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-Structure paper
Title | Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements. |
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Journal, issue, pages | Proc Natl Acad Sci U S A, Vol. 114, Issue 31, Page E6342-E6351, Year 2017 |
Publish date | Aug 1, 2017 |
![]() | Gustavo Fuertes / Niccolò Banterle / Kiersten M Ruff / Aritra Chowdhury / Davide Mercadante / Christine Koehler / Michael Kachala / Gemma Estrada Girona / Sigrid Milles / Ankur Mishra / Patrick R Onck / Frauke Gräter / Santiago Esteban-Martín / Rohit V Pappu / Dmitri I Svergun / Edward A Lemke / ![]() ![]() ![]() ![]() |
PubMed Abstract | Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous ...Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration ( ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance ( ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values and For chemically denatured proteins we obtain mutual consistency in our inferences based on and , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between and that is amplified in the absence of denaturants. Therefore, joint assessments of and combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles. |
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Methods | SAS (X-ray synchrotron) |
Structure data | ![]() SASDE23: Labeled nuclear pore complex protein Nup153 (NUL-Alexa488/Alexa594) without denaturant ![]() SASDE33: ![]() SASDE43: Labeled nuclear pore complex protein Nup153 (NUL-Alexa488/Alexa594) with denaturant ![]() SASDE53: Unlabeled dihydrolipoyllysine-residue succinyltransferase component (BBL) with denaturant ![]() SASDE63: Labeled dihydrolipoyllysine-residue succinyltransferase component (BBL-Alexa488/Alexa594) with denaturant ![]() SASDE73: Unlabeled cold shock protein (CSP) with denaturant ![]() SASDE83: Labeled cold shock protein (CSP-Alexa488/Alexa594) with denaturant ![]() SASDE93: Unlabeled thioredoxin (TRX) with denaturant (Thioredoxin 1, TRX) ![]() SASDEA3: Labeled thioredoxin (TRX-Alexa488/Alexa594) with denaturant ![]() SASDEB3: Unlabeled nuclear pore complex protein Nup98-Nup96 (N98) without denaturant ![]() SASDEC3: Unlabeled nucleoporin NSP1 (NSP) without denaturant ![]() SASDEH2: ![]() SASDEJ2: Labeled nucleoporin NUP49/NSP49 (N49-Alexa488/Alexa594) without denaturant ![]() SASDEK2: ![]() SASDEL2: Labeled nucleoporin NUP49/NSP49 (N49-Alexa488/Alexa594) with denaturant ![]() SASDEM2: ![]() SASDEN2: Labeled Nuclear Localization Signal from the inner nuclear membrane protein HEH2 (NLS-Alexa488/Alexa594) without denaturant ![]() SASDEP2: ![]() SASDEQ2: Labeled Nuclear Localization Signal from the inner nuclear membrane protein HEH2 (NLS-Alexa488/Alexa594) with denaturant ![]() SASDER2: ![]() SASDES2: Labeled Importin Beta Binding Domain (IBB-Alexa488/Alexa594) from importin subunit alpha-1 without denaturant ![]() SASDET2: ![]() SASDEU2: Labeled Importin Beta Binding Domain from importin subunit alpha-1 (IBB-Alexa488/Alexa594) with denaturant ![]() SASDEV2: ![]() SASDEW2: Labeled nuclear pore complex protein Nup153 (NUS-Alexa488/Alexa594) without denaturant ![]() SASDEX2: ![]() SASDEY2: Labeled nuclear pore complex protein Nup153 (NUS-Alexa488/Alexa594) with denaturant ![]() SASDEZ2: |
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