Nc. Kaarsholm et al., ENGINEERING STABILITY OF THE INSULIN MONOMER FOLD WITH APPLICATION TOSTRUCTURE-ACTIVITY-RELATIONSHIPS, Biochemistry, 32(40), 1993, pp. 10773-10778
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.