@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} }