Probing the lower size limit for protein-like fold stability: ten-residue microproteins with specific, rigid structures in water

Abstract

Mutational optimization of two long-range interactions first observed in Ac-WINGKWT-NH2, (a) bifurcated H-bonding involving the threonine amide H(N) and side chain OH and the N-terminal acetyl carbonyl and (b) an H-bond between the entgegen-H(N) of the C-terminal amide and the indole ring of Trp6 that stabilizes a face-to-edge indole/indole interaction between Trp1 and Trp6, has afforded < or = 10 residue systems that yield a remarkably stable fold in water. Optimization was achieved by designing a hydrophobic cluster that sequesters these H-bonds from solvent exposure. The structures and extent of amide H/D exchange protection for CH3CH2CO-WI pGXWTGPS (p = D-Pro, X = Leu or Ile) were determined. These two systems are greater than 94% folded at 298 K (97.5% at 280 K) with melting temperatures > 75 degrees C. The fold appears to display minimal fluxionality; a well-converged NMR structure rationalizes all of the large structuring shifts observed, and we suggest that these designed constructs can be viewed as microproteins.

Figure 1

Tracking the changes in chemical shift structuring diagnostics through N-terminal mutations.

Figure 2

Two time points in the D₂O exchange (pD = 3.84) of a mixture of Pr-WIpGLWTGPS and its unfolded p3P mutant: A, 1.16 h; B, 15.6 h. Resonances of the unfolded control are labeled below the trace in panel A; labels above the trace are for the folded peptide; “x” denotes the location of resonances due to the cis-Gp amide species which is also folded. The Gly8 H_N appearing at 4.57 ppm, not on the spectral segment shown here (see Supporting Information), is protected to comparable degree.

Figure 2

Two time points in the D₂O exchange (pD = 3.84) of a mixture of Pr-WIpGLWTGPS and its unfolded p3P mutant: A, 1.16 h; B, 15.6 h. Resonances of the unfolded control are labeled below the trace in panel A; labels above the trace are for the folded peptide; “x” denotes the location of resonances due to the cis-Gp amide species which is also folded. The Gly8 H_N appearing at 4.57 ppm, not on the spectral segment shown here (see Supporting Information), is protected to comparable degree.

Figure 3

The CD melt of a Trp/Trp exciton couplet: spectra of Pr-WIpGIWTGPS were recorded in 10°C intervals.

Figure 4

CD melting data for Pr-WIpGIWTGPS, adjusted (by subtracting the CD of Ac-WIPGKWTG-NH₂ serving as an unfolded control) so that the zero degree line represents 0% folding, see Supporting Information. The actual melting temperature is expected to be in excess of 85°C, considering baseline effects. In the case of the plot of 213 nm data, a T_m > 80°C is required based on the second derivative method^9c for defining T_m.

Figure 5

The H_N/Hα NOESY spectrum of Pr-WIpGIWTGPS at 270K. The H_N resonance positions are labeled and indicated by solid lines; other resonance positions are shown by gray lines. Peaks due to the minor cis-GP isomer and water suppression artifacts have been deleted to clarify the assignment; the complete spectrum appears in the Supporting Information.

Figure 6

Views of the NMR structure ensemble (backbone RMSD = 0.30 ± 0.13 Å) generated for Pr-WIpGIWTGPS. Panel A presents a view in which the twisted hairpin conformation is apparent; all heavy atoms are shown. This view also shows the I2/I5 alignment. Panel B shows two views of the Pr-C=O/Thr7 relationship in a representative structure. In the bonds-only view (right) the Me group of Thr, as well as the ethyl groups of Ile5 and the propanoyl cap, have been deleted. In the space-filling (CPK, left) view of the same region, these three alkyl groups (which effectively sequester the Thr7 OH proton from water) are included.

Figure 7

Tracking three large structuring shifts and their melting behavior during fold optimization.