Using an amino acid fluorescence resonance energy transfer pair to probe protein unfolding: application to the villin headpiece subdomain and the LysM domain

Abstract

Previously, we have shown that p-cyanophenylalanine (Phe CN) and tryptophan (Trp) constitute an efficient fluorescence resonance energy transfer (FRET) pair that has several advantages over commonly used dye pairs. Here, we aim to examine the general applicability of this FRET pair in protein folding-unfolding studies by applying it to the urea-induced unfolding transitions of two small proteins, the villin headpiece subdomain (HP35) and the lysin motif (LysM) domain. Depending on whether Phe CN is exposed to solvent, we are able to extract either qualitative information about the folding pathway, as demonstrated by HP35, which has been suggested to unfold in a stepwise manner, or quantitative thermodynamic and structural information, as demonstrated by LysM, which has been shown to be an ideal two-state folder. Our results show that the unfolding transition of HP35 reported by FRET occurs at a denaturant concentration lower than that measured by circular dichroism (CD) and that the loop linking helix 2 and helix 3 remains compact in the denatured state, which are consistent with the notion that HP35 unfolds in discrete steps and that its unfolded state contains residual structures. On the other hand, our FRET results on the LysM domain allow us to develop a model for extracting structural and thermodynamic parameters about its unfolding, and we find that our results are in agreement with those obtained by other methods. Given the fact that Phe CN is a non-natural amino acid and, thus, amenable to incorporation into peptides and proteins via existing peptide synthesis and protein expression methods, we believe that the FRET method demonstrated here is widely applicable to protein conformational studies, especially to the study of relatively small proteins.

Figure 1

(a) NMR structure of HP35 (PDB entry 1VII). (b) NMR structure of the LysM domain (PDB entry 1E0G). In both cases, the Trp side chain and the side chain of the residue which was replaced with Phe_CN in this study are shown.

Figure 2

CD spectrum of HP35-P collected at 25 °C. The peptide concentration was 24 μM [in 50 mM phosphate buffe (pH 7)]. The inset shows the C≡N stretching vibration of HP35-P at 26 °C.

Figure 3

Fluorescence spectra of HP35-P at different urea concentrations, as indicated in the figure. λ_ex = 240 nm.

Figure 4

(a) Fluorescence intensities (integrated area) of Phe_CN (blue) and Trp (red) of HP35-P as a function of urea concentration. (b) Ratio of the Phe_CN fluorescence intensity (F_DA) to the Trp fluorescence intensity (F_AD) vs urea concentration. Also shown is the mean residue ellipticity of HP35-P at 222 nm as a function of urea concentration.

Figure 5

Fluorescence spectra of LysM-P at different urea concentrations, as indicated in the figure. λ_ex = 240 nm.

Figure 6

(a) Fluorescence intensity (integrated area) of Phe_CN (blue) and Trp (red) of LysM-P as a function of urea concentration. (b) Ratio of the Phe_CN fluorescence intensity (F_DA) to the Trp fluorescence intensity (F_AD) vs urea concentration. Also shown is the normalized fluorescence intensity of Trp, measured upon direct excitation at 290 nm, as a function of urea concentration. Smooth lines are global fits to these data according to the methods discussed in the text.

Figure 7

Mean residue ellipticity of LysM-P at 222 nm vs temperature. The solid line is the best fit of these data to a two-state model discussed in the text.