Backbone Dynamics of TEM-1 Determined by NMR: Evidence for a Highly Ordered Protein
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- 作者：Pierre-Yves Savard ; Sté ; phane M. Gagné
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：September 26, 2006
- 年：2006
- 卷：45
- 期：38
- 页码：11414 - 11424
- 全文大小：1543K
- 年卷期：v.45,no.38(September 26, 2006)
- ISSN：1520-4995
文摘
Backbone dynamics of TEM-1 
mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-lactamase (263 amino acids, 28.9 kDa) were studied by 15Nnuclear magnetic resonance relaxation at 11.7, 14.1, and 18.8 T. The high quality of the spectra allowedus to measure the longitudinal relaxation rate (R1), the transverse relaxation rate (R2), and the {1H}-15NNOE for up to 227 of the 250 potentially observable backbone amide groups. The model-free formalismwas used to determine internal motional parameters using an axially anisotropic model. TEM-1 exhibitsa small prolate axial anisotropy (Dmages/entities/par.gif">/Dmages/entities/bottom.gif"> = 1.23 ± 0.01) and a global correlation time (mages/gifchars/tau.gif" BORDER=0 >m) of 12.41 ± 0.01ns. The unusually high average generalized order parameter (S2) of 0.90 ± 0.02 indicates that TEM-1 isone of the most ordered proteins studied by liquid-state NMR to date. Although the mages/gifchars/Omega.gif" BORDER=0 >-loop has a highdegree of order in the picosecond-to-nanosecond time scale (mean S2 value of 0.90 ± 0.02), we observedthe presence of microsecond-to-millisecond time scale motions for this loop, as for the vicinity of theactive site. These motions could be relevant for the catalytic function of TEM-1. Amide exchangeexperiments were also performed, and several amide groups were not exchanged after 12 days, an indicationthat global motions in TEM-1 are also very limited. Although detailed dynamics characterization by NMRcannot 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 improvementof future molecular dynamics simulations of TEM-1 in the presence of functionally relevant substrates.