Effect of flexibility and cis residues in single-molecule FRET studies of polyproline.

Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Research Abstract Details 

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Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Abstract Text:

Robert B Best,Kusai A Merchant,Irina V Gopich,Benjamin Schuler,Ad Bax,William A Eaton,Robert B Best,Kusai A Merchant,Irina V Gopich,Benjamin Schuler,Ad Bax,William A Eaton,

Polyproline has recently been used as a spacer between donor and acceptor chromophores to help establish the accuracy of distances determined from single-molecule Förster resonance energy transfer (FRET) measurements. This work showed that the FRET efficiency in water is higher than expected for a rigid spacer and was attributed to the flexibility of the polypeptide. Here, we investigate this issue further, using a combination of single-molecule fluorescence intensity and lifetime measurements, NMR, theory, and molecular dynamics simulations of polyproline-20 that include the dyes and their linkers to the polypeptide. NMR shows that in water approximately 30% of the molecules contain internal cis prolines, whereas none are detectable in trifluoroethanol. Simulations suggest that the all-trans form of polyproline is relatively stiff, with persistence lengths of 9-13 nm using different established force fields, and that the kinks arising from internal cis prolines are primarily responsible for the higher mean FRET efficiency in water. We show that the observed efficiency histograms and distributions of donor fluorescence lifetimes are explained by the presence of multiple species with efficiencies consistent with the simulations and populations determined by NMR. In calculating FRET efficiencies from the simulation, we find that the fluctuations of the chromophores, attached to long flexible linkers, also play an important role. A similar simulation approach suggests that the flexibility of the chromophore linkers is largely responsible for the previously unexplained high value of R(0) required to fit the data in the classic study of Stryer and Haugland.

Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Publishing Authors By Initials

RB Best,KA Merchant,IV Gopich,B Schuler,A Bax,WA Eaton,RB Best,KA Merchant,IV Gopich,B Schuler,A Bax,WA Eaton,

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Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Journal Published:

PUBLICATION TYPE: Research Support, N.I.H., Intr

Journal: Proceedings of the National Academy of Sciences of

VOLUME: 104

Page Numbers: 18964-9

Journal Abbreviation: Proc. Natl. Acad. Sci. U.S.A.

ISSN: 1091-6490

DAY: 20

MONTH: 11

YEAR: 2007

Effect of flexibility and cis residues in single-molecule FRET studies of polyproline. Information

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LANGUAGE: eng

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AFFILIATION: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA. rbb24@cam.ac.uk

Country: United States

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MEDLINETA: Proc Natl Acad Sci U S A

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