Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins

Abstract

We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on single-molecule signals detected within 100 micros to 1 ms. Distance distributions caused by fluctuations faster than 100 micros are studied within these subpopulations by using time-correlated single-photon counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (I) to the -1/2 power (I(-1/2)), and that the average contribution to the contour length per base is 0.40-0.45 nm. We study unfolded chymotrypsin inhibitor 2 (CI2) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded CI2 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state.

Fig. 1.

FRET reveals distance distributions related to polymer flexibility. (a) Distance distributions in polymers are probed by using FRET from a donor fluorophore (D; yellow bulb) to an acceptor fluorophore (A; red bulb). A rigid rod (Upper, black; with contour length, L, much smaller than persistence length, l_P) is distinguished from a flexible Gaussian chain (Lower, green; L » l_P) and intermediate cases (L ∼ l_P; not shown) by differences in the distributions of energy transfer efficiency, E, caused by distributions in D–A distances. Flexible fluorophore linkers and rotational diffusion of the fluorophores on similar time scales to fluorescence also affect the measurements. (b) Experimental data and simulations of E distributions [P(E)] of D–A-labeled polymers summarized as plots of standard deviation ΔE vs. mean efficiency 〈E 〉. Simulations (with R₀ = 6.9 nm): (i) a rigid rod with linkers and fluorophore rotational diffusion lifetime τ_r similar to the donor fluorescence lifetime τ_f (τ_r ≈ τ_f) (solid black); (ii) wormlike chains with L = 12 nm (solid red), L = 16 nm (solid purple), and L = 20 nm (solid cyan), each with varying l_P, τ_r ≈ τ_f, and linkers; (iii) a Gaussian chain with varying L × l_P, τ_r ≈ τ_f, and linkers (solid green); and (iv) a hypothetical polymer with the largest possible fluctuations (solid blue). Data: dsDNA with 7-, 12-, 17-, 22-, and 27-bp D–A separations (black squares); dsDNA with 5-, 15-, and 25-bp D–A separations (gray squares, internally labeled); ssDNA at varying salt concentration [(dT)₃₀, red circles; (dT)₄₀, purple circles; (dT)₅₀, cyan circles]. For all figures, the data points are averages of two or three independent experiments, and error bars are standard errors of the mean.

Fig. 2.

Time-resolved FRET curves for subpopulations extracted by using nsALEX. (a) Photon burst histogram resulting from single molecules of D–A-labeled C1–C53 CI2 mutant diffusing through laser spot in 4 M GdnCl. E and S are calculated for each burst and placed in the histogram. Four species are detected, and their corresponding time-resolved fluorescence decay curves are extracted (shown in b–e): parallel D decay (black); perpendicular D decay (green); parallel A decay (red); and perpendicular A decay (cyan). (b) Proteins with D only emit only after D_exc (leakage of D into A channel removed for clarity). (c) Proteins with A only emit only after A_exc (direct excitation of A by D_exc removed for clarity). (d) Unfolded proteins labeled with D and A emit D and A fluorescence after D_exc pulse (ratio of intensities and lifetimes depend on FRET efficiency) and emit A fluorescence after A_exc. (e) Folded proteins labeled with D and A emit similarly to case d, except with a higher relative intensity of A compared with D after the D_exc pulse, and with a shorter D lifetime, indicating a higher E due to shorter average distance.

Fig. 3.

〈E 〉 vs. distance between attachment points for D and A for dsDNA with 7-, 12-, 17-, 22-, and 27-bp separations (black squares) and dsDNA with 5-, 15-, and 25-bp separations (gray squares). 〈E 〉 calculated from calibrated single-molecule intensity ratios rather than fluorescence lifetime information are shown as cyan crosses (23). FRET model [E = 1/1 + (R/R₀)⁶] with measured R₀ = 69 Å and 〈κ² 〉= 2/3 (solid red) and simulation accounting for linkers and the measured slower rotational diffusion ( ns and ns; solid black) are shown. Simulations were adjusted to R₀ = 62 Å to match gray squares.

Fig. 4.

Effects of ionic strength on ssDNA flexibility. (a) Extracted persistence length, l_P, vs. ionic strength ([NaCl] + [Tris buffer]) for (dT)₃₀ (red circles), (dT)₄₀ (purple circles), and (dT)₅₀ (cyan circles). Solid red, purple, and cyan lines: respective fits to model, with constant, intrinsic persistence length and electrostatic contribution . Dotted lines: respective fits to model, , where the electrostatic contribution varies as . (b) Residuals plotted for fits in a. Fits were weighted by using error value averaged over all data points.

Fig. 5.

Effects of denaturant concentration on distance distributions of unfolded CI2 and ACBP. (a) ΔE vs. 〈E 〉 for the unfolded subpopulation of 1–53 CI2 mutant ([GdnCl] = 3, 3.5, 4, and 5 M; blue diamonds) and 17–86 ACBP mutant ([GdnCl] = 1.4, 1.6, 2, 2.2, 2.5, 3, 4, and 5 M; orange hexagons) at varying denaturant concentrations. Using single-molecule burst analysis, nsALEX excludes contributions from the folded subpopulations of CI2 and ACBP. Simulations for the Gaussian chain with varying L × l_P dye linkers are shown in solid green. Dashed blue and orange lines: outliers denoting maximum possible errors due to photophysical artifacts (see Assessment of Photophysical Artifacts in Supporting Appendix). (b) Same data as in a, plotted as 〈E 〉 (blue diamonds) and ΔE (cyan diamonds) vs. [GdnCl] for unfolded CI2. (c) Same data as in a, plotted as 〈E 〉 (orange hexagons) and ΔE (magenta hexagons) vs. [GdnCl] for unfolded ACBP.