TY - GEN A1 - Best, Robert B. A1 - Zheng, Wenwei A1 - Borgia, Alessandro A1 - Buholzer, Karin A1 - Borgia, Madeleine B. A1 - Hofmann, Hagen A1 - Soranno, Andrea A1 - Nettels, Daniel A1 - Gast, Klaus A1 - Grishaev, Alexander A1 - Schuler, Benjamin T1 - Comment on "Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water" T2 - Science N2 - Riback et al. (Reports, 13 October 2017, p. 238) used small-angle x-ray scattering (SAXS) experiments to infer a degree of compaction for unfolded proteins in water versus chemical denaturant that is highly consistent with the results from Forster resonance energy transfer (FRET) experiments. There is thus no "contradiction" between the two methods, nor evidence to support their claim that commonly used FRET fluorophores cause protein compaction. Y1 - 2018 U6 - https://doi.org/10.1126/science.aar7101 SN - 0036-8075 SN - 1095-9203 VL - 361 IS - 6405 PB - American Assoc. for the Advancement of Science CY - Washington ER - TY - JOUR A1 - Borgia, Alessandro A1 - Zheng, Wenwei A1 - Buholzer, Karin A1 - Borgia, Madeleine B. A1 - Schüler, Anja A1 - Hofmann, Hagen A1 - Soranno, Andrea A1 - Nettels, Daniel A1 - Gast, Klaus A1 - Grishaev, Alexander A1 - Best, Robert B. A1 - Schuler, Benjamin T1 - Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods JF - Journal of the American Chemical Society N2 - There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no: such increase of the radius of gyration (R-g). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination Of single-molecule Forster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius, with denaturant concentration obtained by 2f-FCS,and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant;induced unfolded state expansion, and to identify the Most likely sources of earlier discrepancies. Y1 - 2016 U6 - https://doi.org/10.1021/jacs.6b05917 SN - 0002-7863 VL - 138 SP - 11714 EP - 11726 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Hofmann, Hagen A1 - Soranno, Andrea A1 - Borgia, Alessandro A1 - Gast, Klaus A1 - Nettels, Daniel A1 - Schuler, Benjamin T1 - Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy JF - Proceedings of the National Academy of Sciences of the United States of America N2 - The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 +/- 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 +/- 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the T-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution. KW - protein folding KW - single-molecule FRET KW - coil-globule transition KW - polymer theory Y1 - 2012 U6 - https://doi.org/10.1073/pnas.1207719109 SN - 0027-8424 VL - 109 IS - 40 SP - 16155 EP - 16160 PB - National Acad. of Sciences CY - Washington ER - TY - JOUR A1 - Nettels, Daniel A1 - Müller-Späth, Sonja A1 - Küster, Frank A1 - Hofmann, Hagen A1 - Haenni, Domminik A1 - Rüegger, Stefan A1 - Reymond, Luc A1 - Hoffmann, Armin S. A1 - Kubelka, Jan A1 - Heinz, Benjamin A1 - Gast, Klaus A1 - Best, Robert B. A1 - Schuler, Benjamin T1 - Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins N2 - We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With singlemolecule FRET, this question can be addressed even under nearnative conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperaturedependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse. Y1 - 2009 UR - http://www.pnas.org/content/106/49/20740.full.pdf+html SN - 0027-8424 ER - TY - JOUR A1 - Kane, Avinash S. A1 - Hoffmann, Armin S. A1 - Baumgärtel, Peter A1 - Seckler, Robert A1 - Reichardt, Gerd A1 - Horsley, David A. A1 - Schuler, Benjamin A1 - Bakajin, Olgica T1 - Microfluidic mixers for the investigation of rapid protein folding kinetics using synchrotron radiation circular dichroism spectroscopy N2 - We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 ;s. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible. Y1 - 2008 UR - http://pubs.acs.org/doi/abs/10.1021/ac801764r SN - 0003-2700 ER - TY - JOUR A1 - Hoffmann, Armin S. A1 - Kane, Avinash S. A1 - Nettels, Daniel A1 - Hertzog, David E. A1 - Baumgärtel, Peter A1 - Lengefeld, Jan A1 - Reichardt, Gerd A1 - Horsley, David A. A1 - Seckler, Robert A1 - Bakajin, Olgica A1 - Schuler, Benjamin T1 - Mapping protein collapse with single molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy Y1 - 2007 UR - http://www.mendeley.com/research/mapping-protein-collapse-with-singlemolecule-fluorescence-and-kinetic- synchrotron-radiation-circular-dichroism-spectroscopy/# SN - 0027-8424 ER - TY - JOUR A1 - Goetz, C. A1 - Suopanki, J. A1 - Schuler, Benjamin A1 - Wanker, E. A1 - Herrmann, Andreas T1 - Perturbation of brain lipid membrane by soluble Huntingtin depends on its polyproline tract Y1 - 2005 SN - 0006-3495 ER - TY - JOUR A1 - Schuler, Benjamin A1 - Lipman, Everett A. A1 - Steinbach, P. J. A1 - Kumke, Michael Uwe A1 - Eaton, W. A. T1 - Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence N2 - To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Forster theory, with polyproline treated as a rigid rod. At donor-acceptor distances much less than the Forster radius R-o, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R-0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Forster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor-acceptor distances Y1 - 2005 SN - 0027-8424 ER - TY - GEN A1 - Schuler, Benjamin A1 - Lipman, Everett A. A1 - Steinbach, Peter J. A1 - Kumke, Michael Uwe A1 - Eaton, William A. T1 - Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence N2 - To determine whether Förster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Förster theory, with polyproline treated as a rigid rod. At donor–acceptor distances much less than the Förster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Förster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor–acceptor distances. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 008 KW - Förster resonance energy transfer KW - molecular dynamics KW - polypeptide KW - FRET Y1 - 2005 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-12229 ER - TY - JOUR A1 - Rhoades, E. A1 - Cohen, M. A1 - Gussakovsky, E. A1 - Schuler, Benjamin A1 - Haran, G. T1 - Single molecule protein folding Y1 - 2004 SN - 0006-3495 ER -