Commonly used FRET fluorophores promote collapse of an otherwise disordered protein.

Autor: Riback JA; Graduate Program in Biophysical Sciences, The University of Chicago, Chicago, IL 60637., Bowman MA; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556., Zmyslowski AM; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637., Plaxco KW; Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106., Clark PL; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556; pclark1@nd.edu trsosnic@uchicago.edu., Sosnick TR; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637; pclark1@nd.edu trsosnic@uchicago.edu.; Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637.
Jazyk: angličtina
Zdroj: Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2019 Apr 30; Vol. 116 (18), pp. 8889-8894. Date of Electronic Publication: 2019 Apr 16.
DOI: 10.1073/pnas.1813038116
Abstrakt: The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (R g ) and end-to-end distance (R ee ) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the R g and R ee are "uncoupled" (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.
Competing Interests: The authors declare no conflict of interest.
Databáze: MEDLINE
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