Enhancing the T R Transition of Insulin by Helix-Promoting Sequence Modifications at the N-Terminal B-Chain

Abstract

Structurally, the T-->R transition of insulin mainly consists of a rearrangement of the N-terminal B-chain (residues B1-B8) from extended to helical in one or both of the trimers of the hexamer. The dependence of the transition on the nature of the ligands inducing it, such as inorganic anions or phenolic compounds, as well as of the metal ions complexing the hexamer, has been the subject of extensive investigations. This study explores the effect of helix-enhancing modifications of the N-terminal B-chain sequence where the transition actually occurs, with special emphasis on N-capping. In total 15 different analogues were prepared by semisynthesis. 80% of the hexamers of the most successful analogues with zinc were found to adopt the T3R3 state in the absence of any transforming ligands, as compared to only 4% of wild-type insulin. Transformation with SCN- ions can exceed the T3R3 state where it stops in the case of wild-type insulin. Full transformation to the R6 state can be achieved by only one-tenth the phenol concentration required for wild-type insulin, i.e. almost at the stoichiometric ratio of 6 phenols per hexamer.

Analysis of Insulin Allostery in Solution and Solid State With FTIR

“…The a-helix content is normally monitored by the ellipticity at 222 nm, but for insulin this area is compromised by the contributions from aromatic residues and thus 251 nm is used to monitor the T-R transition. 10 A titration of the insulin hexamer with phenol monitored by CD is shown in Figure 2B. The binding isotherm shows a hyperbolic one-site binding model with a calculated dissociation constant, K D , of 2.7 mM (Tab.…”

“…CD has previously been used to analyse the allosteric changes of insulin in the presence of phenolic ligands where the band at 251 nm in the CD signal has been correlated with the T-R transition. 10,29 This band is commonly assigned to the dichroic absorbance of cysteine residues. The a-helix content is normally monitored by the ellipticity at 222 nm, but for insulin this area is compromised by the contributions from aromatic residues and thus 251 nm is used to monitor the T-R transition.…”

“…[2][3][4] The hexameric structure acts as an allosteric unit and is modulated by homotropic and heterotropic interactions. [5][6][7][8][9][10] Three global conformations of the insulin hexamer have been identified by X-ray crystallography 2,[11][12][13][14][15] and in solution [16][17][18] and are designated T 6 , R 3 T 3 and R 6 . The structural transition from T to R form involves rearrangement of the first eight amino acid residues in the B-chain, from an extended conformation in the T state to a-helix in the R state.…”

“…The conformational state is traditionally analysed with UV absorbing probes having a distinct binding affinity to the T 6 and R 6 state, 5,[7][8][9]19,27,28 or by near UV circular dichroism (CD) monitoring the tertiary conformational state of insulin. 3,8,10,28,29 Both techniques have several drawbacks with respect to pharmaceutical formulations including low compatibility with excipients, the need for relatively well defined conditions, and they are only applicable in solution. Several calorimetric studies have been carried out including ITC and DSC.…”

Ligand Migration in the Gaseous Insulin-CB7 Complex—A Cautionary Tale About the Use of ECD-MS for Ligand Binding Site Determination

“…Several N-terminal mutations, including the Phe B1 Gly mutation, appear to have only a minor effect of the conformation of the B-chain [34]. This may be attributable to the flexibility of the N-terminal region of the B-chain observed in nuclear magnetic resonance (NMR) experiments under a range of solution conditions [35].…”