Structure and dynamics of des-pentapeptide-insulin in solution: the molten-globule hypothesis.

Hua, Qing; Kochoyan, Michel; Weiss, Michael A.

doi:10.1073/pnas.89.6.2379

Cited by 40 publications

(56 citation statements)

References 25 publications

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“…Thus, the lack of correlation between ELISA and RBA for the measurement of IAA is not a general feature of the assays, but appears to reflect intrinsic characteristics of insulin-IAA interaction. Studies employing NMR spectroscopy demonstrated that insulin functions as a 'molten globule' whose conformation -owing to molecular interaction -changes when binding to the insulin receptor [215]. Conformational changes of insulin may also influence binding to antibodies, contributing to stabilisation of the antigen -antibody complex, as suggested by an analysis of the binding of monoclonal antibodies (mAb) to alanine scanning mutants of rat proinsulin I [216].…”

Section: Assay Techniques For Iaa and Their Limitationsmentioning

confidence: 98%

Developments in the prediction of type 1 diabetes mellitus, with special reference to insulin autoantibodies

Franke

Galloway

Wilkin

2005

Diabetes Metab. Res. Rev.

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The prodromal phase of type 1 diabetes is characterised by the appearance of multiple islet-cell related autoantibodies (Aab). The major target antigens are islet-cell antigen, glutamic acid decarboxylase (GAD), protein-tyrosine phosphatase-2 (IA-2) and insulin. Insulin autoantibodies (IAA), in contrast to the other autoimmune markers, are the only beta-cell specific antibodies. There is general consensus that the presence of multiple Aab (> or = 3) is associated with a high risk of developing diabetes, where the presence of a single islet-cell-related Aab has usually a low predictive value. The most commonly used assay format for the detection of Aab to GAD, IA-2 and insulin is the fluid-phase radiobinding assay. The RBA does not identify or measure Aab, but merely detects its presence. However, on the basis of molecular studies, disease-specific constructs of GAD and IA-2 have been employed leading to somewhat improved sensitivity and specificity of the RBA. Serological studies have shown epitope restriction of IAA that can differentiate diabetes-related from unrelated IAA, but current assays do not distinguish between disease-predictive and non-predictive IAA or between IAA and insulin antibodies (IA). More recently, phage display technology has been successful in identifying disease-specific anti-idiotopes of insulin. In addition, phage display has facilitated the in vitro production of antibodies with high affinity. Identification of disease-specific anti-idiotopes of insulin should enable the production of a high affinity reagent against the same anti-idiotope. Such a development would form the basis of a disease-specific radioimmunoassay able to identify and measure particular idiotypes, rather than merely detect and titrate IAA.

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Section: Assay Techniques For Iaa and Their Limitationsmentioning

confidence: 98%

Developments in the prediction of type 1 diabetes mellitus, with special reference to insulin autoantibodies

Franke

Galloway

Wilkin

2005

Diabetes Metab. Res. Rev.

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“…Although studies of truncated insulin analogs have revealed only T-like features (18,19,53,54), a variety of theoretical and experimental studies indicate that the monomer is flexible (41,46,(55)(56)(57), including at the N-terminal segment of the B-chain (residues B1-B8). Stabilization of its T-state conformation has been achieved by substitution of Gly B8 by D-amino acids.…”

Section: Discussionmentioning

confidence: 99%

The Structure of a Mutant Insulin Uncouples Receptor Binding from Protein Allostery

Wan

Huang

Whittaker

et al. 2008

Journal of Biological Chemistry

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The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T 6 , T 3 R 3 f , and R 6 . Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of His B5 in wild-type structures: on the T 6 surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in His B5 pK a , in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of His B5 (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of Arg B5 -insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T 6 hexamers rather than R 6 as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3 Å , closely resembles the wild-type T 6 hexamer. Whereas Arg B5 is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving His B5 in wild-type T-states. The substantial receptor-binding activity of Arg B5 -insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers.Insulin is a small globular protein containing two chains, A (21 residues) and B (30 residues). In pancreatic ␤-cells, the hormone is stored as Zn 2ϩ -stabilized hexamers, which form microcrystalline arrays within specialized secretory granules (1). The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. Structure-function relationships have been inferred from patterns of sequence conservation (2) and extensively probed by mutagenesis (2-10). A variety of evidence suggests that insulin undergoes a change in conformation on binding to the insulin receptor (IR) 2 (11). A model for induced fit is provided by the TR transition, a long range allosteric reorganization of zinc insulin hexamers (12). The structural basis of the TR transition has been extensively investigated by x-ray crystallography (13-15). In this paper, we investigate the relationship between such allosteric reorganization and biological activity. Experimental design exploits the contrasting structures of classical hexamers to introduce an electrostatic block to the TR transition.The TR transition encompasses three families of zinc insulin hexamers, designated T 6 , T 3 R 3 f , and R 6 (Fig. 1). Interconversion among these families is regulated by ionic strength (13, 16...

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“…Of these helices, the A2-A8 helix is not well folded like other two helics (Baker et al, 1988;Hua et al, 1996aHua et al, , b, 2002a. Insulin is a molten-globule like small protein in some situations, as observed by NMR and other biophysical chemical methods (Hua et al, 1992a).…”

Section: Structural Featuresmentioning

confidence: 99%

Insulin: a small protein with a long journey

Hua

2010

Protein Cell

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Insulin is a hormone that is essential for regulating energy storage and glucose metabolism in the body. Insulin in liver, muscle, and fat tissues stimulates the cell to take up glucose from blood and store it as glycogen in liver and muscle. Failure of insulin control causes diabetes mellitus (DM). Insulin is the unique medicine to treat some forms of DM. The population of diabetics has dramatically increased over the past two decades, due to high absorption of carbohydrates (or fats and proteins), lack of physical exercise, and development of new diagnostic techniques. At present, the two largest developing countries (India and China) and the largest developed country (United States) represent the top three countries in terms of diabetic population. Insulin is a small protein, but contains almost all structural features typical of proteins: α-helix, β-sheet, β-turn, high order assembly, allosteric T®R-transition, and conformational changes in amyloidal fibrillation. More than ten years' efforts on studying insulin disulfide intermediates by NMR have enabled us to decipher the whole picture of insulin folding coupled to disulfide pairing, especially at the initial stage that forms the nascent peptide. Two structural switches are also known to regulate insulin binding to receptors and progress has been made to identify the residues involved in binding. However, resolving the complex structure of insulin and its receptor remains a challenge in insulin research. Nevertheless, the accumulated knowledge of insulin structure has allowed us to specifically design a new ultra-stable and active single-chain insulin analog (SCI-57), and provides a novel way to design super-stable, fast-acting and cheaper insulin formulations for DM patients. Continuing this long journey of insulin study will benefit basic research in proteins and in pharmaceutical therapy.

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Structure and dynamics of des-pentapeptide-insulin in solution: the molten-globule hypothesis.

Cited by 40 publications

References 25 publications

Developments in the prediction of type 1 diabetes mellitus, with special reference to insulin autoantibodies

Developments in the prediction of type 1 diabetes mellitus, with special reference to insulin autoantibodies

The Structure of a Mutant Insulin Uncouples Receptor Binding from Protein Allostery

Insulin: a small protein with a long journey

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