Structural properties of apocytochrome b5: presence of a stable native core

Abstract

Upon removal of the heme group, the water-soluble fragment of cytochrome b5 adopts a conformation less stable and compact than that of the holoprotein [Huntley, T. E., & Strittmatter, P. (1972) J. Biol. Chem. 247, 4641-4647]. This conformation, imposed by the amino acid sequence alone, has not been described in detail. One- and two-dimensional proton nuclear magnetic resonance spectroscopy techniques were applied to the apoprotein of the soluble fragment of rat liver cytochrome b5 in an effort to characterize the structure of the apoprotein. Nuclear Overhauser spectroscopy revealed a number of short interresidue distances and demonstrated that, in spite of the increased flexibility, at least one cluster of side chains exists on a time scale long enough for study. Several residues participating in the cluster, in particular the only Trp (Trp 22), were identified. Similarities with the spectrum of the reduced holoprotein were observed that led to the inspection of the cytochrome b5 crystal structure for assigning resonances. It appeared that the environment of this residue maintains its integrity in the apoprotein. Since in the holoprotein Trp 22 belongs to a hydrophobic core formed in part by beta-strands, it is proposed that some of this beta-structure is stable in the absence of the heme-protein interactions. Implications for structure and folding are discussed.

Thermodynamics of apocytochrome b5 unfolding

“…Furthermore, on the tryptic fragment it was shown by NMR (Moore & Lecomte, 1990Moore et al, 1991) that apocytochrome b5 has a hydrophobic core region. Current interest is focused on protein folding concerns, in particular the stage at which intermediates occur that are able to undergo a cooperative transition.…”

A chromatographic approach to the determination of relative free energies of interaction between hydrophobic and amphiphilic amino acid side chains

“…Experimental results of many workers indicate that aromatic residues may be important in the formation of "islands" of stable structure in partially folded and/or nonnative states (Moore & LeComte, 1990). Regions of cytochrome c showing the highest protection factors in the acid-denatured A form are those that contribute to the hydrophobic core of the native protein and contain several aromatic side chains (Jeng et al, 1990).…”

Folding energy landscape of cytochrome cb 562

“…The covalently bound porphyrin plays an integral role in cyt c folding, possibly as a hydrophobic nucleation site, but does not lead to nonnative clusters and misfolded traps (10). Many b-type cytochromes (e.g., cyt b 5 and cyt b 562 ), however, adopt native or near-native structures in the absence of their noncovalently bound porphyrins (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). On the basis of these observations, it has been suggested that folding precedes heme incorporation in the b-type cytochromes, whereas heme attachment is a prerequisite for folding in the c-type proteins.…”