A test of the relationship between sequence and structure in proteins: Excision of the heme binding site in apocytochrome b₅

Abstract

The water-soluble domain of rat hepatic holocytochrome 6, is an ap protein containing elements of secondary structure in the sequence . The heme group is enclosed by four helices, a2, a3, a4, and a5. To test the hypothesis that a small 6 hemoprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to @l-al-P4-P3-A-P2-a6 was prepared, where A is a sevenresidue linker bypassing the heme binding site. The fragment ("abridged b5") was found to contain a and / 3 secondary structure by circular dichroism spectroscopy and tertiary structure by Trp fluorescence emission spectroscopy. NMR data revealed a species with spectral properties similar to those of the full-length apoprotein. This folded form is in slow equilibrium on the chemical shift time scale with other less folded species. Thermal denaturation, as monitored by circular dichroism, absorption, and fluorescence spectroscopy, as well as size-exclusion chromatography-fast protein liquid chromatography (SEC-FPLC), confirmed the coexistence of at least two distinct conformational ensembles. It was concluded that the protein fragment is capable of adopting a specific fold likely related to that of cytochrome 65, but does not achieve high thermodynamic stability and cooperativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the dominating influence to restrict the conformational space searched by the chain is structural propensities at a local level rather than internal packing. The sequence also holds the properties necessary to generate a barrier to unfolding.

A physical basis for protein secondary structure

“…In this view, such subdomains are merely ''polypeptide micelles'' with an intrinsic chain bias. Indeed, many examples in the literature are consistent with this interpretation (52)(53)(54)(55).…”

The progressive development of structure and stability during the equilibrium folding of the α subunit of tryptophan synthase from Escherichia coli

“…or of engineered sequences Oas & Kim, 1988;Peng & Kim, 1994;Gegg et al, 1997;Constans et al, 1998!. These incomplete polypeptides can expose autonomously folding domains that are obscured in the folding of the full-length protein.…”

Insertion of the cytochrome b5 heme‐binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture

“…The thermal and chemical denaturations of apocytochrome b 5 from various sources are cooperative, two-state processes (Pfeil 1993;Constans et al 1998;Storch et al 1999a,b;Cowley et al 2002). Specifically, the Gibbs free energy of denaturation for rat microsomal apocytochrome b 5 is only ∼6 kJ/mole at room temperature and neutral pH, such that a small fraction of the molecules sample the unfolded state under those conditions.…”

“…In the apoprotein, core 1 can be viewed as a large, flexible loop inserted into a stable scaffold (Falzone et al 1996). In previous studies aimed at analyzing the structural determinants of apocytochrome b 5 , an abridged version of the protein was prepared that contained core 2 and a short linker in place of the heme-binding segment (Constans et al 1998). The main tertiary features of core 2 appeared to be specified by the shortened sequence, in support of a certain degree of structural independence of the two regions.…”