Off-pathway status for the alkali molten globule of horse ferricytochrome C.
The Na(+)-stabilized alkali molten globule (B-state) of horse ferricytochrome c produced at pH 12.9 in 2 M NaCl has been studied to find out its relevance to the kinetic folding pathway of the protein. Details of stopped-flow kinetics indicate that the B-state when driven to fold at pH 11.5 goes through a submillisecond burst expansion to a state B' which must unfold to the base-denatured U(B)-state before folding to the alkaline native state, N(B). This folding hallmark suggests that the B-state is a dead-end or off-pathway species, playing apparently an unclear role in the folding kinetics. Interestingly, the folding kinetics of the B-state at a final pH of 7 is very similar to that observed for the guanidinium-unfolded ferricytochrome c (U) at pH 7. Both B and U exhibit a submillisecond burst phase followed by three observable phases, fast, medium, and slow, with matching rate constants for the fast phase, suggesting that B- and U-states share a common folding mechanism. Even the minima in the folding chevrons for the B- and U-states appear at the same denaturant concentration, but the former is shifted vertically upward and has shallower limbs, suggesting that the transition state relevant for the B-state folding is relatively more compact with greater surface burial allaying large-scale diffusive migration of chain segments. It is concluded that the B-state is not a good model for the kinetic molten globule of cytochrome c. Part of the reason for such atypical response of a typical molten globule may possibly be related to misligation of the ferric heme with lysyl side chains at the extreme alkaline pH required to produce the B-state. To eliminate this possibility, the companion paper [Bhuyan, A. K. (2010) Biochemistry (DOI 10.1021/bi100881n )] studies the B-state of ferrocytochrome c where ligation of the heme with any intrapolypeptide side chain is completely suppressed. The study concludes that the B-state is kinetically an abortive species.
Bhuyan
Abani K
AK
School of Chemistry, University of Hyderabad, Hyderabad 500046, India. akbsc@uohyd.ernet.in
D000468:Alkalies / Q000378:metabolism; D000818:Animals; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017510:Protein Folding; D013816:Thermodynamics
D000468:Alkalies; D045304:Cytochromes c