ENGINEERING STABILITY OF THE INSULIN MONOMER FOLD WITH APPLICATION TOSTRUCTURE-ACTIVITY-RELATIONSHIPS

Citation
Nc. Kaarsholm et al., ENGINEERING STABILITY OF THE INSULIN MONOMER FOLD WITH APPLICATION TOSTRUCTURE-ACTIVITY-RELATIONSHIPS, Biochemistry, 32(40), 1993, pp. 10773-10778
Citations number
29
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
32
Issue
40
Year of publication
1993
Pages
10773 - 10778
Database
ISI
SICI code
0006-2960(1993)32:40<10773:ESOTIM>2.0.ZU;2-6
Abstract
To evaluate the possible relationship between biological activity and structural stability in selected regions of the insulin molecule, we h ave analyzed the guanidine hydrochloride induced reversible unfolding of a series of mutant insulins using a combination of near- and far-UV circular dichroism (CD). The unfolding curves are reasonably describe d on the basis of a two-state denaturation scheme; however, the observ ation of subtle differences between near- and far-UV CD detected unfol ding indicates that intermediates may be present. Three regions of the insulin molecule are analyzed in detail with respect to their contrib ution to folding stability, i.e., the central B-chain helix, the NH2-t erminal A-chain helix, and the B25-B30 extended chain region. Consider able enhancement of folding stability is engineered by mutations at th e N-cap of the central B-chain helix and at the C-cap of the NH2-termi nal A-chain helix. Mutations that confer increased stability in these regions are identical to those that lead to enhanced biological activi ty. In contrast, for insulin species modified in the B25-B30 region of the molecule, we observe no correlation between global folding stabil ity and bioactivity. Mutations in the three regions examined are found to affect stability in a nearly independent fashion, and stabilizing mutations are generally found to enhance the cooperativity of the unfo lding transition. We conclude that highly potent insulins (i.e., HisA8 , ArgA8, GluB10, and AspB10) elicit enhanced activity because these mu tations stabilize structural motifs of critical importance for recepto r recognition.