Two‐dimensional NMR studies on des‐pentapeptide‐insulin

Abstract

The shortened analogue of insulin, des-(B26 -B30)-pentapeptide insulin, has been characterized by twodimensional 'H NMR. The 'H resonance assignments and the secondary structure in water solution are discussed. The results indicate that the secondary structure in solution is very similar to that reported for the crystalline state. A high flexibility of both A and B chains is observed. Of the two conformations seen in the 2-Zn insulin crystals and indicated as molecules 1 and 2 (Chinese nomenclature), the structure of the analogue is more similar to that of molecule 1There is a long standing interest in insulin on account of its extreme importance as a drug for the treatment of diabetes [l]. In particular, several recent studies have been reported on insulins modified by site-directed mutagenesis, which have altered aggregation properties [2]. We have started a highresolution NMR study of insulin with the aim of elucidating the solution structure of the hormone and of its various modified forms. Here we report the 'H resonance assignments for des-(B26 -B30)-pentapeptide insulin (DPI) and discuss some structural aspects. This should provide the basis for a full structure determination of DPI in solution and should facilitate the interpretation of the NMR spectra and structural comparisons of modified insulins.Insulin consists of two polypeptide chains, called the A and B chains, which are linked by two disulfide bridges (see Fig. 1). The crystal structures of insulin [3--81 and of the monomeric des-(B26 -B30)-pentapeptide insulin [9 -1 I] are known. From X-ray crystallography [12,13] and CD measurements [14], it was found that the insulin monomer can adopt a number of different conformations. Careful analysis of the different X-ray crystal forms showed that these conformations can be grouped into two classes which are sometimes referred to as molecule 1 and molecule 2 (Chinese nomenclature) [6]. The major differences between the two forms are found at the end of the B chain (B25-B30) and the first part of the A The shortened analogue DPI obtained by removal of five C-terminal amino acids from the B chain has a relative molecular mass of about 5200 and differs from insulin in several aspects. Normal insulin can occur in various aggregation states in solution : monomeric, dimeric, hexameric and even polymeric states have been identified by a variety of methods [3, 13, 151. However, it is known that DPI has much less tendency to form such aggregates. A rationale for this comes from the X-ray structure which shows that the residues B26 ~ B30 of two insulin monomers form an antiparallel P-sheet. In the blood, insulin is present in very low concentrations which ensures that it circulates and brings about its biological effects as a monomer [3]. Monomeric DPI still has full biological activity provided that the carboxy-terminus of the B chain is amidated [17].The crystal structure of DPI has been solved at a resolution of 0.12 nm [9]. Furthermore, MD simulations of DPI in the crystal have been reported [18]. Several au...

The solution structure of a monomeric insulin

“…As pointed out previously, des-(B26 -B30)-insulin is a rather flexible molecule [25]. When it is dissolved in 'H,O, all amide protons exchange rapidly with the solvent.…”

“…We shall compare the NMR structure with molecules 1 and 2 in the porcine insulin X-ray structure and its own averaged crystal structure from an RMD trajectory. Our previous results [25] suggested that the protein appears to be rather flexible, on the basis of the sensitivity of the chemical shifts to changes in temperature and pH. This conclusion is reinforced by analysis of the NOES.…”

“…The 2D NOE [27] spectra were recorded as described previously [25]. The water signal was suppressed by presaturation.…”

“…Three restraints corresponding to disulphide bridges were added. Furthermore 10 restraints between the carbonyloxygen atoms of residue i and the amide-hydrogen atoms of residue i + 4 in the previously identified helices were included for DG [25]. Structures embedded without these extra helix restraints showed also the presence of helices in the predicted regions, but they were more irregular.…”

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

“…We and others have recently described the two-dimensional NMR resonance assignment of a monomeric insulin analogue, des-pentapeptide-(B26-B30)-insulin (DPI) (1,2), which provides a favorable system for NMR studies due to reduced aggregation (3). DPI retains sufficient structural information for proper function and folding (4)(5)(6)(7)(8)(9)(10)(11)(12)(13), and its crystal structure is similar to the corresponding portion ofthe intact protein (6,7).…”