Kinetic Measurements of T→ R Structural Transitions in Insulin

Karatas, Yunus; Krüger, Peter; Wollmer, Axel

doi:10.1515/bchm3.1991.372.2.1035

Cited by 8 publications

(6 citation statements)

References 2 publications

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“…Whereas the T 3 -related site is predominantly octahedral, the R 3 -related site is tetrahedral. This difference has been exploited to monitor the TR transition in Co 2ϩ -substituted hexamers in wildtype insulin (wt) and among insulin analogs (32,72). T 3 R 3 f hexamers contain one octahedral (T3) and one tetrahedral (R 3 f ) metal-ion binding site.…”

Section: Figure 7 Substitution Of Hismentioning

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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Section: Figure 7 Substitution Of Hismentioning

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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“…It hence seems obvious that ligand binding and release do not require the hexamer to dissociate into subunits, but can be achieved by rather local fluctuations of one or a few ''gatekeeper'' residues (Jacoby et al, 1996a). According to all the results of kinetic experiments, the dissociation is also much faster than the T $ R transition (Karatas et al, 1991;Birnbaum et al, 1997) and therefore must occur within the intact R 6 hexamer. Because information on the kinetics of phenol (un)binding is not available, experimental studies can only provide an upper limit of the phenol lifetime in the hexamer, which according to its NMR chemical shifts is less than 10 ms (Roy et al, 1989).…”

Section: Introductionmentioning

confidence: 99%

MD Simulation of Protein-Ligand Interaction: Formation and Dissociation of an Insulin-Phenol Complex

Swegat

Schlitter

Krüger

et al. 2003

Biophysical Journal

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Complexes of proteins with small ligands are of utmost importance in biochemistry, and therefore equilibria, formation, and decay have been investigated extensively by means of biochemical and biophysical methods. Theoretical studies of the molecular dynamics of such systems in solution are restricted to 10 ns, i.e., to fast processes. Only recently new theoretical methods have been developed not to observe the process in real time, but to explore its pathway(s) through the energy landscape. From the profiles of free energy, equilibrium and kinetic quantities can be determined using transition-state theory. This study is dedicated to the pharmacologically relevant insulin-phenol complex. The distance of the center of mass chosen as a reaction coordinate allows a reasonable description over most of the pathway. The analysis is facilitated by analytical expressions we recently derived for distance-type reaction coordinates. Only the sudden onset of rotations at the very release of the ligand cannot be parameterized by a distance. They obviously require a particular treatment. Like a preliminary study on a peptide, the present case emphasizes the contribution of internal friction inside a protein, which can be computed from simulation data. The calculated equilibrium constant and the friction-corrected rates agree well with experimental data.

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“…The insulin hexamers behave as a dimer of two cooperative trimers which are linked by a negative cooperativity [6,7,8]. The binding of SCNions only transforms one trimer and so the transition does not exceed the T3R3-state, whereas titration with phenol achieves complete transformation which, however, proceeds in two consecutive steps: T6 T3R3 R6 [5,6,7,8,9,10].…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Characterization of New N-Terminal Bchain Modified Human Insulin Analogues

Shneine¹

2010

JNUS

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New human insulin analogues were synthesized by fragment condensation reactions of N A1 , N B29 Msc protected native human insulin of different B-chain lengths with the dipeptide Boc-Asn-Thr-OH. Edman degradation was used to obtain shortened B-chain insulin intermediates. Purification of the products was achieved using gel filtration, ion exchange chromatography and RP-MPLC chromatography. Analysis of synthesized analogues accomplished by RP-HPLC chromatography and amino acid analysis. The modified insulin analogues with the amino acids Asparagine in the first position and Threonine in the second position of the N-terminal B-chain of insulin would be expected to show an enhanced T R* transition compared with that of wild-type structure.

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Kinetic Measurements of T→ R Structural Transitions in Insulin

Cited by 8 publications

References 2 publications

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

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

MD Simulation of Protein-Ligand Interaction: Formation and Dissociation of an Insulin-Phenol Complex

Synthesis and Characterization of New N-Terminal Bchain Modified Human Insulin Analogues

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