Experimental Evidence for Millisecond–Timescale Structural Evolution Following the Microsecond–Timescale Folding of a Small Protein

Prior work has shown that small proteins can fold (i.e., convert from unstructured to structured states) within $10\mathrm{\mu }\mathrm{s}$. Here we use time-resolved solid state nuclear magnetic resonance (ssNMR) methods to show that full folding of the 35-residue villin headpiece subdomain (HP35) requires a slow annealing process that has not been previously detected. ${}^{13}\mathrm{C}$ ssNMR spectra of frozen HP35 solutions, acquired with a variable time ${\tau }_e$ at $30°\mathrm{C}$ after rapid cooling from $95°\mathrm{C}$ and before rapid freezing, show changes on the 3–10 ms timescale, attributable to slow rearrangements of protein sidechains during ${\tau }_e$.

Figure 1

(a) CD spectra of HP35 as a function of temperature, with 2.5 mM HP35 in 20 mM sodium acetate buffer, pH 5, with 20% $\mathrm{v}/\mathrm{v}$ glycerol. (b) CD signal at 222 nm, showing $T_{\mathrm{mid}}\approx 68°\mathrm{C}$. (c) ${}^{13}\mathrm{C}$ ssNMR spectra of HP35 solutions, rapidly frozen with the indicated evolution times ${\tau }_e$ following a rapid temperature jump from 95 to $30°\mathrm{C}$. All carbon sites of V50 and G52 were ${}^{13}\mathrm{C}$-labeled, as were the L69 backbone carbons. (d) First and second principal components (PC1 and PC2) from singular value decomposition of the aliphatic region of the spectra in panel (c). (e) PC2 weight $w_{PC2}$ as a function of ${\tau }_e$. Dashed line is a fit with the form $w_{PC2}=a\exp (-{\tau }_e/{\tau }_{PC2})+b$.

Figure 2

(a) L69 ${\mathrm{C}}_{\alpha }\text{−}\mathrm{CO}$ crosspeaks from 2D ${}^{13}\mathrm{C}\text{−}{}^{13}\mathrm{C}$ ssNMR spectra of rapidly frozen HP35 solutions with indicated ${\tau }_e$ values. (b) Crosspeak from a slowly frozen HP35 solution containing 40% $\mathrm{v}/\mathrm{v}$ glycerol. (c) Difference spectra obtained by subtracting the crosspeak in panel (b) from crosspeaks in panel (a). Purple arrow indicates a signal attributable to partially disordered HP35 molecules. (d) Crosspeak volumes $v$ in difference spectra as a function of ${\tau }_e$. Dashed line is a fit with the form $v=a\exp (-{\tau }_e/{\tau }_{\mathrm{\Delta }})+b$. (e) Examples of 2D spectral regions from which L69 ${\mathrm{C}}_{\alpha }\text{−}\mathrm{CO}$ crosspeaks were extracted.

Figure 3

(a) 2D ${}^{13}\mathrm{C}\text{−}{}^{13}\mathrm{C}$ ssNMR spectrum of a rapidly frozen HP35 solution (${\tau }_e=1.6\mathrm{ms}$) with ${}^{13}\mathrm{C}$ labeling of all carbon sites of M53, S56, F58, and G74. (b),(c),(d) 1D slices at positions indicated by dotted lines in panel (a), from 2D spectra with indicated ${\tau }_e$ values. These slices show crosspeaks from F58 aromatic carbon sites, the M53 ${\mathrm{C}}_{\gamma }$ site, and the combined CO sites of S56 and F58, respectively.

Figure 4

(a) Schematic illustration of HP35 folding. During the inverse $T$ jump to $30°\mathrm{C}$, HP35 converts from an unfolded ensemble to an ensemble with a well-ordered backbone structure but partially disordered sidechain conformations (represented by a superposition of 8 structures from the MD trajectory reported by Piana et al. [27]). The final structure (represented by coordinates from Protein Data Bank file 1YRF) forms through an annealing process on the 3–10 ms timescale. (b) Views of the annealing process in the $\alpha$-helical segments of HP35.