A TEST OF THE RELATIONSHIP BETWEEN SEQUENCE AND STRUCTURE IN PROTEINS- EXCISION OF THE HEME-BINDING SITE IN APOCYTOCHROME B(5)

Citation
Aj. Constans et al., A TEST OF THE RELATIONSHIP BETWEEN SEQUENCE AND STRUCTURE IN PROTEINS- EXCISION OF THE HEME-BINDING SITE IN APOCYTOCHROME B(5), Protein science, 7(9), 1998, pp. 1983-1993
Citations number
75
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
ISSN journal
0961-8368
Volume
7
Issue
9
Year of publication
1998
Pages
1983 - 1993
Database
ISI
SICI code
0961-8368(1998)7:9<1983:ATOTRB>2.0.ZU;2-0
Abstract
The water-soluble domain of rat hepatic holocytochrome b(5) is an alph a beta protein containing elements of secondary structure in the seque nce beta-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5- beta 2-alpha 6. The heme group is enclosed by four helices, alpha 2, a lpha 3, alpha 4, and alpha 5. To test the hypothesis that a small b he moprotein can be constructed in two parts, one forming the heme site, the other an organizing scaffold, a protein fragment corresponding to beta 1-alpha 1-beta 4-beta 3-Lambda-beta 2-alpha 6 was prepared, where Lambda is a seven-residue linker bypassing the heme binding site. The fragment (''abridged b5'') was found to contain alpha and beta second ary structure by circular dichroism spectroscopy and tertiary structur e by Trp fluorescence emission spectroscopy. NMR data revealed a speci es with spectral properties similar to those of the full-length apopro tein. This folded form is in slow equilibrium on the chemical shift ti me scale with other less folded species. Thermal denaturation, as moni tored by circular dichroism, absorption, and fluorescence spectroscopy , as well as size-exclusion chromatography-fast protein liquid chromat ography (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 cytoc hrome b(5) but does not achieve high thermodynamic stability and coope rativity. Abridged b5 demonstrates that the spliced sequence contains the information necessary to fold the protein. It suggests that the do minating influence to restrict the conformational space searched by th e chain is structural propensities at a local level rather than intern al packing. The sequence also holds the properties necessary to genera te a barrier to unfolding.