LOCAL DYNAMICS AND STABILITY OF APOCYTOCHROME B(562) EXAMINED BY HYDROGEN-EXCHANGE

Cytochrome b(562) is a heme-binding protein consisting of four helices folded into a classic helix bundle motif, Though retaining much of th e topology of the holoprotein, apocytochrome b(562) displays physical features commonly associated with so-called protein molten globules, H ere, the stability and dynamics of this ''structured'' molten globule are probed by examination of the dependence of its hydrogen exchange b ehavior upon the presence of a chemical denaturant. Compared to other systems studied in this manner, apocytochrome b(562) displays a limite d dynamic range of hydrogen exchange rates and the analysis required t he development of a quantitative approach, The protein is found to hav e three regions of subglobal cooperative stability, The most stable re gion, or core, is composed of the central two helices of the bundle, w ith the N- and C-terminal helices being of independent and lower stabi lity. The dependence of the global unfolding free energy upon denatura nt concentration indicates the applicability of a binding model and ex plains the observed difference between global unfolding free energies obtained by the linear extrapolation method and those obtained by calo rimetry and hydrogen exchange, These observations place a significant restraint upon the type of folding pathway that is operative for this protein and suggest that that the N-and C-terminal helices fold and un fold independently of the core of the molecule.