Backbone Dynamics of a Free and a Phosphopeptide-Complexed Src Homology 2 Domain Studied by 15N NMR Relaxation

Abstract

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

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

“…All the NMR experiments were performed at 298 K. The T 1 , T 2 , and 15 N{ 1 H}-NOE measurements were performed at 1 H frequency of 600 MHz at 298 K. The T 1 ( 15 N) relaxation times were determined with the following T 1 relaxation delays: 0, 50, 100, 150, 200, 300, and 500 ms [22,23]. The T 2 ( 15 N) relaxation times were obtained based on a CMPG-type sequence with the interval of 625 ls between 15 N 180°pulse in the CPMG cycles with the following T 2 relaxation delays: 10, 30, 50, 70, 90, 110, 150, and 190 ms. 15 N{ 1 H}-NOEs were determined by the comparison of the peak volumes between the HSQC spectra with and without 1 H saturation of 2.5 s with special care for water-suppression [22,23,34]. The volumes of the peaks were analyzed and fitted by the program ANAL-YSIS [24].…”

Backbone dynamics of the channel‐forming antibiotic zervamicin IIB studied by 15N NMR relaxation

“…The pulse sequences of Farrow et al [14] were used. Temperature calibration and control in all experiments were carried out as described in Orekhov et al [15].…”

The counterreceptor binding site of human CD2 exhibits an extended surface patch with multiple conformations fluctuating with millisecond to microsecond motions

“…The rates RN(N,) and RN(N,) were measured using similar pulse sequences as described in Dayie and Wagner (1994), and the heteronuclear NOE enhancements were measured using the pulse sequence described in Farrow et al (1994). Delays and gradient strengths and durations were optimized to minimize losses due to relaxation.…”