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. 2014 Oct 7;107(7):1654-60.
doi: 10.1016/j.bpj.2014.07.071.

Modest influence of FRET chromophores on the properties of unfolded proteins

Gül H Zerze  1 , Robert B Best  2 , Jeetain Mittal  3
Affiliations

Modest influence of FRET chromophores on the properties of unfolded proteins

Gül H Zerze et al. Biophys J. .

Abstract

Single-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor κ(2), has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances.

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Figures

Figure 1
Figure 1
Contact maps of ensembles without (upper left triangle) and with (lower right triangle) extrinsic chromophores for three proteins (left column) along with their native contacts (right column). (A) CSP, (B) LR, and (C) IN. Folded structures of each protein are shown in the left corner of native contacts maps; (yellow and magenta) secondary structures; and (gray sphere) C-terminal α-carbon atom of each protein. For native contact maps and structures, the Protein Data Bank entries PDB:1G6P (41), PDB:1LMB (42), and PDB:1WJA (43) were used for CSP, LR, and IN, respectively. To see this figure in color, go online.
Figure 2
Figure 2
Per-residue secondary structure propensities calculated based on the DSSP definition. (A) CSP, (B) LR, (C) IN. (Black curves) Averages of ensembles without dyes; (red curves) averages of ensembles in presence of dyes. Standard errors are calculated using block averaging (44). (Vertical green lines) Residues to which the dyes are attached. To see this figure in color, go online.
Figure 3
Figure 3
Distance distributions of proteins with and without dyes and orientational factor distribution of dyes. (From top to bottom) Radius of gyration (Rg) with and without dyes, End-to-end (E2E) distance without dyes, E2E distance with dyes, dye-to-dye (D2D) distance, and orientational factor κ2 distributions of our data and the isotropic case. (Red curves) E2E and D2D distance distributions are Gaussian chain fits; (green curves) self-avoiding chain fits. To see this figure in color, go online.
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References

    1. Förster T. Intermolecular energy migration and fluorescence [Zwischenmolekulare energiewanderung und fluoreszenz] Ann. Phys. 1948;2:55–75.
    1. Förster T. Delocalized excitation and excitation transfer. In: Sinanoglu O., editor. Modern Quantum Chemistry: Istanbul Lectures. Part III, Action of Light and Organic Crystals. Academic Press; New York: 1965.
    1. Kuzmenkina E.V., Heyes C.D., Nienhaus G.U. Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions. Proc. Natl. Acad. Sci. USA. 2005;102:15471–15476. - PMC - PubMed
    1. Kuzmenkina E.V., Heyes C.D., Nienhaus G.U. Single-molecule FRET study of denaturant induced unfolding of RNase H. J. Mol. Biol. 2006;357:313–324. - PubMed
    1. Sherman E., Haran G. Coil-globule transition in the denatured state of a small protein. Proc. Natl. Acad. Sci. USA. 2006;103:11539–11543. - PMC - PubMed

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