@misc{BestZhengBorgiaetal.2018, author = {Best, Robert B. and Zheng, Wenwei and Borgia, Alessandro and Buholzer, Karin and Borgia, Madeleine B. and Hofmann, Hagen and Soranno, Andrea and Nettels, Daniel and Gast, Klaus and Grishaev, Alexander and Schuler, Benjamin}, title = {Comment on "Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water"}, series = {Science}, volume = {361}, journal = {Science}, number = {6405}, publisher = {American Assoc. for the Advancement of Science}, address = {Washington}, issn = {0036-8075}, doi = {10.1126/science.aar7101}, pages = {2}, year = {2018}, abstract = {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.}, language = {en} } @article{BorgiaZhengBuholzeretal.2016, author = {Borgia, Alessandro and Zheng, Wenwei and Buholzer, Karin and Borgia, Madeleine B. and Sch{\"u}ler, Anja and Hofmann, Hagen and Soranno, Andrea and Nettels, Daniel and Gast, Klaus and Grishaev, Alexander and Best, Robert B. and Schuler, Benjamin}, title = {Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods}, series = {Journal of the American Chemical Society}, volume = {138}, journal = {Journal of the American Chemical Society}, publisher = {American Chemical Society}, address = {Washington}, issn = {0002-7863}, doi = {10.1021/jacs.6b05917}, pages = {11714 -- 11726}, year = {2016}, abstract = {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.}, language = {en} } @article{HofmannSorannoBorgiaetal.2012, author = {Hofmann, Hagen and Soranno, Andrea and Borgia, Alessandro and Gast, Klaus and Nettels, Daniel and Schuler, Benjamin}, title = {Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy}, series = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {109}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, number = {40}, publisher = {National Acad. of Sciences}, address = {Washington}, issn = {0027-8424}, doi = {10.1073/pnas.1207719109}, pages = {16155 -- 16160}, year = {2012}, abstract = {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.}, language = {en} } @article{HoffmannKaneNettelsetal.2007, author = {Hoffmann, Armin S. and Kane, Avinash S. and Nettels, Daniel and Hertzog, David E. and Baumg{\"a}rtel, Peter and Lengefeld, Jan and Reichardt, Gerd and Horsley, David A. and Seckler, Robert and Bakajin, Olgica and Schuler, Benjamin}, title = {Mapping protein collapse with single molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy}, issn = {0027-8424}, year = {2007}, language = {en} } @article{NettelsMuellerSpaethKuesteretal.2009, author = {Nettels, Daniel and M{\"u}ller-Sp{\"a}th, Sonja and K{\"u}ster, Frank and Hofmann, Hagen and Haenni, Domminik and R{\"u}egger, Stefan and Reymond, Luc and Hoffmann, Armin S. and Kubelka, Jan and Heinz, Benjamin and Gast, Klaus and Best, Robert B. and Schuler, Benjamin}, title = {Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins}, issn = {0027-8424}, year = {2009}, abstract = {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.}, language = {en} } @article{KaneHoffmannBaumgaerteletal.2008, author = {Kane, Avinash S. and Hoffmann, Armin S. and Baumg{\"a}rtel, Peter and Seckler, Robert and Reichardt, Gerd and Horsley, David A. and Schuler, Benjamin and Bakajin, Olgica}, title = {Microfluidic mixers for the investigation of rapid protein folding kinetics using synchrotron radiation circular dichroism spectroscopy}, issn = {0003-2700}, year = {2008}, abstract = {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.}, language = {en} } @article{LipmanSchulerBakajinetal.2003, author = {Lipman, Everett A. and Schuler, Benjamin and Bakajin, Olgica and Eaton, William A.}, title = {Single-molecule measurement of protein folding kinetics}, issn = {0036-8075}, year = {2003}, abstract = {In order to investigate the behavior of single molecules under conditions far from equilibrium, we have coupled a microfabricated laminar-flow mixer to a confocal optical system. This combination enables time-resolved measurement of Foerster resonance energy transfer after an abrupt change in solution conditions. Observations of a small protein show the evolution of the intramolecular distance distribution as folding progresses. This technique can expose subpopulations, such as unfolded protein under conditions favoring the native structure, that would be obscured in equilibrium experiments.}, language = {en} } @article{TolanSchulerBeerninketal.2003, author = {Tolan, Dean R. and Schuler, Benjamin and Beernink, Peter T. and Jaenicke, Rainer}, title = {Thermodynamic analysis of the dissociation of the aldolase tetramer substituted at one or both of the subunit interfaces}, year = {2003}, abstract = {The fructose-1,6-bis(phosphate) aldolase isologous tetramer tightly associates through two different subunit interfaces defined by its 222 symmetry. Both single- and double-interfacial mutant aldolases have a destabilized quaternary structure, but there is little effect on the catalytic activity. These enzymes are however thermolabile. This study demonstrates the temperature-dependent dissociation of the mutant enzymes and determines the dissociation free energies of both mutant and native aldolase. Subunit dissociation is measured by sedimentation equilibrium in the analytical ultracentrifuge. At 25C the tetramerdimer dissociation constants for each single-mutant enzyme are similar, about 10 -6 M. For the double-mutant enzyme, sedimentation velocity experiments on sucrose density gradients support a tetramermonomer equilibrium. Furthermore, sedimentation equilibrium experiments determined a dissociation constant of 10- 15 M3 for the double-mutant enzyme. By the same methods the upper limit for the dissociation constant of wild-type aldolase A is approximately 10-28 M3, which indicates an extremely stable tetramer. The thermodynamic values describing monomer-tetramer and dimer-tetramer equilibria are analyzed with regard to possible cooperative interaction between the two subunit interfaces.}, language = {en} } @article{SchulerSeckler1998, author = {Schuler, Benjamin and Seckler, Robert}, title = {P22 tailspike folding mutants revisited : effects on thermodynamic stability of the isolated beta-helix domain}, year = {1998}, language = {en} } @article{MillerSchulerSeckler1998, author = {Miller, Stefan and Schuler, Benjamin and Seckler, Robert}, title = {A reversibly unfolding fragment of P22 tailspike protein with native structure : the isolated beta-helix domain}, year = {1998}, language = {en} }