Backbone dynamics of TEM-1 determined by NMR:: Evidence for a highly ordered protein

Backbone dynamics of TEM-1, beta-lactamase ( 263 amino acids, 28.9 kDa) were studied by N-15 nuclear magnetic resonance relaxation at 11.7, 14.1, and 18.8 T. The high quality of the spectra allowed us to measure the longitudinal relaxation rate (R-1), the transverse relaxation rate (R-2), and the {H-1}- N-15 NOE for up to 227 of the 250 potentially observable backbone amide groups. The model-free formalism was used to determine internal motional parameters using an axially anisotropic model. TEM-1 exhibits a small prolate axial anisotropy (D-vertical bar vertical bar/D-perpendicular to 1.23 +/- 0.01) and a global correlation time (tau(m)) of 12.41 +/- 0.01 ns. The unusually high average generalized order parameter (S-2) of 0.90 +/- 0.02 indicates that TEM-1 is one of the most ordered proteins studied by liquid-state NMR to date. Although the Omega-loop has a high degree of order in the picosecond-to-nanosecond time scale (mean S2 value of 0.90 +/- 0.02), we observed the presence of microsecond-to-millisecond time scale motions for this loop, as for the vicinity of the active site. These motions could be relevant for the catalytic function of TEM-1. Amide exchange experiments were also performed, and several amide groups were not exchanged after 12 days, an indication that global motions in TEM-1 are also very limited. Although detailed dynamics characterization by NMR cannot be readily applied to TEM-1 in the presence of relevant substrates, the unusual picosecond-to-nanosecond dynamics behavior of TEM-1 presented here will be essential to the validation and improvement of future molecular dynamics simulations of TEM-1 in the presence of functionally relevant substrates.