Fast and faster: a designed variant of the B-domain of protein A folds in 3 microsec

Abstract

We have introduced the mutation glycine 29 to alanine, designed to increase the rate of protein folding, into the B-domain of protein A (BdpA). From NMR lineshape analysis, we find the G29A mutation increases the folding rate constant by threefold; the folding time is 3 microsec. Although wild-type BdpA folds extremely fast, simple-point mutations can still speed up the folding; thus, the folding rate is not evolutionarily maximized. The short folding time of G29A BdpA (the shortest time yet reported) makes it an attractive candidate for an all-atom molecular dynamics simulation that could potentially show a complete folding reaction starting from an extended chain. We also constructed a fluorescent variant of BdpA by mutating phenylalanine 13 to tryptophan, allowing fluorescence-based time-resolved temperature-jump measurements. Temperature jumps and NMR complement each other, and give a very complete picture of the folding kinetics.

Figure 1.

Ribbon diagram of G29A/F13W BdpA showing the helical architecture of the domain and the location of the point mutations studied in this work, based on the NMR structure of the Z-domain (PDB ID 2spz; Tashiro et al. 1997).

Figure 2.

Guanidine hydrochloride (GuHCl) denaturation of WT (circles), G29A (squares), and G29A/F13W (triangles) BdpA followed by circular dichroism at 222 nm. Solid lines are fits to a two-state denaturation model (Pace and Scholtz 1997).

Figure 3.

NMR resonance of the G29A/F13W His 18 Hɛ at different concentrations of denaturant (thiourea + urea in a 1 : 3.33 molar ratio). The shifting of the signal as the population of denatured protein increases is clearly visible, along with the broadening present when both native and denatured states are populated and undergoing rapid exchange. The number alongside each peak gives the denaturant concentration, and the data have been moved along the Y-axis for convenient viewing.

Figure 4.

Folding (filled symbols) and unfolding (open symbols) rate constants vs. denaturant for G29A (squares) and G29A/F13W (triangles), from lineshape analysis of the His 18 Hɛ. The extrapolated rate constants for WT from Myers and Oas (2001) are shown as circles for comparison.