Comparative 2D NMR studies of human insulin and despentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition

Hua, Qing‐Xin; Weiss, Michael A.

doi:10.1021/bi00236a025

Cited by 134 publications

(131 citation statements)

References 39 publications

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“…When it is dissolved in 'H,O, all amide protons exchange rapidly with the solvent. Recently Hua et al [37] reported that slowly exchanging amide protons were observed for des-(B26 -B30)-insulin under conditions similar to ours. In our 2D experiments using fresh material in 'H20, which take about 12-24 h at 300 K, no cross-peaks corresponding to amide resonances were observed.…”

Section: Local Mobilitysupporting

confidence: 86%

The solution structure of a monomeric insulin

Knegtel

Boelens

Ganadu

et al. 1991

European Journal of Biochemistry

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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...

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Section: Local Mobilitysupporting

confidence: 86%

The solution structure of a monomeric insulin

Knegtel

Boelens

Ganadu

et al. 1991

European Journal of Biochemistry

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“…Because the amide line widths of 3G-DKP-insulin are appropriate for a small globular protein, less extensive anti- Table V. phase cancellation is observed in DQF-COSY spectra (37) and more efficient total correlation spectroscopy transfer is achieved. Similar technical improvement in NMR quality was observed on deleting residues B26 -B30 in NMR studies of DPI (38,39). We suggest that the detached (A1-A5) or deleted (B26 -B30) elements are coupled to non-local motions in the core of insulin; in native insulin and DKP-insulin, the millisecond time scale of such motions leads to incomplete averaging of NMR chemical shifts ("conformational broadening"; Refs.…”

Section: Discussionsupporting

confidence: 65%

“…By conventional criteria of sensitivity and resolution, the quality of the variant spectra is superior to that of DKP-insulin or native insulin (1,39). This "improvement" (a seeming paradox in light of the partial fold of the analog and its very low activity) reflects a reduction in the extent of anomalous broadening otherwise characteristic of insulin and insulin analogs (43,44).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Insulin Chain Combination

Hua

Chen

Jia

et al. 2002

Journal of Biological Chemistry

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The A and B chains of insulin combine to form native disulfide bridges without detectable isomers. The fidelity of chain combination thus recapitulates the folding of proinsulin, a precursor protein in which the two chains are tethered by a disordered connecting peptide. We have recently shown that chain combination is blocked by seemingly conservative substitutions in the C-terminal ␣-helix of the A chain. Such analogs, once formed, nevertheless retain high biological activity. By contrast, we demonstrate here that chain combination is robust to non-conservative substitutions in the Nterminal ␣-helix. Introduction of multiple glycine substitutions into the N-terminal segment of the A chain (residues A1-A5) yields analogs that are less stable than native insulin and essentially without biological activity.1 H NMR studies of a representative analog lacking invariant side chains Ile A2 and Val A3 (A chain sequence GGGEQCCTSICSLYQLENYCN; substitutions are italicized and cysteines are underlined) demonstrate local unfolding of the A1-A5 segment in an otherwise nativelike structure. That this and related partial folds retain efficient disulfide pairing suggests that the native Nterminal ␣-helix does not participate in the transition state of the reaction. Implications for the hierarchical folding mechanisms of proinsulin and insulin-like growth factors are discussed.

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“…Effects of Urea on Monomeric Insulin Fibrillation-Insulin exists as a monomer in 20% acetic acid (30,45,52). The near-UV CD spectra of insulin measured in the presence of 20% acetic acid and various urea concentrations are represented in Fig.…”

Section: Kinetics Of Fibrillation For Hexameric Insulin In the Presenmentioning

confidence: 99%

Stimulation of Insulin Fibrillation by Urea-induced Intermediates

Ahmad

Millett

Doniach

et al. 2004

Journal of Biological Chemistry

107

105

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Fibrillar deposits of insulin cause serious problems in implantable insulin pumps, commercial production of insulin, and for some diabetics. We performed a systematic investigation of the effect of urea-induced structural perturbations on the mechanism of fibrillation of insulin. The addition of as little as 0.5 M urea to zincbound hexameric insulin led to dissociation into dimers. Moderate concentrations of urea led to accumulation of a partially unfolded dimer state, which dissociates into an expanded, partially folded monomeric state. Very high concentrations of urea resulted in an unfolded monomer with some residual structure. The addition of even very low concentrations of urea resulted in increased fibrillation. Accelerated fibrillation correlated with population of the partially folded intermediates, which existed at up to 8 M urea, accounting for the formation of substantial amounts of fibrils under such conditions. Under monomeric conditions the addition of low concentrations of urea slowed down the rate of fibrillation, e.g. 5-fold at 0.75 M urea. The decreased fibrillation of the monomer was due to an induced non-native conformation with significantly increased ␣-helical content compared with the native conformation. The data indicate a close-knit relationship between insulin conformation and propensity to fibrillate. The correlation between fibrillation and the partially unfolded monomer indicates that the latter is a critical amyloidogenic intermediate in insulin fibrillation.

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Comparative 2D NMR studies of human insulin and despentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition

Cited by 134 publications

References 39 publications

The solution structure of a monomeric insulin

The solution structure of a monomeric insulin

Mechanism of Insulin Chain Combination

Stimulation of Insulin Fibrillation by Urea-induced Intermediates

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