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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 conformation of des-(B26-B30)-insulin (DPI) has been investigated by 'H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in VUL'UO and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20-ps RMD. The final structure was compared with the des-(B26 -B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26 -B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A l , Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.Due to its biological importance as a hormone and drug, insulin has been the subject of numerous studies regarding its structure and function [l]. Several crystal structures of insulins have been reported [2 -61 and a detailed picture of the spatial structure of insulins has emerged.The insulin molecule consists of two polypeptide chains, A and B, of 21 and 30 residues respectively (cf. Fig. 1). The horse-shoe-shaped A chain contains two helices (residues A2-A8 and A13-A20) and the B chain one helix (residues B9 -BI 9). These chains are connected by two disulphide bridges (A7 -B7 and A20 -B19) which, together with a third one (A6 -A1 I), help stabilize the folded form of insulin over a broad range of temperatures and pH values. In the crystal structures the last six residues of the B chain form an antiparallel fl-sheet with another insulin molecule. Insulin is a rather flexible molecule, as evidenced by large structural differences found in different crystal structures. The major differences between various forms of insulin are in the N-terminal of the B chain. For instance, the porcine 4-Zn insulin structure has an additional helical region running from B1 to B8 [4], whereas in 2-Zn insulin this fragment is in a random coil conformation. In fact, a variety of solution and crystal studies showed that the conversion of the 2-Zn to the 4-Zn structure can be induced by high concentrations of anions [7]. Adding Correspondence to R . Boelens, Department of Chemistry, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The NetherlandsAhbveviutions. Des-(3326 -B30)-insulin, insulin lacking the C-te...

BackgroundThe development and improvement of reliable computational methods designed to evaluate the quality of protein models is relevant in the context of protein structure refinement, which has been recently identified as one of the bottlenecks limiting the quality and usefulness of protein structure prediction.ResultsIn this contribution, we present a computational method (Artificial Intelligence Decoys Evaluator: AIDE) which is able to consistently discriminate between correct and incorrect protein models. In particular, the method is based on neural networks that use as input 15 structural parameters, which include energy, solvent accessible surface, hydrophobic contacts and secondary structure content. The results obtained with AIDE on a set of decoy structures were evaluated using statistical indicators such as Pearson correlation coefficients, Znat, fraction enrichment, as well as ROC plots. It turned out that AIDE performances are comparable and often complementary to available state-of-the-art learning-based methods.ConclusionIn light of the results obtained with AIDE, as well as its comparison with available learning-based methods, it can be concluded that AIDE can be successfully used to evaluate the quality of protein structures. The use of AIDE in combination with other evaluation tools is expected to further enhance protein refinement efforts.