High Precision FRET to Determine Dynamic Protein Structures

Abstract

Measuring in solution and utilizing the single-molecule advantage of fluorescence detection we established a toolbox to generate FRET-constrained structure models of biomolecules which can also show their heterogeneity and flexibility. Our approach comprises seven steps: (1) Quantitative measurement of FRET by multiparameter fluorescence detection of single molecules [1]; (2) Rigorous analysis and error determination of FRET derived donor-acceptor distances by analyzing the photon distributions and time resolved anisotropies of the dyes; (3) Appropriate description for the spatial distribution of the fluorophore by fast accessible volume (AV) simulations [2] to determine the dye positions relative to the biomolecule; (4) Search for possible structures via a FRET positioning system using a spring-network algorithm. Possible structures are generated either by a model-based approach with rigid body docking or model free by selecting suitable models from a huge structure library; (5) Docking is repeated many times to find all possible arrangements and assure the completeness of generated structural ensemble; (6) The obtained models are ranked according to their violation of FRET constraints and steric clashes. Then they are assigned to clusters of related structural organization in order to judge the uniqueness of structural models; (7) The precision (RMSD) of the structure models is determined using a bootstrapping procedure. We demonstrate the accuracy of high-precision (hp) FRET in two experiments - determination of the DNA position in HIV-1 reverse transcriptase:primer/template complexes and arrangement of a primer/template DNA bound by HIV-1 reverse transcriptase and analysis of the internal structural heterogeneity of human guanylate binding protein 1 (hGBP1). These studies show that hpFRET studies are valuable tool to complement the structure information obtained by classical methods.[1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010).[2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011).