LONGITUDINAL AND TRANSVERSE H-1-N-15 DIPOLAR N-15 CHEMICAL-SHIFT ANISOTROPY RELAXATION INTERFERENCE - UNAMBIGUOUS DETERMINATION OF ROTATIONAL DIFFUSION TENSORS AND CHEMICAL-EXCHANGE EFFECTS IN BIOLOGICAL MACROMOLECULES

High-resolution proton-detected heteronuclear correlation NMR spectros copy allows the measurement of N-15 spin relaxation rates at multiple sites throughout a biological macromolecule. The rate constants are de termined by stochastic internal motions on time scales of picoseconds to nanoseconds, overall molecular rotational diffusion on time scales of nanoseconds, and chemical exchange rates on time scales of microsec onds to milliseconds. A new method has been developed for distinguishi ng the contributions of chemical exchange from the contributions due t o anisotropic rotational diffusion by measuring both longitudinal and transverse interference between the H-1-N-15 dipolar and N-15 chemical shift anisotropy interactions. The spectroscopic experiment for measu ring the longitudinal cross-correlation rate constant for H-1-N-15 dip olar/N-15 chemical shift anisotropy interference is based on the appro ach for measuring the transverse cross-correlation rate constant (Tjan dra, N,; Szabo, A.; Bar, A. J. Am. Chem. Sec. 1996, 118, 6986-6991)but incorporates a novel method for averaging the relaxation rates of lon gitudinal magnetization and two spin order. Application of this techni que to Escherichia coli ribonuclease H affords an improved description of rotational diffusion anisotropy and permits a more accurate assess ment of chemical exchange in this molecule. The results definitively d emonstrate that amino acid residues K60 and W90 are subject to conform ational exchange processes, whereas increased transverse relaxation ra tes for residues in the helix alpha(D) arise from anisotropic rotation al diffusion.