Carbonyl 13C NMR spectrum of basic pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding.
The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering.
Tüchsen
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Institute of Life Sciences and Chemistry, University of Roskilde, Denmark.
Hansen
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PE
D000577:Amides; D007611:Aprotinin; D002247:Carbon Isotopes; D003903:Deuterium; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011487:Protein Conformation
D000577:Amides; D002247:Carbon Isotopes; D006859:Hydrogen; D007611:Aprotinin; D003903:Deuterium