NMR EXPERIMENTS FOR THE MEASUREMENT OF CARBON RELAXATION PROPERTIES IN HIGHLY ENRICHED, UNIFORMLY C-13,N-15-LABELED PROTEINS - APPLICATION TO C-13(ALPHA) CARBONS

New two-dimensional NMR experiments with high sensitivity and resoluti on are presented for the measurement of T-1, T-1 rho, and steady-state H-1-C-13 NOE values for CH C-13(alpha) spin systems in highly enriche d, uniformly C-13-labeled proteins. Using a sample consisting of appro ximately equimolar amounts of 99% C-13(alpha)-alanine and 99% uniforml y C-13-labeled alanine (C-13(3)-alanine) dissolved in perdeuterated gl ycerol, high signal-to-noise C-13(alpha) relaxation measurements of bo th singly and uniformly C-13-labeled alanine have been made. This allo ws an investigation of the influence of both carbon-carbon scalar coup ling effects and dipolar relaxation effects on the measurement of rela xation properties of carbon spins. T-1, T-1 rho, and steady-state H-1( alpha)-C-13(alpha) NOE values have been measured over a range of tempe ratures from 10 degrees C to 40 degrees C, with the correlation time f or molecular tumbling varying from similar to 17 to similar to 1 ns. T he results indicate that, for macromolecules, the contributions to the longitudinal carbon relaxation from neighboring carbons must be inclu ded in the interpretation of T-1 data in terms of motional models. The H-1(alpha)-C-13(alpha) steady-state NOE can be influenced significant ly by C-13(alpha)-C-13 beta cross relaxation, and because of the small H-1(alpha)-C-13(alpha) NOE in proteins, it may not be possible to mea sure H-1(alpha)-C-13(alpha) NOE values with high accuracy. Theoretical results are presented which indicate that it is possible to measure a ccurate (CT1 rho)-C-13-T-alpha values in all residues, with the except ion of serine and threonine when the C-13(alpha) and C-13(beta) chemic al shifts are nearly equivalent, and experimental verification is prov ided for the case of alanine. A strategy is proposed for obtaining acc urate dynamics of C-13(alpha) carbons based on the measurement of C-13 (alpha) T-1 values using at least two field strengths and T-1 rho valu es measured at a single field.