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 submicrosecond folding time constants. The mutational studies revealed that ultrafast folding requires both some prestructuring of the loop and a favorable interaction between the chain termini in the transition state. In the case of YY-DPETGT-WY, another submicrosecond folding species [Davis, C. M., Xiao, S., Raleigh, D. P., and 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 retardation of the folding rate. 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 analogues 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.

Figure 1

Nomenclature for HP7-related hairpins.

Figure 2

Packing motif for the turn-flanking indole rings (Trp3 and Trp10) and the backbone conformation of HP7. The W3-Hε3 location is highlighted.

Figure 3

The aromatic region of 750 MHz NMR spectra of HP7 and a loop region mutant in D₂O (pD 6). The colored peaks are the Hε3 resonances of the two Trp indole rings: red for W3 and green for W10 (which served as the control for no exchange broadening). Even though the NAAAKK loop mutant displays a somewhat larger upfield shift for W3-Hε3 and a comparable shift melt, the extensive line broadening indicates much slower folding dynamics.

Figure 4

Arrhenius plots for HP7 (diamonds, ◆), [K1A,E12A]-HP7 (open square, □), [E12A-NH₂]-HP7 (△), and the NAAAKT loop analog at pH 6 (filled circles, ●) and pH 3 (open circles, ○). The folding rate constant plot appears as the top panel. The unfolding rate plots appear in the lower panel. All lines are least squares fits.