Mutational Effects on the Folding Dynamics of a Minimized Hairpin

Abstract

The fold stabilities and folding dynamics of a series of mutants of a model hairpin, KTW-NPATGK-WTE (HP7), are reported. The parent system and the corresponding DPATGK loop species display sub-μs folding time constants. The mutational studies revealed that ultrafast folding requires both some pre-structuring of the loop and a favorable interaction between the chain termini at the transition state. In the case of YY-DPETGT-WY, another sub-μs folding species [Davis, C. M.; Xiao, S.; Raleigh, D. P.; Dyer, R. B. (2012) J. Am. Chem. Soc. 134, 14476–14482], a hydrophobic cluster provides the latter. In the case of HP7, the Coulombic interaction between the terminal NH3+ and CO2− units provides this; a C-terminal Glu to amidated Ala mutation results in a 5-fold folding rate retardation. The effects of mutations within the reversing loop indicate the balance between loop flexibility (favoring fast conformational searching) and turn-formation in the unfolded state is a major factor in determining the folding dynamics. The –NAAAKX- loops examined display no detectable turn formation propensity in other hairpin constructs, but do result in stable analogs of HP7. Peptide KTW-NAAAKK-WTE displays the same fold stability as HP7 but both the folding and unfolding time constants are greater by a factor of 20.

Circular Permutation of a WW Domain: Folding Still Occurs after Excising the Turn of the Folding-Nucleating Hairpin

“…In addition, the lineshapes of the 2.5 ppm upfield-shifted edge-Trp-Hε3 (Fig. S3), reflecting exchange broadening due to the folded/unfolded-state dynamics (18, 21), could be used to derive the folding rate constants. In the case of hairpin st HP1, replacing the RGSG turn locus with PATG increased the Tm (as measured by CD) by at least 16 °C.…”

“…We have reported folding times (at 300K) as short as 0.4 μs with the longest folding timescale for species that have comparable or higher fold stability on the order of 18 μs (18). The fold population (χ F ) for st HP1 in H 2 O is 0.72 at 320K; the native hairpin of Pin1 is much less stable (Tm ≪ 0 °C).…”

“…(18,21) We did not want to resort to denaturant (e.g., urea) or co-solvent addition for the standard topology construct. Since residues at the very ends of the wild-type WW domain (+KLPPG…SG-) provide some fold stability enhancement, but are not required for folding, we explored the effects of truncation as a way of lowering thermal stability of WW st 34 while preserving its core structure.…”

“…(18,20) The resins used for the synthesis were Wang resins preloaded with the C-terminal amino acid. Peptides were cleaved from the resin using a 95:2.5:2.5 trifluoroacetic acid (TFA): triisopropylsilane: water mixture.…”

“…S3). The folding rates were calculated as previously described (18,21) from k F = 4πχ U (χ F ) 2 (Δv) 2 (Δ ex ) −1 . Common values of χ U and χ F (= 1 - χ U ) were employed for all probes in each system examined; these were the average values from the shift melts of all probes.…”