Engineering Out Motion:  A Surface Disulfide Bond Alters the Mobility of Tryptophan 22 in Cytochrome b 5 As Probed by Time-Resolved Fluorescence and 1H NMR Experiments

• Published in: Biochemistry (Easton) 1999-04, Vol.38 (16), p.5065-5075
• Main Authors: Storch, Elizabeth M, Grinstead, Jeffrey S, Campbell, A. Patricia, Daggett, Valerie, Atkins, William M
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi982159i

In the accompanying paper [Storch et al. (1999) Biochemistry 38, 5054−5064] equilibrium denaturation studies and molecular dynamics (MD) simulations were used to investigate localized dynamics on the surface of cytochrome b 5 (cyt b 5) that result in the formation of a cleft. In those studies, an S18C:R47C disulfide mutant was engineered to inhibit cleft mobility. Temperature- and urea-induced denaturation studies revealed significant differences in Trp 22 fluorescence between the wild-type and mutant proteins. On the basis of the results, it was proposed that wild type populates a conformational ensemble that is unavailable to the disulfide mutant and is mediated by cleft mobility. As a result, the solvent accessibility of Trp 22 is decreased in S18C:R47C, suggesting that the local environment of this residue is less mobile due to the constraining effects of the disulfide on cleft dynamics. To further probe the structural effects on the local environment of Trp 22 caused by inhibition of cleft formation, we report here the results of steady-state and time-resolved fluorescence quenching, differential phase/modulation fluorescence anisotropy, and 1H NMR studies. In Trp fluorescence experiments, the Stern−Volmer quenching constant increases in wild type versus the oxidized disulfide mutant with increasing temperature. At 50 °C, K SV is nearly 1.5-fold greater in wild type compared to the oxidized disulfide mutant. In the reduced disulfide mutant, K SV was the same as wild type. The bimolecular collisional quenching constant, k q, for acrylamide quenching of Trp 22 increases 2.7-fold for wild type and only 1.8-fold for S18C:R47C, upon increasing the temperature from 25 to 50 °C. The time-resolved anisotropy decay at 25 °C was fit to a double-exponential decay for both the wild type and S18C:R47C. Both proteins exhibited a minor contribution from a low-amplitude fast decay, consistent with local motion of Trp 22. This component was more prevalent in the wild type, and the fractional contribution increased significantly upon raising the temperature. The fast rotational component of the S18C:R47C mutant was less sensitive to increasing temperature. A comparison of the 1H NMR monitored temperature titration of the δ-methyl protons of Ile 76 for wild type and oxidized disulfide mutant, S18C:R47C, showed a significantly smaller downfield shift for the mutant protein, suggesting that Trp 22 in the mutant protein experiences comparatively decreased cleft dynamics in