Detection of an equilibrium intermediate in the folding of a monomeric insulin analog.


To determine the conformational properties of the C-terminal region of the insulin B-chain relative to the helical core of the molecule, we have investigated the fluorescence properties of an insulin analog in which amino acids B28 and B29 have been substituted with a tryptophan and proline residue respectively, ([WB28,PB29]insulin). The biological properties and far-UV circular dichroism (CD) spectrum of the molecule indicate that the conformation is similar to that of native human insulin. Guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation of the analog as monitored by CD intensity at 224 nm indicates a single cooperative transition with a midpoint of 4.9 M GdnHCl. In contrast, when the equilibrium denaturation is observed by steady-state fluorescence emission intensity at 350 nm, two distinct transitions are observed. The first transition accounts for 60% of the observed signal and has a midpoint of 1.5 M GdnHCl. The second transition roughly parallels that observed by CD measurements with an approximate midpoint of 4.5 M GdnHCl. The near-UV CD spectrum, size-exclusion, and ultracentrifugation properties of [WB28,PB29]insulin indicate that this analog does not self-associate in a concentration-dependent manner as does human insulin. Thus, the observed fluorescence changes must be due to specific conformational transitions which occur upon unfolding of the insulin monomer with the product of the first transition representing a stable folding intermediate of this molecule. Study holds ProTherm entries: 4801 Extra Details: additive : EDTA(1 mM), helical core; proline; single cooperative transition;,conformational transitions; folding intermediate

Submission Details


Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Bryant C;Strohl M;Green LK;Long HB;Alter LA;Pekar AH;Chance RE;Brems DN,Biochemistry (1992) Detection of an equilibrium intermediate in the folding of a monomeric insulin analog. PMID:1610818
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