This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (10(8)-10(12) s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.

Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease.

Rapid recording of 2D NMR spectra without phase cycling. Application to the study of hydrogen exchange in proteins

Sensitivity-enhanced two-dimensional heteronuclear shift correlation NMR spectroscopy

Ab Initio Methods for the Calculation of NMR Shielding and Indirect Spin-Spin Coupling Constants

13C NMR spectra of pentacyclic triterpenoids--a compilation and some salient features

The DEPT pulse sequence (π/2)(H,y)−(2J)−1−π(H), (π/2)(C,x)−(2J)−1 −ϑ(H,x)π(C)−(2J)−1−(acquire 13C) is analyzed theoretically for a variable ϑ pulse for three spin systems: CH, CH2, and CH3. It is shown that the pulse train produces an enhanced distortion‐free 13C signal which has the following characteristics: (a) there is phase coherency within and between the components of the 13C multiplets; (b) the enhancements vary with ϑ as (γH/γC)sin ϑ for CH, (γH/γC)sin 2ϑ for CH2, and (3γH/4γC) (sin ϑ+sin 3ϑ) for CH3. Experimental evidence is provided for these predictions. An important application of the DEPT pulse train is for the generation of both individual proton‐coupled and proton‐decoupled 13C methine (CH), methylene (CH2), and methyl (CH3) subspectra. This can be readily achieved by forming suitable combinations of DEPT spectra determined at ϑ = (π/4), (π/2), and (3π/4). Such spectral editing is less sensitive to variations in J values than the INEPT pulse sequence. Signal enhancement for 195Pt and 29Si ...

Proton‐polarization transfer enhancement of a heteronuclear spin multiplet with preservation of phase coherency and relative component intensities

Depth and refocusing pulses designed for multipulse NMR with surface coils

Uniform sample excitation with surface coils for in vivo spectroscopy by adiabatic rapid half passage

Complete accurate editing of decoupled 13C spectra using DEPT and a quaternary-only sequence

Pulse sequences utilizing the correlated motion of coupled heteronuclei in the transverse plane of the doubly rotating frame

M.Robin Bendall

Papers

Proton‐polarization transfer enhancement of a heteronuclear spin multiplet with preservation of phase coherency and relative component intensities

Depth and refocusing pulses designed for multipulse NMR with surface coils

Uniform sample excitation with surface coils for in vivo spectroscopy by adiabatic rapid half passage

Complete accurate editing of decoupled 13C spectra using DEPT and a quaternary-only sequence

Pulse sequences utilizing the correlated motion of coupled heteronuclei in the transverse plane of the doubly rotating frame