Solution conformation of endothelin and point mutants by nuclear magnetic resonance spectroscopy.
Two-dimensional NMR techniques were utilized to determine the secondary structural elements of endothelin-1 (ET-1), a potent vasoconstrictor peptide, and two of its point mutants, Met-7 to Ala-7 (ETM7A), and Asp-8 to Ala-8 (ETD8A) in acetic acid-d3/water solution. Sequence specific NMR assignments were determined for all three peptides, as well as chemical shifts and NOE connectivity patterns. The chemical shifts of ET-1 and ETM7A are identical (+/- 0.05 ppm) except for the site of substitution, whereas marked shift changes were detected between ET-1 and ETD8A. These chemical shift differences imply that the Asp-8 to Ala-8 mutation has induced a conformational change relative to the parent conformation. All three molecules show the same basic nuclear Overhauser effect (NOE) pattern, which suggests that the gross conformation of all three molecules is the same. Small changes in sequential NOE intensities and changes in medium-range NOE patterns indicate that there are subtle conformational differences between ET-1 and ETD8A.
Dalgarno
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Schering-Plough Research Institute, Bloomfield, New Jersey.
Slater
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Chackalamannil
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Senior
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MM
D000085:Acetates; D019342:Acetic Acid; D000595:Amino Acid Sequence; D003903:Deuterium; D016232:Endothelins / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012996:Solutions; D013057:Spectrum Analysis / Q000379:methods
D000085:Acetates; D016232:Endothelins; D012996:Solutions; D003903:Deuterium; D019342:Acetic Acid