Structure-function relationships in the free insulin monomer

Hefford, Mary Alice; Oda, G.; Kaplan, Harvey

doi:10.1042/bj2370663

Cited by 14 publications

(13 citation statements)

References 20 publications

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“…The observed precipitation is sensitive to salt and critically dependent on the localization of conserved glutamate residues on the N‐ and C‐terminal sides of C‐peptide, and therefore appears to result from specific charge interactions between the two peptides. Here, the positive charges of arginine B22 and lysine B29, known to be important for insulin oligomerization , of two insulin molecules could be neutralized by binding to a single C‐peptide, thus lowering the solubility of the complex. If, on the other hand, a C‐peptide variant with a reduced number of charges is present or the insulin excess is higher, a complex with zero net charge is not promoted and no precipitation is observed (Figs and ).…”

Section: Discussionmentioning

confidence: 99%

Insulin solubility transitions by pH‐dependent interactions with proinsulin C‐peptide

et al. 2012

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Proinsulin processing into insulin and C-peptide in the secretory granules of the pancreatic b-cells occurs under mildly acidic conditions and at high peptide concentrations (> 10 mM). Mature insulin has reduced solubility and a propensity to adopt an amyloid-like structure, but is physiologically released as a mixture of a zinc-containing core and a zinc-free, C-peptiderich fluid phase. C-peptide is known to function in the insulin secretion, but its exact mode of interaction is not established. We now demonstrate that C-peptide in sub-stoichiometric amount versus insulin coprecipitates with insulin at the pH found in secretory vesicles. Precipitation is reversible and the precipitate is dissolved by elevation of the pH. This effect was found to be dependent on relatively conserved glutamate residues in the otherwise poorly conserved C-peptide. Together, the data show that C-peptide has the ability to influence insulin solubility. The physiological pH changes between insulin processing and release sites may therefore affect the quaternary structure of insulin, as well as the phase transitions during insulin sorting and secretion. Structured digital abstractInsulin and C-peptide bind by molecular sieving (View Interaction: 1, 2) C-peptide and Insulin bind by dynamic light scattering (View Interaction: 1, 2) C-peptide and Insulin bind by fluorescence technology (View Interaction: 1,2,3,4)

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Section: Discussionmentioning

confidence: 99%

Insulin solubility transitions by pH‐dependent interactions with proinsulin C‐peptide

et al. 2012

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“…The monomeric insulin molecule, thought to be the functional form in solution [1], is known to exist in equilibrium with oligomeric forms in vitro, as determined using different methods: (a) Sedimentation equilibrium [2–6], (b) Static and Dynamic Light scattering [7–10] (c) Small angle neutron and X-ray scattering [11–13], (d) Osmotic pressure [14], (e) mass-spectrometry and NMR [15,16], and (f) Circular dichroism [17]. …”

Section: Introductionmentioning

confidence: 99%

pH-dependent self-association of zinc-free insulin characterized by concentration-gradient static light scattering

Attri

Fernández

Minton

2010

Biophysical Chemistry

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Insulin self-association at pH 1.85, 1.95, 3.0, 7.2, 8.0 and 10 was studied via composition gradient light scattering (CG-SLS). At pH 1.95 in acetic acid, insulin was found to exist as a monomer, and in pH 1.85 HCl as a dimer. At pH values of 3.0 -8.0, the dependence of scattering intensity upon total insulin concentration at concentrations of up to 1.5 mg/mL may be quantitatively accounted for by a simple isodesmic association equilibrium scheme requiring only a single association constant for addition of monomer to monomer or any oligomer. At pH 10, the association constant for addition of monomer to monomer was found to be smaller than the association constant for addition of monomer to all higher oligomers by a factor of approximately five. The isodesmic association scheme was also found to quantitatively account for the concentration dependence of the weight-average molecular weight derived from previously published sedimentation equilibrium measurements made at pH 7.0, and the best-fit value of the stepwise equilibrium constant obtained therefrom was in excellent agreement with that obtained from analysis of the light scattering data obtained at pH 7.2

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“…Natively folded insulin, both bovine and human, is predominately α-helical; its threedimensional structure was solved by X-ray crystallography already in 1969 (Adams et al (1969)). Insulin functionality is dependent on its quaternary structure; the folded protein can assemble into different oligomers, with the monomer as the biologically active form (Hefford et al 1986). In aqueous solution, natively folded insulin monomers exist in equilibrium with dimers, tetramers, and hexamers (Brange and Langkjoer 1993;Nettleton et al 2000;Pekar and Frank 1972).…”

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confidence: 99%

Amyloid formation of bovine insulin is retarded in moderately acidic pH and by addition of short-chain alcohols

et al. 2020

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Protein aggregation and amyloid formation are associated with multiple human diseases, but are also a problem in protein production. Understanding how aggregation can be modulated is therefore of importance in both medical and industrial contexts. We have used bovine insulin as a model protein to explore how amyloid formation is affected by buffer pH and by the addition of short-chain alcohols. We find that bovine insulin forms amyloid fibrils, albeit with different rates and resulting fibril morphologies, across a wide pH range (2-7). At pH 4.0, bovine insulin displayed relatively low aggregation propensity in combination with high solubility; this condition was therefore chosen as basis for further exploration of how bovine insulin's native state can be stabilized in the presence of short-chain alcohols that are relevant because of their common use as eluents in industrial-scale chromatography purification. We found that ethanol and isopropanol are efficient modulators of bovine insulin aggregation, providing a three to four times retardation of the aggregation kinetics at 30-35% (vol/vol) concentration; we attribute this to the formation of oligomers, which we detected by AFM. We discuss this effect in terms of reduced solvent polarity and show, by circular dichroism recordings, that a concomitant change in α-helical packing of the insulin monomer occurs in ethanol. Our results extend current knowledge of how insulin aggregates, and may, although bovine insulin serves as a simplistic model, provide insights into how buffers and additives can be fine-tuned in industrial production of proteins in general and pharmaceutical insulin in particular.

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Structure-function relationships in the free insulin monomer

Cited by 14 publications

References 20 publications

Insulin solubility transitions by pH‐dependent interactions with proinsulin C‐peptide

Insulin solubility transitions by pH‐dependent interactions with proinsulin C‐peptide

pH-dependent self-association of zinc-free insulin characterized by concentration-gradient static light scattering

Amyloid formation of bovine insulin is retarded in moderately acidic pH and by addition of short-chain alcohols

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