A Protein Caught in a Kinetic Trap:  Structures and Stabilities of Insulin Disulfide Isomers

Hua, Qing; Jia, Wenhua; Frank, Bruce H.; Phillips, Nelson B.; Weiss, Michael A.

doi:10.1021/bi0202981

Cited by 78 publications

(93 citation statements)

References 63 publications

(169 reference statements)

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“…The disulfide linkage patterns and native ␣-helix content of the partially structured intermediates of PIP and HPI are listed in Table IV, and the peptide models of the insulin folding intermediates whose structures have been studied by NMR are listed in Table V (7,9,10,12). By comparing the peptide models with our captured intermediates here, we could infer that all of the partially structured intermediates of PIP and HPI retain helix 1.…”

Section: Formation Of the A20 -B19 Bridge Is The Most Importantmentioning

confidence: 99%

“…The three disulfide bonds are important in maintaining the native conformation and biological activities of the insulin molecular. The contributions of these bridges to the structure, stability, and activity of hormone have been investigated in analogues lacking selected disulfide bridges (7)(8)(9)(10)(11)(12). Internal cystine A6 -A11 is close to the hydrophobic core and its substitution with Ser or Ala resulted in the unfolding of helix 2 (10,11).…”

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confidence: 99%

“…Disulfide isomers retaining this core disulfide bridge exhibit native-like partial folds with nonnegligible biological activity. Such isomers provide examples of kinetic traps in an energy landscape (7)(8)(9)14).…”

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confidence: 99%

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In Vitro Refolding of Human Proinsulin

Qiao

Min

Hua

et al. 2003

Journal of Biological Chemistry

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Human insulin is a double-chain peptide that is synthesized in vivo as a single-chain human proinsulin (HPI). We have investigated the disulfide-forming pathway of a single-chain porcine insulin precursor (PIP). Here we further studied the folding pathway of HPI in vitro. While the oxidized refolding process of HPI was quenched, four obvious intermediates (namely P1, P2, P3, and P4, respectively) with three disulfide bridges were isolated and characterized. Contrary to the folding pathway of PIP, no intermediates with one-or two-disulfide bonds could be captured under different refolding conditions. CD analysis showed that P1, P2, and P3 retained partially structural conformations, whereas P4 contained little secondary structure. Based on the timedependent distribution, disulfide pair analysis, and disulfide-reshuffling process of the intermediates, we have proposed that the folding pathway of HPI is significantly different from that of PIP. These differences reveal that the C-peptide not only facilitates the folding of HPI but also governs its kinetic folding pathway of HPI. Detailed analysis of the molecular folding process reveals that there are some similar folding mechanisms between PIP and HPI. These similarities imply that the initiation site for the folding of PIP/HPI may reside in the central ␣-helix of the B-chain. The formation of disulfide A20 -B19 may guide the transfer of the folding information from the B-chain template to the unstructured A-chain. Furthermore, the implications of this in vitro refolding study on the in vivo folding process of HPI have been discussed.

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Section: Formation Of the A20 -B19 Bridge Is The Most Importantmentioning

confidence: 99%

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confidence: 99%

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In Vitro Refolding of Human Proinsulin

Qiao

Min

Hua

et al. 2003

Journal of Biological Chemistry

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“…Non-native disulfides can act as kinetic traps in protein folding and misfolding (30)(31)(32)(33). Hence, some pathways of protein aggregation depend strongly on the redox environment.…”

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confidence: 99%

An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human Gamma-D Crystallin

Serebryany

Woodard

Adkar

et al. 2017

Biophysical Journal

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Considerable mechanistic insight has been gained into amyloid aggregation; however, a large class of non-amyloid protein aggregates are considered "amorphous," and in most cases little is known about their mechanisms. Amorphous aggregation of γ-crystallins in the eye lens causes a widespread disease of aging, cataract. We combined simulations and experiments to study the mechanism of aggregation of two γD-crystallin mutants, W42R and W42Q -the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Twochain all-atom simulations predicted that one nonnative disulfide in particular, between Cys32 and Cys41, was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro. Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal β-hairpin and rearrangement of the native β-sheet topology.

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“…1 H NMR studies have demonstrated that IGF-swap possesses a well organized three-dimensional structure with salient differences from that of native IGF-I (26,28). The alternative structure is as stable (or more stable) than native IGF-I as probed by thiol-catalyzed disulfide exchange (26) and chemical denaturation (29). Proinsulin in contrast refolds to form a unique ground state (30); disulfide isomers exist only as metastable kinetic traps (29,31,32).…”

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Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

Sohma

Hua

Liu

et al. 2010

Journal of Biological Chemistry

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Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6 -A11, A7-B7, and A20 -B19) maintained by IGFbinding proteins; IGF-swap has alternative pairing (A7-A11, A6 -B7, and A20 -B19) and impaired activity. Studies of minidomain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His The vertebrate insulin-related superfamily consists of insulin and insulin-related growth factors (IGF-I 5 and IGF-II) (1, 2), relaxin (3-5), and relaxin-related factors (6 -9). Insulin and IGFs function as ligands for receptor tyrosine kinases (the insulin receptor and type 1 IGF receptor; IR and IGF-1R) (10), whereas relaxin and related factors bind to G-protein-coupled receptors (11). Interest in the evolution and folding properties of insulin-related polypeptides has recently been invigorated by the discovery of mutations in the human insulin gene associated with permanent neonatal-onset diabetes mellitus (12). These dominant mutations impair the foldability of variant and (in trans) wild-type proinsulin, leading to ␤-cell dysfunction, endoplasmic reticular (ER) stress, and impaired ␤-cell viability (13). One such mutation occurs at position B5 (12, 14). Insulin contains a conserved His at B5, whereas IGF-I contains a conserved Thr (Table 1). Here, we investigate reciprocal substitutions in these proteins, Thr B5 in insulin and His B5 in IGF-I, as probes of competing evolutionary constraints among otherwise homologous sequences. 6IGFs are single-chain polypeptides containing A-and B-domains, an intervening connecting (C)-domain, and a C-terminal D-domain (Fig. 1A) (1, 2); insulin (like relaxin and related factors) contains two chains (designated A and B; Fig. 1B) as a consequence of proteolytic processing in the trans-Golgi network (3-5, 15). Crystal structures of IGF-I and insulin exhibit similar ␣-helical domains (Fig. 1, A and B) (1-9). Protein folding is linked to specific disulfide pairing. The canonical cystines in insulin are A6 -A11, A7-B7, and A20 -B19, and the corresponding cystines in IGF-I are at polypeptide positions 47-52,

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A Protein Caught in a Kinetic Trap: Structures and Stabilities of Insulin Disulfide Isomers

Cited by 78 publications

References 63 publications

In Vitro Refolding of Human Proinsulin

In Vitro Refolding of Human Proinsulin

An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human Gamma-D Crystallin

Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I

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