Two-dimensional NMR studies of Des-(B26-B30)-insulin: sequence-specific resonance assignments and effects of solvent composition.
Des-pentapeptide-insulin (DPI), a monomeric analogue which lacks the C-terminal five residues of the B-chain, provides a tractable model for 2D-NMR studies of insulin under a variety of solvent conditions. In this paper we present the sequential assignment of DPI at pH 1.8 and 25 degrees C in 10% deuterated DMSO/90% H2O; the chemical shifts are in general similar to those recently described in the absence of an organic cosolvent [1], in 20% acetic acid [2] and (for intact insulin) in 35% acetonitrile [3]. Under each of these solvent conditions qualitative analysis of the 2D-NMR data indicates that the major elements of secondary structure observed in the crystal state (three alpha-helices and B-chain beta-turn) are retained in solution. However, there is disagreement in the literature regarding the stability of the insulin fold, as monitored by amide-proton exchange rates and long-range nuclear Overhauser enhancements [1-3]. In contrast to a previous study [1], we observe slowly exchanging amide resonances (in freshly prepared D2O solutions) and nonlocal NOEs under each of the solvent conditions described, implying the existence of a stably folded secondary structure and hydrophobic core. The slowly-exchanging resonances are assigned to the central alpha-helix of the B-chain, the ends of the adjoining beta-turn, and the two A-chain alpha-helices. Qualitative analysis of long-range NOEs indicates that the major features of the crystal state are retained under these solvent conditions.
Hua
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QX
Institute of Biophysics, Academia Sinica, Beijing, China.
Weiss
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MA
D000595:Amino Acid Sequence; D004121:Dimethyl Sulfoxide; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011522:Protons; D012996:Solutions; D012997:Solvents
D007328:Insulin; D011522:Protons; D012996:Solutions; D012997:Solvents; C007285:insulin, despentapeptide(B26-B30)-; D004121:Dimethyl Sulfoxide