Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy

Abstract

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

Figure 1

Schematic representation of the folding of GCN4-Pf. (Right) Structure of folded GCN4-P1 from x-ray diffraction.(13). A hypothetical unfolded structure is shown at Left. The peptide adheres to the positively charged surface by electrostatic interaction with the negatively charged glutamic acids at the C terminus of the peptide. Conformational fluctuations cause changes in the donor–acceptor distance, resulting in an anticorrelated modulation in the donor and acceptor fluorescence intensities.

Figure 2

Typical time-resolved signals along the donor (white line) and acceptor (black line) fluorescence channels from a single GCN4-Pf at pH 6.1.

Figure 3

Probability distributions P(Ø_ET) represented by sticks under urea concentrations as labeled measured on freely diffusing GCN4-Pf. The black-and-white solid lines in the 0- and 7-M sections are Gaussian representations of the distributions of Ø_ET with the contribution of the shot noise deconvolved. The thick solid line in the 3-M section urea is a fit of the 3-M data to 45% of the 0-M and 55% of the 7-M distributions. The thin lines are the contributions of the 0- and 8-M urea distributions needed to fit the 3-M distribution.

Figure 4

(Left) Probability distributions of the average donor acceptor separation measured on the aminosilanized cover slips from optimally filtered trajectories. (Right) Probability distributions under different urea concentrations measured on freely diffusing GCN4-Pf with shot noise broadening of a single value overlaid for comparison.

Figure 5

Signal correlation functions, C_k,l(t), and their dependence on urea concentration for immobilized GCN4-Pf. A, acceptor autocorrelation; D, donor autocorrelation; C, crosscorrelation. (Top) A fit to the 3-M crosscorrelation function with the contributions from the conformational fluctuations because of the folded and unfolded states removed by subtracting the mean of the 0- and 7-M data from the 3-M urea data. The three panels (Bottom) were calculated from Eq. 1 with urea concentrations as labeled.

Figure 6

(Top) Mean instantaneous diffusion coefficient as a function of the effective folding coordinate R computed from r by using R₀ = 44 Å. (Bottom) Potentials of mean force calculated from the probability distributions from optimally filtered trajectories of immobilized GCN4-Pf by using G(R) = −kT lnP(R). Concentrations of urea are as labeled.