title: The pKa of His-24 in the folding transition state of apomyoglobin.
abstract: In native apomyoglobin, His-24 cannot be protonated, although at pH 4 the native protein forms a molten globule folding intermediate in which the histidine residues are readily protonated. The inability to protonate His-24 in the native protein dramatically affects the unfolding/refolding kinetics, as demonstrated by simulations for a simple model. Kinetic data for wild type and for a mutant lacking His-24 are analyzed. The pK(a) values of histidine residues in native apomyoglobin are known from earlier studies, and the average histidine pK(a) in the molten globule is determined from the pH dependence of the equilibrium between the native and molten globule forms. Analysis of the pH-dependent unfolding/refolding kinetics reveals that the average pK(a) of the histidine residues, including His-24, is closely similar in the folding transition state to the value found in the molten globule intermediate. Consequently, protonation of His-24 is not a barrier to refolding of the molten globule to the native protein. Instead, the normal pK(a) of His-24 in the transition state, coupled with its inaccessibility in the native state, promotes fast unfolding at low pH. The analysis of the wild-type results is confirmed and extended by using the wild-type parameters to fit the unfolding kinetics of a mutant lacking His-24.
authors.affiliation: Department of Biochemistry, Beckman Center, Room B400, Stanford University Medical Center, Stanford, CA 94305-5307, USA.
authors.forename: M
authors.initials: M
authors.lastname: Jamin
authors.forename: B
authors.initials: B
authors.lastname: Geierstanger
authors.forename: R L
authors.initials: RL
authors.lastname: Baldwin
mesh_terms: D000085:Acetates; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D002021:Buffers; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D009211:Myoglobin / Q000737:chemistry*; D017510:Protein Folding; D014907:Whales
chemical_list: D000085:Acetates; D001059:Apoproteins; D002021:Buffers; D009211:Myoglobin; C010857:apomyoglobin; D006639:Histidine