cm-1. Both proteins exhibit also a weak component band near 1690 cm-1. Calculations of the amide I spectrum indicate that the absorption at high wavenumbers is not caused by the parallel β-sheets but by the bends between the β-strands. We therefore suggest to modify the interpretation of the amide I spectrum as follows: a high wavenumber band near 1690 cm-1 may be caused by other structures than antiparallel β-sheets. However, when the spectrum consists of only two distinct bands, one near 1690 cm-1 and one near 1630 cm-1, then an assignment to antiparallel β-sheets is consistent with the literature.

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  • 17.
    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.))
  • 18.
    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.))
  • 19.
    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.))
  • 20.
    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, p. 1-52Chapter in book (Other (popular science, discussion, etc.))
    Abstract [en]

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

  • 21.
    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, p. 1073-101Article, 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.

  • 22.
    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, p. 1036-1043Article 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.

  • 23.
    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, p. 63-82Article, 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.

  • 24.
    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, p. 53-78Chapter 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.

  • 25.
    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, p. 101-114Article 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.

  • 26.
    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)
  • 27.
    Berntsson, Elina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Tallinn University of Technology, Estonia.
    Paul, Suman
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vosough, Faraz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sholts, Sabrina B.
    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.
    Roos, Per M.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wärmländer, Sebastian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lithium ions display weak interaction with amyloid-beta (Aβ) peptides and have minor effects on their aggregation2021In: Acta Biochimica Polonica, ISSN 0001-527X, E-ISSN 1734-154X, Vol. 68, no 2, p. 169-179Article in journal (Refereed)
    Abstract [en]

    Alzheimer’s disease (AD) is an incurable disease and the main cause of age-related dementia worldwide, despite decades of research. Treatment of AD with lithium (Li) has shown promising results, but the underlying mechanism is unclear. The pathological hallmark of AD brains is deposition of amyloid plaques, consisting mainly of amyloid-β (Aβ) peptides aggregated into amyloid fibrils. The plaques contain also metal ions of e.g. Cu, Fe, and Zn, and such ions are known to interact with Aβ peptides and modulate their aggregation and toxicity. The interactions between Aβ peptides and Li+ions have however not been well investigated. Here, we use a range of biophysical techniques to characterize in vitro interactions between Aβ peptides and Li+ions. We show that Li+ions display weak and non-specific interactions with Aβ peptides, and have minor effects on Aβ aggregation. These results indicate that possible beneficial effects of Li on AD pathology are not likely caused by direct interactions between Aβ peptides and Li+ions.

  • 28.
    Berntsson, Elina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vosough, Faraz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Noormagi, Andra
    Padari, Kärt
    Asplund, Fanny
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gielnik, Maciej
    Paul, Suman
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jarvet, Jüri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tõugu, Vello
    Roos, Per M.
    Kozak, Maciej
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. CellPept Sweden AB, Sweden.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pooga, Margus
    Palumaa, Peep
    Wärmländer, Sebastian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. CellPept Sweden AB, Sweden.
    Characterization of Uranyl (UO22+) Ion Binding to Amyloid Beta (Aβ) Peptides: Effects on Aβ Structure and Aggregation2023In: ACS Chemical Neuroscience, E-ISSN 1948-7193, Vol. 14, no 15, p. 2618-2633Article in journal (Refereed)
    Abstract [en]

    Uranium (U) is naturally present in ambient air, water, and soil, and depleted uranium (DU) is released into the environment via industrial and military activities. While the radiological damage from U is rather well understood, less is known about the chemical damage mechanisms, which dominate in DU. Heavy metal exposure is associated with numerous health conditions, including Alzheimer’s disease (AD), the most prevalent age-related cause of dementia. The pathological hallmark of AD is the deposition of amyloid plaques, consisting mainly of amyloid-β (Aβ) peptides aggregated into amyloid fibrils in the brain. However, the toxic species in AD are likely oligomeric Aβ aggregates. Exposure to heavy metals such as Cd, Hg, Mn, and Pb is known to increase Aβ production, and these metals bind to Aβ peptides and modulate their aggregation. The possible effects of U in AD pathology have been sparsely studied. Here, we use biophysical techniques to study in vitro interactions between Aβ peptides and uranyl ions, UO22+, of DU. We show for the first time that uranyl ions bind to Aβ peptides with affinities in the micromolar range, induce structural changes in Aβ monomers and oligomers, and inhibit Aβ fibrillization. This suggests a possible link between AD and U exposure, which could be further explored by cell, animal, and epidemiological studies. General toxic mechanisms of uranyl ions could be modulation of protein folding, misfolding, and aggregation. 

  • 29. Carissimi, Guzmán
    et al.
    Baronio, Cesare M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Montalbán, Mercedes G.
    Víllora, Gloria
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    On the Secondary Structure of Silk Fibroin Nanoparticles Obtained Using Ionic Liquids: An Infrared Spectroscopy Study2020In: Polymers, E-ISSN 2073-4360, Vol. 12, no 6, article id 1294Article in journal (Refereed)
    Abstract [en]

    Silk fibroin from Bombyx mori caterpillar is an outstanding biocompatible polymer for the production of biomaterials. Its impressive combination of strength, flexibility, and degradability are related to the protein’s secondary structure, which may be altered during the manufacture of the biomaterial. The present study looks at the silk fibroin secondary structure during nanoparticle production using ionic liquids and high-power ultrasound using novel infrared spectroscopic approaches. The infrared spectrum of silk fibroin fibers shows that they are composed of 58% β-sheet, 9% turns, and 33% irregular and/or turn-like structures. When fibroin was dissolved in ionic liquids, its amide I band resembled that of soluble silk and no β-sheet absorption was detected. Silk fibroin nanoparticles regenerated from the ionic liquid solution exhibited an amide I band that resembled that of the silk fibers but had a reduced β-sheet content and a corresponding higher content of turns, suggesting an incomplete turn-to-sheet transition during the regeneration process. Both the analysis of the experimental infrared spectrum and spectrum calculations suggest a particular type of β-sheet structure that was involved in this deficiency, whereas the two other types of β-sheet structure found in silk fibroin fibers were readily formed.

  • 30. Carissimi, Guzmán
    et al.
    Montalbán, Mercedes G.
    Villora, Gloria
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Direct Quantification of Drug Loading Content in Polymeric Nanoparticles by Infrared Spectroscopy2020In: Pharmaceutics, ISSN 1999-4923, E-ISSN 1999-4923, Vol. 12, no 10, article id 912Article in journal (Refereed)
    Abstract [en]

    Nanotechnology has enabled the development of novel therapeutic strategies such as targeted nanodrug delivery systems, control and stimulus-responsive release mechanisms, and the production of theranostic agents. As a prerequisite for the use of nanoparticles as drug delivery systems, the amount of loaded drug must be precisely quantified, a task for which two approaches are currently used. However, both approaches suffer from the inefficiencies of drug extraction and of the solid-liquid separation process, as well as from dilution errors. This work describes a new, reliable, and simple method for direct drug quantification in polymeric nanoparticles using attenuated total reflection Fourier transform infrared spectroscopy, which can be adapted for a wide variety of drug delivery systems. Silk fibroin nanoparticles and naringenin were used as model polymeric nanoparticle carrier and drug, respectively. The specificity, linearity, detection limit, precision, and accuracy of the spectroscopic approach were determined in order to validate the method. A good linear relation was observed within 0.00 to 7.89% of naringenin relative mass with an R-2 of 0.973. The accuracy was determined by the spike and recovery method. The results showed an average 104% recovery. The limit of detection and limit of quantification of the drug loading content were determined to be 0.3 and 1.0%, respectively. The method's robustness is demonstrated by the notable similarities between the calibrations carried out using two different equipment setups at two different institutions.

  • 31. 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, p. 1089-1096Article 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.

  • 32. 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, p. 84-97Article 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.

  • 33.
    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, p. 14967-14972Article 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.

  • 34.
    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, p. 932-943Article 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.

  • 35.
    Gerdes, Zandra
    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 AB, Sweden; Swedish University of Agricultural Sciences, Sweden.
    Nybom, Inna
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Ek, Caroline
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Adolfsson-Erici, Margaretha
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gorokhova, Elena
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Microplastic-mediated transport of PCBs? A depuration study with Daphnia magna2019In: PLOS ONE, E-ISSN 1932-6203, Vol. 14, no 2, article id e0205378Article in journal (Refereed)
    Abstract [en]

    The role of microplastic (MP) as a carrier of persistent organic pollutants (POPs) to aquatic organisms has been a topic of debate. However, the reverse POP transport can occur if relative contaminant concentrations are higher in the organism than in the microplastic. We evaluated the effect of microplastic on the PCB removal in planktonic animals by exposing the cladoceran Daphnia magna with a high body burden of polychlorinated biphenyls (PCB 18, 40, 128 and 209) to a mixture of microplastic and algae; daphnids exposed to only algae served as the control. As the endpoints, we used PCB body burden, growth, fecundity and elemental composition (%C and %N) of the daphnids. In the daphnids fed with microplastic, PCB 209 was removed more efficiently, while there was no difference for any other congeners and Sigma PCBs between the microplastic-exposed and control animals. Also, higher size-specific egg production in the animals carrying PCB and receiving food mixed with micro-plastics was observed. However, the effects of the microplastic exposure on fecundity were of low biological significance, because the PCB body burden and the microplastic exposure concentrations were greatly exceeding environmentally relevant concentrations.

  • 36.
    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, p. 292-302Article 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.

  • 37. Gonska, Nathalie
    et al.
    Lopez, Patricia A.
    Lozano-Picazo, Paloma
    Thorpe, Michael
    Guinea, Gustavo
    Johansson, Jan
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Perez-Rigueiro, Jose
    Rising, Anna
    Structure-Function Relationship of Artificial Spider Silk Fibers Produced by Straining Flow Spinning2020In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 21, no 6, p. 2116-2124Article in journal (Refereed)
    Abstract [en]

    The production of large quantities of artificial spider silk fibers that match the mechanical properties of the native material has turned out to be challenging. Recent advancements in the field make biomimetic spinning approaches an attractive way forward since they allow the spider silk proteins to assemble into the secondary, tertiary, and quaternary structures that are characteristic of the native silk fiber. Straining flow spinning (SFS) is a newly developed and versatile method that allows production under a wide range of processing conditions. Here, we use a recombinant spider silk protein that shows unprecedented water solubility and that is capable of native-like assembly, and we spin it into fibers by the SFS technique. We show that fibers may be spun using different hydrodynamical and chemical conditions and conclude that these spinning conditions affect fiber mechanics. In particular, it was found that the addition of acetonitrile and polyethylene glycol to the collection bath results in fibers with increased beta-sheet content and improved mechanical properties.

  • 38. Greco, Gabriele
    et al.
    Arndt, Tina
    Schmuck, Benjamin
    Francis, Juanita
    Bäcklund, Fredrik G.
    Shilkova, Olga
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gonska, Nathalie
    Seisenbaeva, Gulaim
    Kessler, Vadim
    Johansson, Jan
    Pugno, Nicola M.
    Rising, Anna
    Tyrosine residues mediate supercontraction in biomimetic spider silk2021In: Communications materials, ISSN 2662-4443, Vol. 2, no 1, article id 43Article in journal (Refereed)
    Abstract [en]

    Exposing spider silk to wet conditions can cause supercontraction. Here, tyrosine amino acid residues within the amorphous regions are found to contribute to supercontraction, which can be controlled by protein engineering. Water and humidity severely affect the material properties of spider major ampullate silk, causing the fiber to become plasticized, contract, swell and undergo torsion. Several amino acid residue types have been proposed to be involved in this process, but the complex composition of the native fiber complicates detailed investigations. Here, we observe supercontraction in biomimetically produced artificial spider silk fibers composed of defined proteins. We found experimental evidence that proline is not the sole residue responsible for supercontraction and that tyrosine residues in the amorphous regions of the silk fiber play an important role. Furthermore, we show that the response of artificial silk fibers to humidity can be tuned, which is important for the development of materials for applications in wet environments, eg producing water resistant fibers with maximal strain at break and toughness modulus.

  • 39. Greco, Gabriele
    et al.
    Francis, Juanita
    Arndt, Tina
    Schmuck, Benjamin
    Backlund, Fredrik G.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Jan
    Pugno, Nicola M.
    Rising, Anna
    Properties of Biomimetic Artificial Spider Silk Fibers Tuned by PostSpin Bath Incubation2020In: Molecules, ISSN 1431-5157, E-ISSN 1420-3049, Vol. 25, no 14, article id 3248Article in journal (Refereed)
    Abstract [en]

    Efficient production of artificial spider silk fibers with properties that match its natural counterpart has still not been achieved. Recently, a biomimetic process for spinning recombinant spider silk proteins (spidroins) was presented, in which important molecular mechanisms involved in native spider silk spinning were recapitulated. However, drawbacks of these fibers included inferior mechanical properties and problems with low resistance to aqueous environments. In this work, we show that >= 5 h incubation of the fibers, in a collection bath of 500 mM NaAc and 200 mM NaCl, at pH 5 results in fibers that do not dissolve in water or phosphate buffered saline, which implies that the fibers can be used for applications that involve wet/humid conditions. Furthermore, incubation in the collection bath improved the strain at break and was associated with increased beta-sheet content, but did not affect the fiber morphology. In summary, we present a simple way to improve artificial spider silk fiber strain at break and resistance to aqueous solvents.

  • 40. 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, p. 3259-70Article 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.

  • 41.
    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, p. 2580-7Article 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.

  • 42. Jafari, Mohammad Javad
    et al.
    Backlund, Fredrik G.
    Arndt, Tina
    Schmuck, Benjamin
    Greco, Gabriele
    Rising, Anna
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ederth, Thomas
    Force-Induced Structural Changes in Spider Silk Fibers Introduced by ATR-FTIR Spectroscopy2023In: ACS applied polymer materials, ISSN 2637-6105, Vol. 5, no 11, p. 9433-9444Article in journal (Refereed)
    Abstract [en]

    Silk fibers have unique mechanical properties, and many studies of silk aim at understanding how these properties are related to secondary structure content, which often is determined by infrared spectroscopy. We report significant method-induced irreversible structural changes to both natural and synthetic spider silk fibers, derived from the widely used attenuated total reflection Fourier-transform infrared (ATR-FTIR) technique. By varying the force used to bring fibers into contact with the internal reflection elements of ATR-FTIR accessories, we observed correlated and largely irreversible changes in the secondary structure, with shape relaxation under pressure occurring within minutes. Fitting of spectral components shows that these changes agree with transformations from the alpha-helix to the beta-sheet secondary structure with possible contributions from other secondary structure elements. We further confirm the findings with IR microspectroscopy, where similar differences were seen between the pressed and unaffected regions of spider silk fibers. Our findings show that ATR-FTIR spectroscopy requires care in its use and in the interpretation of the results.

  • 43. 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, p. 550-562Article 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.

  • 44.
    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, p. 4448-4456Article 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

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  • 45.
    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, p. 4831-4842Article 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.

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  • 46.
    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, p. 2282-2289Article 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.

  • 47.
    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, p. 1310-1318Article 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.

  • 48.
    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, p. 749-757Article 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.

  • 49.
    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, p. 114-23Article 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.

  • 50.
    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, p. 89-94Article 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.

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