Engineering out motion: Introduction of a de novo disulfide bond and a salt bridge designed to close a dynamic cleft on the surface of cytochrome b(5)

A previous molecular dynamics (MD) simulation of cytochrome bs (cyt bs) at 25 degrees C displayed localized dynamics on the surface of the protein giv ing rise to the periodic formation of a cleft that provides access to the h eme through a protected hydrophobic channel [Storch and Daggett (1995) Bioc hemistry 34, 9682]. Here we describe the production and testing of mutants designed to prevent the cleft from opening using a combination of experimen tal and theoretical techniques. Two mutants have been designed to close the surface cleft: S18D to introduce a salt bridge and S18C:R47C to incorporat e a disulfide bond. The putative cleft forms between two separate cores of the protein: one is structural in nature and can be monitored through the f luorescence of Trp 22, and the other binds the heme prosthetic group and ca n be tracked via heme absorbance. An increase in motion localized to the cl eft region was observed for each protein, except for the disulfide-containi ng variant, in MD simulations at 50 degrees C compared to simulations at 25 degrees C. For the disulfide-containing variant, the cleft remained closed . Both urea and temperature denaturation curves were nearly identical for w ild-type and mutant proteins when heme absorbance was monitored. In contras t, fluorescence studies revealed oxidized S18C:R47C to be considerably more stable based on the midpoints of the denaturation transitions, T-m and U-1 /2. Moreover, the fluorescence changes for each protein were complete at si milar to 50 degrees C and a urea concentration of similar to 3.9 M, signifi cantly below the temperature and urea concentration (62 degrees C, 5 M urea ) required to observe heme release. In addition, solvent accessibility base d on acrylamide quenching of Trp 22 was lower in the S18C:R47C mutant, part icularly at 50 degrees C, before heme release [presented in the accompanyin g paper (58)]. The results suggest that a constraining disulfide bond can b e designed to inhibit dynamic cleft formation on the surface of cyt b(5) Lo cated near the heme, the native dynamics of the cleft may be functionally i mportant for protein-protein recognition and/or complex stabilization.