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Papers by Reinhard Schweitzer-Stenner

The Journal of Physical Chemistry B, 2013

Several lines of evidence now well establish that unfolded peptides in general, and alanine in sp... more Several lines of evidence now well establish that unfolded peptides in general, and alanine in specific, have an intrinsic preference for the polyproline II (pPII) conformation. Investigation of local order in the unfolded state is, however, complicated by experimental limitations and the inherent dynamics of the system, which has in some cases yielded inconsistent results from different types of experiments. One method of studying these systems is the use of short model peptides, and specifically short alanine peptides, known for predominantly sampling pPII structure in aqueous solution. Recently, He et al. (J. Am. Chem. Soc. 2012, 134, 1571−1576 proposed that unblocked tripeptides may not be suitable models for studying conformational propensities in unfolded peptides due to the presence of end effect, that is, electrostatic interactions between investigated amino acid residues and terminal charges. To determine whether changing the protonation states of the N-and C-termini influence the conformational manifold of the central amino acid residue in tripeptides, we have examined the pH-dependence of unblocked trialanine and the conformational preferences of alanine in the alanine dipeptide. To this end, we measured and globally analyzed amide I′ band profiles and NMR J-coupling constants. We described conformational distributions as the superposition of two-dimensional Gaussian distributions assignable to specific subspaces of the Ramachandran plot. Results show that the conformational ensemble of trialanine as a whole, and the pPII content (χ pPII = 0.84) in particular, remains practically unaffected by changing the protonation state. We found that compared to trialanine, the alanine dipeptide has slightly lower pPII content (χ pPII = 0.74) and an ensemble more reminiscent of the unblocked Gly-Ala-Gly model peptide. In addition, a two-state thermodynamic analysis of the conformational sensitive Δε(T) and 3 J(H N H α )(T) data obtained from electronic circular dichroism and H NMR spectra indicate that the free energy landscape of trialanine is similar in all protonation states. MD simulations for the investigated peptides corroborate this notion and show further that the hydration shell around unblocked trialanine is unaffected by the protonation/deprotonation of the C-terminal group. In contrast, the alanine dipeptide shows a reduced water density around the central residue as well as a less ordered hydration shell, which decreases the pPII propensity and reduces the lifetime of sampled conformations.

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2005

Several recent reports have suggested that binding monomeric IgE (mIgE) to its type 1 receptor, F... more Several recent reports have suggested that binding monomeric IgE (mIgE) to its type 1 receptor, Fc epsilon RI, on mast cells induces important responses. These observations contradict the notion that it is the aggregation of this receptor that is essential for initiating mast cell response. In the present study, we suggest that the most probable causes for the reported observations are the experimental protocol used combined with the high expression levels of the Fc epsilon RI by mast cells. Specifically, we suggest using the published data and physicochemical calculations that the exceptionally high number of cell surface Fc epsilon RI-bound monoclonal IgE yields, in the two-dimensions of the cells' membranes, a situation where even a low affinity of these mIgE for epitopes on their own structure or on another cell surface component may lead to their aggregation. Hence, we hypothesize that the reported response to mIgE binding is a result of such an Fc epsilon RI-IgE induced aggregation.

Biophysical Journal, 2009

Biochemistry, 2008

The oxidized state of cytochrome c is a subject of continuous interest, owing to the multitude of... more The oxidized state of cytochrome c is a subject of continuous interest, owing to the multitude of conformations which the protein can adopt in solution and on surfaces of artificial and cell membranes. The structural diversity corresponds to a variety of functions in electron transfer, peroxidase and apoptosis processes. In spite of numerous studies, a comprehensive analysis and comparison of native and nonnative states of ferricytochrome c has thus far not been achieved. This results in part from the fact that the influence of solvent conditions (i.e., ionic strength, anion concentration, temperature dependence of pH values) on structure, function and equilibrium thermodynamics has not yet been thoroughly assessed. The current study is a first step in this direction, in that it provides the necessary experimental data to compare different non-native states adopted at high temperature and alkaline pH. To this end, we employed visible electronic circular dichroism (ECD) and absorption spectroscopy to probe structural changes of the heme environment in bovine and horse heart ferricytochrome c as a function of temperature between 278 and 363 K at different neutral and alkaline pH values. A careful selection of buffers enabled us to monitor the partial unfolding of the native state at room temperature while avoiding a change to an alkaline state at high temperatures. We found compelling evidence for the existence of a thermodynamic intermediate of the thermal unfolding/folding process, termed III h , which is structurally different from the alkaline states, IV 1 and IV 2 , contrary to current belief. At neutral or slightly acidic pH, III h is populated in a temperature region between 320 and 345 K. The unfolded state of the protein becomes populated at higher temperatures. The ECD spectra of the B-bands of bovine and horse heart cytochrome c (pH 7.0) exhibit a pronounced couplet that is maintained below 343 K, before protein unfolding replaces it by a rather strong positive Cotton band. A preliminary vibronic analysis of the B-band profile reveals that the couplet reflects a B-band splitting of 350 cm -1 , which is mostly of electronic origin, due to the internal electric field in the heme cavity. Our results suggest that the conformational transition from the native state, III, into a thermally activated intermediate state, III h , does not substantially affect the internal electric field and causes only moderate rearrangements of the heme pocket, which involves changes, rather than a rupture, of the Fe 3+ -M80 linkage. In the unfolded state, as well as in the alkaline states IV and V, the band splitting is practically eliminated, but the positive Cotton effect observed for the B-band suggests that the proximal environment, encompassing H18 and the two cysteine residues 14 and 17, is most likely still intact and covalently bound to the heme chromophore. Both alkaline states IV and V were found to melt via intermediate states. Unfolded states probed at neutral and alkaline pH can be discriminated, owing to the different intensities of the Cotton bands of the respective B-band transitions. Differences between the ECD intensities of the B-bands of the different unfolded states and alkaline states most likely reflect different degrees of openness of the corresponding heme crevice.

Methods in enzymology, 2009

Over the last 50 years cytochrome c has been used as a model system for studying electron transfe... more Over the last 50 years cytochrome c has been used as a model system for studying electron transfer and protein folding processes. Recently, convincing evidence has been provided that this protein is also involved in other biological processes such as the apoptosis and α-synuclein aggregation. Numerous lines of evidence suggest that the diversity of the functional properties of cytochrome c is linked to its conformational plasticity. This chapter introduces circular dichroism and absorption spectroscopy, as an ideal tool to explore this protein's conformational in solution. Besides assisting in distinguishing different conformations and in obtaining the equilibrium thermodynamics of the transitions between them, the two spectroscopies can also be used to explore details of heme-protein interaction, for example, the influence of the external electric field on the prosthetic heme group.

total PSI effort. These eukaryotic targets frequently present unique challenges. All CESG protein... more total PSI effort. These eukaryotic targets frequently present unique challenges. All CESG protein production protocols and Technology Dissemination Reports are accessible through the PSI Knowledgebase: http://kb-psi-structuralgenomics.org/KB/ and CESG's website. Selected technology developments are presented here. These include advances in expression vector design, enhanced methodology for cell based and wheat germ cell-free expression systems, new software to improve the quality and reduce time for structure determination by X-ray crystallography and NMR, and optimized techniques for the production of TEV protease for use in our protein production platform. We actively share our advances with the biotechnology, pharmaceutical, and academic communities through collaborations, oral presentations, peer-reviewed articles, submissions to public databases and material distribution channels, including PepcDB, PDB, BMRB, PSI Materials Repository, and technology transfer workshops. Supported by NIH/NIGMS Protein Structure Initiative grants GM074901 and GM064598. We acknowledge ALL members of the CESG team for their dedicated work.

Title: Conformational Discrepancies Between Molecular Dynamics Force Fields and Vibrational Spect... more Title: Conformational Discrepancies Between Molecular Dynamics Force Fields and Vibrational Spectroscopy in Short Alanine-Based Peptides. Authors: Verbaro, Daniel; Gosh, Indrajit; Nau, Werner; Schweitzer-Stenner, Reinhard. Publication: Biophysical Journal, vol. ...

domain and several long intrinsically disordered regions containing tandem short repeats. The 150... more domain and several long intrinsically disordered regions containing tandem short repeats. The 1500 residue BLUE region consists of 92 copies of 16-residue repeats, each with two octads of 8 residues. The octads in BLUE motifs form unstable a-helix-like coils in aqueous solution and negligible heptadbased, a-helical coiled-coils. The a-helix-like conformation is highly temperature dependent. The a-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface( Forbes et al., (2010) J. Mol. .The conformation of BLUE octads was investigated further by NMR and molecular dynamics simulations. Molecular dynamics simulations of a 16-residue repeat indicated different behaviors of the helical state depending upon the force field used and the charge state of the peptide termini. Multidimensional NMR of the same peptide sequence indicated a helical conformation with a break in the helicity in the middle of the peptide consistent with one of the simulations. Interestingly, some of the simulations indicate the transient formation of 4.4 residue per turn left-handed p-helices, at the expense of a helices in the octads of BLUE. These observations suggest an intriguing possibility of an a-p conversion that alters reversibly the net contour length of the BLUE peptides, effecting protein elasticity in a heretofore unexpected mechanism.

The Journal of Physical Chemistry B, 2013

Several lines of evidence now well establish that unfolded peptides in general, and alanine in sp... more Several lines of evidence now well establish that unfolded peptides in general, and alanine in specific, have an intrinsic preference for the polyproline II (pPII) conformation. Investigation of local order in the unfolded state is, however, complicated by experimental limitations and the inherent dynamics of the system, which has in some cases yielded inconsistent results from different types of experiments. One method of studying these systems is the use of short model peptides, and specifically short alanine peptides, known for predominantly sampling pPII structure in aqueous solution. Recently, He et al. (J. Am. Chem. Soc. 2012, 134, 1571−1576 proposed that unblocked tripeptides may not be suitable models for studying conformational propensities in unfolded peptides due to the presence of end effect, that is, electrostatic interactions between investigated amino acid residues and terminal charges. To determine whether changing the protonation states of the N-and C-termini influence the conformational manifold of the central amino acid residue in tripeptides, we have examined the pH-dependence of unblocked trialanine and the conformational preferences of alanine in the alanine dipeptide. To this end, we measured and globally analyzed amide I′ band profiles and NMR J-coupling constants. We described conformational distributions as the superposition of two-dimensional Gaussian distributions assignable to specific subspaces of the Ramachandran plot. Results show that the conformational ensemble of trialanine as a whole, and the pPII content (χ pPII = 0.84) in particular, remains practically unaffected by changing the protonation state. We found that compared to trialanine, the alanine dipeptide has slightly lower pPII content (χ pPII = 0.74) and an ensemble more reminiscent of the unblocked Gly-Ala-Gly model peptide. In addition, a two-state thermodynamic analysis of the conformational sensitive Δε(T) and 3 J(H N H α )(T) data obtained from electronic circular dichroism and H NMR spectra indicate that the free energy landscape of trialanine is similar in all protonation states. MD simulations for the investigated peptides corroborate this notion and show further that the hydration shell around unblocked trialanine is unaffected by the protonation/deprotonation of the C-terminal group. In contrast, the alanine dipeptide shows a reduced water density around the central residue as well as a less ordered hydration shell, which decreases the pPII propensity and reduces the lifetime of sampled conformations.

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2005

Several recent reports have suggested that binding monomeric IgE (mIgE) to its type 1 receptor, F... more Several recent reports have suggested that binding monomeric IgE (mIgE) to its type 1 receptor, Fc epsilon RI, on mast cells induces important responses. These observations contradict the notion that it is the aggregation of this receptor that is essential for initiating mast cell response. In the present study, we suggest that the most probable causes for the reported observations are the experimental protocol used combined with the high expression levels of the Fc epsilon RI by mast cells. Specifically, we suggest using the published data and physicochemical calculations that the exceptionally high number of cell surface Fc epsilon RI-bound monoclonal IgE yields, in the two-dimensions of the cells' membranes, a situation where even a low affinity of these mIgE for epitopes on their own structure or on another cell surface component may lead to their aggregation. Hence, we hypothesize that the reported response to mIgE binding is a result of such an Fc epsilon RI-IgE induced aggregation.

Biophysical Journal, 2009

Biochemistry, 2008

The oxidized state of cytochrome c is a subject of continuous interest, owing to the multitude of... more The oxidized state of cytochrome c is a subject of continuous interest, owing to the multitude of conformations which the protein can adopt in solution and on surfaces of artificial and cell membranes. The structural diversity corresponds to a variety of functions in electron transfer, peroxidase and apoptosis processes. In spite of numerous studies, a comprehensive analysis and comparison of native and nonnative states of ferricytochrome c has thus far not been achieved. This results in part from the fact that the influence of solvent conditions (i.e., ionic strength, anion concentration, temperature dependence of pH values) on structure, function and equilibrium thermodynamics has not yet been thoroughly assessed. The current study is a first step in this direction, in that it provides the necessary experimental data to compare different non-native states adopted at high temperature and alkaline pH. To this end, we employed visible electronic circular dichroism (ECD) and absorption spectroscopy to probe structural changes of the heme environment in bovine and horse heart ferricytochrome c as a function of temperature between 278 and 363 K at different neutral and alkaline pH values. A careful selection of buffers enabled us to monitor the partial unfolding of the native state at room temperature while avoiding a change to an alkaline state at high temperatures. We found compelling evidence for the existence of a thermodynamic intermediate of the thermal unfolding/folding process, termed III h , which is structurally different from the alkaline states, IV 1 and IV 2 , contrary to current belief. At neutral or slightly acidic pH, III h is populated in a temperature region between 320 and 345 K. The unfolded state of the protein becomes populated at higher temperatures. The ECD spectra of the B-bands of bovine and horse heart cytochrome c (pH 7.0) exhibit a pronounced couplet that is maintained below 343 K, before protein unfolding replaces it by a rather strong positive Cotton band. A preliminary vibronic analysis of the B-band profile reveals that the couplet reflects a B-band splitting of 350 cm -1 , which is mostly of electronic origin, due to the internal electric field in the heme cavity. Our results suggest that the conformational transition from the native state, III, into a thermally activated intermediate state, III h , does not substantially affect the internal electric field and causes only moderate rearrangements of the heme pocket, which involves changes, rather than a rupture, of the Fe 3+ -M80 linkage. In the unfolded state, as well as in the alkaline states IV and V, the band splitting is practically eliminated, but the positive Cotton effect observed for the B-band suggests that the proximal environment, encompassing H18 and the two cysteine residues 14 and 17, is most likely still intact and covalently bound to the heme chromophore. Both alkaline states IV and V were found to melt via intermediate states. Unfolded states probed at neutral and alkaline pH can be discriminated, owing to the different intensities of the Cotton bands of the respective B-band transitions. Differences between the ECD intensities of the B-bands of the different unfolded states and alkaline states most likely reflect different degrees of openness of the corresponding heme crevice.

Methods in enzymology, 2009

Over the last 50 years cytochrome c has been used as a model system for studying electron transfe... more Over the last 50 years cytochrome c has been used as a model system for studying electron transfer and protein folding processes. Recently, convincing evidence has been provided that this protein is also involved in other biological processes such as the apoptosis and α-synuclein aggregation. Numerous lines of evidence suggest that the diversity of the functional properties of cytochrome c is linked to its conformational plasticity. This chapter introduces circular dichroism and absorption spectroscopy, as an ideal tool to explore this protein's conformational in solution. Besides assisting in distinguishing different conformations and in obtaining the equilibrium thermodynamics of the transitions between them, the two spectroscopies can also be used to explore details of heme-protein interaction, for example, the influence of the external electric field on the prosthetic heme group.

total PSI effort. These eukaryotic targets frequently present unique challenges. All CESG protein... more total PSI effort. These eukaryotic targets frequently present unique challenges. All CESG protein production protocols and Technology Dissemination Reports are accessible through the PSI Knowledgebase: http://kb-psi-structuralgenomics.org/KB/ and CESG's website. Selected technology developments are presented here. These include advances in expression vector design, enhanced methodology for cell based and wheat germ cell-free expression systems, new software to improve the quality and reduce time for structure determination by X-ray crystallography and NMR, and optimized techniques for the production of TEV protease for use in our protein production platform. We actively share our advances with the biotechnology, pharmaceutical, and academic communities through collaborations, oral presentations, peer-reviewed articles, submissions to public databases and material distribution channels, including PepcDB, PDB, BMRB, PSI Materials Repository, and technology transfer workshops. Supported by NIH/NIGMS Protein Structure Initiative grants GM074901 and GM064598. We acknowledge ALL members of the CESG team for their dedicated work.

Title: Conformational Discrepancies Between Molecular Dynamics Force Fields and Vibrational Spect... more Title: Conformational Discrepancies Between Molecular Dynamics Force Fields and Vibrational Spectroscopy in Short Alanine-Based Peptides. Authors: Verbaro, Daniel; Gosh, Indrajit; Nau, Werner; Schweitzer-Stenner, Reinhard. Publication: Biophysical Journal, vol. ...

domain and several long intrinsically disordered regions containing tandem short repeats. The 150... more domain and several long intrinsically disordered regions containing tandem short repeats. The 1500 residue BLUE region consists of 92 copies of 16-residue repeats, each with two octads of 8 residues. The octads in BLUE motifs form unstable a-helix-like coils in aqueous solution and negligible heptadbased, a-helical coiled-coils. The a-helix-like conformation is highly temperature dependent. The a-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface( Forbes et al., (2010) J. Mol. .The conformation of BLUE octads was investigated further by NMR and molecular dynamics simulations. Molecular dynamics simulations of a 16-residue repeat indicated different behaviors of the helical state depending upon the force field used and the charge state of the peptide termini. Multidimensional NMR of the same peptide sequence indicated a helical conformation with a break in the helicity in the middle of the peptide consistent with one of the simulations. Interestingly, some of the simulations indicate the transient formation of 4.4 residue per turn left-handed p-helices, at the expense of a helices in the octads of BLUE. These observations suggest an intriguing possibility of an a-p conversion that alters reversibly the net contour length of the BLUE peptides, effecting protein elasticity in a heretofore unexpected mechanism.