Local dynamics and stability of apocytochrome b562 examined by hydrogen exchange.
Cytochrome b562 is a heme-binding protein consisting of four helices folded into a classic helix bundle motif. Though retaining much of the topology of the holoprotein, apocytochrome b562 displays physical features commonly associated with so-called protein molten globules. Here, the stability and dynamics of this "structured" molten globule are probed by examination of the dependence of its hydrogen exchange behavior upon the presence of a chemical denaturant. Compared to other systems studied in this manner, apocytochrome b562 displays a limited dynamic range of hydrogen exchange rates and the analysis required the development of a quantitative approach. The protein is found to have three regions of subglobal cooperative stability. The most stable region, or core, is composed of the central two helices of the bundle, with the N- and C-terminal helices being of independent and lower stability. The dependence of the global unfolding free energy upon denaturant concentration indicates the applicability of a binding model and explains the observed difference between global unfolding free energies obtained by the linear extrapolation method and those obtained by calorimetry 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 unfold independently of the core of the molecule.
Fuentes
E J
EJ
Department of Chemistry, State University of New York at Buffalo 14260-3000, USA.
Wand
A J
AJ
D000595:Amino Acid Sequence; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D019791:Guanidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D011522:Protons; D013816:Thermodynamics
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D011522:Protons; C017850:cytochrome b562, E coli; D019791:Guanidine