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

Abstract

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.

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

“…Data were processed with NMRPipe/NMRDRAW and analysed with CCPN software (Delaglio et al , 1995; Vranken et al , 2005). 15 N relaxation measurements and 1 H‐ 15 N heteronuclear NOE were collected as previously described (Kay et al , 1989). R1 and R2 values were determined for each residue by fitting an exponential decay to the peak intensity of data collected in an interleaved manner to minimize time‐dependent temperature or stability effects with delay times in random sequence.…”

Solution structure of DnaE intein from Nostoc punctiforme: Structural basis for the design of a new split intein suitable for site‐specific chemical modification

“…Nuclear spin relaxation measurement of 15 N spins of proteins is a popular approach for studies of global protein motions and internal backbone mobility [31][32][33][34]. We hypothesized that the residues indicating higher internal mobility are suitable locations for a new split site and recorded a set of 15 N relaxation measurements (Fig.…”

An Improved Picture of Methyl Dynamics in Proteins from Slowly Relaxing Local Structure Analysis of 2H Spin Relaxation

“…12 and 43 based on 15 N T 1 /T 2 ratios. 44 When β MQ is set equal to zero then the SRLS spectral density is formally analogous with the original MF spectral density (eq 1).…”