Simultaneous Determination of Two Subdomain Folding Rates Using the "Transfer-Quench" Method

Abstract

The investigation of the mechanism of protein folding is complicated by the context dependence of the rates of intramolecular contact formation. Methods based on site-specific labeling and ultrafast spectroscopic detection of fluorescence signals were developed for monitoring the rates of individual subdomain folding transitions in situ, in the context of the whole molecule. However, each site-specific labeling modification might affect rates of folding of near-neighbor structural elements, and thus limit the ability to resolve fine differences in rates of folding of these elements. Therefore, it is highly desirable to be able to study the rates of folding of two or more neighboring subdomain structures using a single mutant to facilitate resolution of the order and interdependence of such steps. Here, we report the development of the "Transfer-Quench" method for measuring the rate of formation of two structural elements using a single triple-labeled mutant. This method is based on Förster resonance energy transfer combined with fluorescence quenching. We placed the donor and acceptor at the loop ends, and a quencher at an α-helical element involved in the node forming the loop. The folding of the triple-labeled mutant is monitored by the acceptor emission. The formation of nonlocal contact (loop closure) increases the time-dependent acceptor emission, while the closure of the labeled helix turn reduces this emission. The method was applied in a study of the folding mechanism of the common model protein, the B domain of staphylococcal protein A. Only natural amino acids were used as probes, and thus possible structural perturbations were minimized. Tyr and Trp residues served as donor and acceptor at the ends of a long loop between helices I and II, and a Cys residue as a quencher for the acceptor. We found that the closure of the loop (segment 14-33) occurs with the same rate constant as the nucleation of helix HII (segment 33-29), in line with the nucleation-condensation model.

Figure 1

Ribbon structure of BdpA (Protein Data Bank entry: 1BDD) showing helix H1 (blue), helix H2 (green), and helix H3 (red). Residues 14, 29, and 33 were substituted to Tyr (yellow), Cys (brown) and Trp (purple), respectively.

Figure 2

Scheme of the Transfer-Quench method. The protein is labeled with donor (D), acceptor (A), and a quencher (Q). After initiation of folding, the loop is closed with rate constant k_f and the helix is formed with rate constant k_q. Two cases are shown for following the trace of the acceptor fluorescence. In Case 1, where k_f ≫ k_q, the trace will start with an increase due to energy transfer from the donor, and will then decrease due to formation of the helix and the contact with the quencher. In Case 2, where k_q ≫ k_f, due to small contribution of direct excitation of the acceptor, the signal decays very fast to a final intensity which is reduced due to the close contact with the quencher. The intensity of the residual acceptor emission depends on the efficiency of the quencher. Both scenarios represent ideal situations of very low direct excitation of the acceptor and very high quenching efficiency.

Figure 3

Folding rates determination process. The rate of the local change, k_A, is obtained by monitoring the change of the acceptor emission in the acceptor only mutant (AO). Next, the rate of the helix formation, k_q, is obtained by monitoring the change of the acceptor emission during folding in a mutant labeled with donor, acceptor, and a quencher (DAX). In this measurement the acceptor is excited directly (excitation wavelength 290 nm) to avoid excitation of the donor and the data is fitted to Eq. 1. Next, the rate of the loop closure, k_f, is obtained by monitoring the change of the acceptor emission during folding in a DAX mutant while exciting the donor (excitation wavelength 280 nm) to include the energy transfer and the data is fitted to Eq. 2. The normalization factor (Eq. 3) includes correction of the change in the acceptor emission intensity, as mentioned in the text.

Figure 4

Folded and unfolded state equilibrium fluorescence measurements of the mutants BdpA-(14f33w29a) (AO, blue) and BdpA-(14y33w29c) (DAX, green). (A and B), Excitation spectra monitored at the acceptor emission wavelength, 350 nm, in 0 M GndHcl (Folded, A) and 6 M GndHcl (Unfolded, B). The reference (purple) is the spectrum obtained for the reference peptide, Tyr-Trp, which exhibits high and solvent-independent FRET efficiency. Both excitation spectra of the reference (purple) and the DAX (green) mutant were normalized to the value of the excitation spectrum of the AO mutant (blue) at the excitation wavelength 300 nm. Because at 300 nm there is no Tyr absorption, the difference intensity at 280 nm is the result of energy transfer. The high intensity observed for the DAX mutant in the folded state (A) is due to high transfer efficiency, which is very low in the unfolded state (B), where the intensity observed for the DAX mutant is close to that observed for the AO mutant, i.e., very low transfer efficiency. (C and D) Emission spectra of the acceptor (Trp) under excitation at 300 nm (no Tyr absorption) where both mutants were at the same concentration. (C) Emission of the DAX mutant (green) and the AO mutant (blue) at 0 M GndHcl (Folded). (D) Emission of the same mutants dissolved in 6 M GndHcl (unfolded, D). Under these conditions, emission of the DAX does not overlap that of the AO mutant due to partial quenching by the Cys residue, which is only four residues from the Trp.

Figure 5

Refolding kinetics of BdpA mutants monitored via the acceptor time-dependent emission intensity. (A) Change in the acceptor fluorescence in the AO (acceptor only, no FRET contribution) mutant 14f33w29a under excitation at 280 nm. The red traces represent curve fitting analysis using Eq. 1 (where A_q = 0) to resolve the rate of the change in the acceptor local environment (k_A). (B) Change in the acceptor fluorescence in the triple-labeled mutant 14y33w29c under excitation at 280 nm (blue, both donor and acceptor absorb) and at 290 nm (green, very low donor absorption). The green trace is the result of normalization according to Eq. 3. The red traces represent curve fitting analysis using Eq. 1 for the green trace to resolve the helix rate formation (while using k_A from the acceptor only mutant measurement) and Eq. 2 for the blue trace to resolve the loop closure rate. The larger difference in the 280- and 290-nm traces is due to decrease in the distance between 14 and 33 residues (loop closure) due to energy transfer. In both (A) and (B), emission intensity was monitored at 350 nm. Refolding was initiated by reduction of the denaturant concentration (GndHCl) from 6 to 2 M at 8°C.