Protein structure. Direct observation of structure-function relationship in a nucleic acid-processing enzyme

Abstract

The relationship between protein three-dimensional structure and function is essential for mechanism determination. Unfortunately, most techniques do not provide a direct measurement of this relationship. Structural data are typically limited to static pictures, and function must be inferred. Conversely, functional assays usually provide little information on structural conformation. We developed a single-molecule technique combining optical tweezers and fluorescence microscopy that allows for both measurements simultaneously. Here we present measurements of UvrD, a DNA repair helicase, that directly and unambiguously reveal the connection between its structure and function. Our data reveal that UvrD exhibits two distinct types of unwinding activity regulated by its stoichiometry. Furthermore, two UvrD conformational states, termed "closed" and "open," correlate with movement toward or away from the DNA fork.

Fig. 1. Experimental layout

Structure of UvrD monomer in the ‘open’ (upper right, PDB ID 3LFU, (16)) and ‘closed’ (lower left, PDB ID 2IS2, (14)) conformations with single fluorophore location. Two microspheres (gray) in dual optical traps (orange cones) are tethered together by a DNA hairpin. One or more UvrD monomers bind ssDNA and unwind the hairpin in the presence of ATP. A confocal microscope (green cone) detects the configuration of the same fluorescently labeled UvrD unwinding complex.

Fig. 2. Effect of UvrD oligomeric state on DNA unwinding activity

Representative time traces of unwinding activity for a UvrD monomer (A) and dimer (B), respectively. Upper panels: Fluorescence photobleaching from a monomer (A) and a dimer (B) (240 and 120 ms per point respectively). Lower panels: Simultaneous measurements of hairpin unwinding and rezipping. The UvrD monomer displays ‘frustrated’ unwinding (A) (15 bp unwound) whereas the dimer displays long-distance unwinding (72 bp) (B). (C) Histogram of the maximum number of base pairs unwound per unwinding attempt (N = 401) showing frustrated (<20 bp) and long-distance (>20 bp) unwinding. Inset: Distribution of fluorophore count for frustrated (blue) or long-distance (red) unwinding attempts.

Fig. 3. Effect of ‘open’ vs. ‘closed’ UvrD conformation on unwinding activity

(A) Location of donor and acceptor fluorophores for FRET measurement and model of UvrD conformational switching. Upper (lower) orange arrows denote 2B (1A-2A) domain orientation. See text and (19) for details on the model. (B) and (C) Representative time traces of monomeric UvrD conformation and activity. Upper panels: donor (green) and acceptor (red) fluorescence intensity. Middle panels: corresponding FRET efficiency showing UvrD reversibly switching between ‘open’ (low FRET) and ‘closed’ (high FRET, dashed red lines) conformations. Fluorescence and FRET data are integrated to 60 and 50 ms per point for (B) and (C) respectively. Shaded and unshaded areas denote low and high FRET intervals, respectively. Lower panels: simultaneous measurements of unwinding and rezipping of the DNA hairpin. Fluorescence intensity and ‘frustrated’ unwinding activity are consistent with monomeric UvrD-DNA complexes (which dissociate at t = 6.2 and 1.8 s). (D) Correlation between UvrD activity and conformation. The mean FRET efficiency and mean UvrD velocity determined over each time interval are plotted (white points; N = 109 intervals, 15 molecules). The colormap represents the probability distribution of FRET state and velocity (19).