Spectroscopic signatures of the T to R conformational transition in the insulin hexamer

Roy, M.; Brader, Mark L.; Lee, R. W.-K.; Kaarsholm, Niels C.; Hansen, Jørgen Fischer; Dunn, Michael F.

doi:10.1016/s0021-9258(19)47269-4

Cited by 88 publications

(98 citation statements)

References 28 publications

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“…3, C and D). This increase is remarkable given that the difference between the half-lives of a rapidly dissociating analog in clinical use (lispro) 5 (27)(28)(29) and WT is Ͻ2-fold (Table 1).…”

Section: Trp B26 Analog Exhibited Markedly Decreased Hexamer Dissociation Ratementioning

confidence: 99%

“…In brief, WT insulin or variants were made 0.6 mM in buffer containing 50 mM Tris-HCl (pH 7.4), 50 mM phenol, and 0.2 mM CoCl 2 (18) and incubated overnight at room temperature to attain conformational equilibrium. Spectra (400 -750 nm) were obtained to monitor tetrahedral Co 2ϩ coordination (27) through its signature absorption band at 574 nm (27). Co 2ϩ sequestration was initiated by addition of EDTA to a concentration of 2 mM.…”

Section: Hexamer Disassembly Assaysmentioning

confidence: 99%

“…Co 2ϩ sequestration was initiated by addition of EDTA to a concentration of 2 mM. Dissociation was probed via attenuation of a 574-nm band (27); data were fit to a monoexponential decay equation (29).…”

Section: Hexamer Disassembly Assaysmentioning

confidence: 99%

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Structure-based stabilization of insulin as a therapeutic protein assembly via enhanced aromatic–aromatic interactions

Rege

Wickramasinghe

Tustan

et al. 2018

Journal of Biological Chemistry

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Key contributions to protein structure and stability are provided by weakly polar interactions, which arise from asymmetric electronic distributions within amino acids and peptide bonds. Of particular interest are aromatic side chains whose directional π-systems commonly stabilize protein interiors and interfaces. Here, we consider aromatic-aromatic interactions within a model protein assembly: the dimer interface of insulin. Semi-classical simulations of aromatic-aromatic interactions at this interface suggested that substitution of residue Tyr by Trp would preserve native structure while enhancing dimerization (and hence hexamer stability). The crystal structure of a [Trp]insulin analog (determined as a TR zinc hexamer at a resolution of 2.25 Å) was observed to be essentially identical to that of WT insulin. Remarkably and yet in general accordance with theoretical expectations, spectroscopic studies demonstrated a 150-fold increase in the lifetime of the variant hexamer, a critical pharmacokinetic parameter influencing design of long-acting formulations. Functional studies in diabetic rats indeed revealed prolonged action following subcutaneous injection. The potency of the Trp-modified analog was equal to or greater than an unmodified control. Thus, exploiting a general quantum-chemical feature of protein structure and stability, our results exemplify a mechanism-based approach to the optimization of a therapeutic protein assembly.

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Section: Trp B26 Analog Exhibited Markedly Decreased Hexamer Dissociation Ratementioning

confidence: 99%

Section: Hexamer Disassembly Assaysmentioning

confidence: 99%

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Structure-based stabilization of insulin as a therapeutic protein assembly via enhanced aromatic–aromatic interactions

Rege

Wickramasinghe

Tustan

et al. 2018

Journal of Biological Chemistry

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“…Metal-bound hexamers are known to exist in essentially three conformational states, T 6 , T 3 R 3 , and R 6 (for the nomenclature see Kaarsholm et al, 1989), all defined by x-ray crystallography (Baker et al, 1988;Bentley et al, 1976;Derewenda et al, 1989;Smith and Dodson, 1992). In solution these states are related by dynamic equilibria that can be shifted from T 6 to T 3 R 3 by the addition of inorganic anions (Bentley et al, 1975;De Graaf et al, 1981;Renscheidt et al, 1984;Ramesh and Bradbury, 1986;Kaarsholm et al, 1989) or adequately moderate concentrations of phenol-like molecules (Wollmer et al, 1987;Roy et al, 1989;Kru ¨ger et al, 1990;Gross and Dunn, 1992). Higher concentrations of the latter can shift the conformational state completely to R 6 : T 6 7 T 3 R 3 7 R 6 (Wollmer et al, 1987;Roy et al, 1989;Thomas and Wollmer, 1989;Kru ¨ger et al, 1990), which further stabilizes the hexamer.…”

Section: Introductionmentioning

confidence: 99%

The Lifetime of Insulin Hexamers

Hassiepen

Federwisch

Mülders

et al. 1999

Biophysical Journal

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The kinetic stability of insulin hexamers containing two metal ions was investigated by means of hybridization experiments. Insulin was covalently labeled at the N(epsilon)-amino group of Lys(B29) by a fluorescence donor and acceptor group, respectively. The labels neither affect the tertiary structure nor interfere with self-association. Equimolar solutions of pure donor and acceptor insulin hexamers were mixed, and the hybridization was monitored by fluorescence resonance energy transfer. With the total insulin concentration remaining constant and the association/dissociation equilibria unperturbed, the subunit interchange between hexamers is an entropy-driven relaxation process that ends at statistical distribution of the labels over 16 types of hexamers differing by their composition. The analytical description of the interchange kinetics on the basis of a plausible model has yielded the first experimental values for the lifetime of the hexamers. The lifetime is reciprocal to the product of the concentration of the exchanged species and the interchange rate constant: tau = 1/(c. k). Measured for different concentrations, temperatures, metal ions, and ligand-dependent conformational states, the lifetime was found to cover a range from minutes for T(6) to days for R(6) hexamers. The approach can be used under an unlimited variety of conditions. The information it provides is of obvious relevance for the handling, storage, and pharmacokinetic properties of insulin preparations.

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“…The T 6 hexamer has no phenolic ligands bound, the T 3 R 3 hexamer has three ligands bound within the same trimer and the R 6 hexamer has a total of six ligands bound (three per trimer, Figure 1). The three conformational hexamers are in dynamic equilibrium in solution and the R 6 hexamer is the most chemically and physically stable (Brange and Langkjñr, 1992;Brader and Dunn, 1991;Kru Èger et al, 1990;Roy et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

Global stochastic optimization in hierarchical modeling of ligand/protein binding profiles

Varshavsky

Birnbaum²,

Beals³

et al. 2000

Kybernetes

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We present a detailed explanation of a mathematical method and numerical technique applied to solve an irregular non-linear fitting problem that results from attempts to model the calorimetric profiles generated by the binding of phenolic ligands to the insulin hexamer. The method employed uses a non-traditional approach to modeling data. Rather than start with a simplified model, we use a hierarchical tree of physical models with different degrees of sophistication. Starting with the model of highest dimension, we work our way to an optimum model which is of a lower dimension and is less complex. The algorithm uses two complementary techniques. First, a sensitivity analysis in the vicinity of the optimal point for each model is used to estimate errors in the parameters; that, in turn, provides the user with insight for model simplification. Second, we utilize the optimized model in the prediction of new experimental curves. The core of the method combines a strategy based on the proper split of the initial global numerical task into three locally independent subtasks, and induces a specific split in the search space. The application of three different optimization techniques (two parametric and one variational) with an alternating objective function defined in corresponding subspaces, in combination with the search along the hierarchical tree of mathematical models, enables us to overcome difficult computational problems, including over-parametrization. We have obtained very accurate fits to a number of calorimetric curves, resulting in a quantitative description of intrinsic functional (free ligand concentration) and vector (equilibrium coefficients and enthalpies of binding) parameters. These quantitative results can now be used to improve the stability of insulin formulations. We believe that, with small modifications to the model, the method and algorithms presented in this article can be applied to other protein-ligand systems.

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Spectroscopic signatures of the T to R conformational transition in the insulin hexamer

Cited by 88 publications

References 28 publications

Structure-based stabilization of insulin as a therapeutic protein assembly via enhanced aromatic–aromatic interactions

Structure-based stabilization of insulin as a therapeutic protein assembly via enhanced aromatic–aromatic interactions

The Lifetime of Insulin Hexamers

Global stochastic optimization in hierarchical modeling of ligand/protein binding profiles

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