ASSIGNMENT OF THE NATURAL ABUNDANCE C-13 SPECTRUM OF PROTEINS USING C-13 H-1-DETECTED HETERONUCLEAR MULTIPLE-BOND CORRELATION NMR-SPECTROSCOPY - STRUCTURAL INFORMATION AND STEREOSPECIFIC ASSIGNMENTS FROM 2-BOND AND 3-BOND CARBON HYDROGEN COUPLING-CONSTANTS

Proton-detected heteronuclear multiple-bond H-1-C-13 correlations (HMBC) previously have been used for assignment purposes in a variety of isotopically enriched proteins. In the present study it is demonstrated that the technique yields an almost complete assignment of the natural abundance C-13 Spectrum of the protein basic pancreatic trypsin inhibitor (BPTI). In addition, the technique permits additional H-1 assignments to be made for this well-studied protein. The intensities of observed correlations permit rough estimates to be made of 2J(C,H) and 3J(C,H) coupling constants. These couplings can be used for conformational studies of both the side chains and the backbone. Intra- and interresidue coupling between C-alpha-H and the carbonyl carbon provides information about the backbone angles-psi and phi. Side-chain conformations can be determined from both two- and three-bond carbon-hydrogen coupling constants. The present study of BPTI together with its known high-precision solution structure yields an experimental correlation between resonance intensities and secondary structure. The spectra show the potential of the method in analyzing C-13 NMR spectra of nonenriched proteins. The method yields C-13 NMR chemical shifts, which are versatile parameters to be used to monitor structural changes, titrations, etc.