Letter to the Editor: 1H, 13C and 15N resonance assignments and secondary structure of the human protein tyrosine phosphatase, PRL-2

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• 作者：Zhou ; Hongjun ; Gallina ; Marijo ; Mao ; Hongyuan ; Nietlispach ; Daniel ; Betz ; Stephen F. ; Fetrow ; Jacquelyn S. ; Domaille ; Peter J.
• 刊名：Journal of Biomolecular NMR
• 出版年：2003
• 出版时间：2003
• 年：2003
• 卷：27
• 期：4
• 页码：397-398
• 全文大小：60 KB
• 刊物类别：Physics and Astronomy
• 刊物主题：Physics
  Biophysics and Biomedical Physics
  Polymer Sciences
  Biochemistry
  
• 出版者：Springer Netherlands
• ISSN：1573-5001

文摘

The usual analysis of ¹⁵N relaxation data of proteins is straightforward as long as the assumption can be made that the backbone of most residues only undergoes fast (ps), small amplitude internal motions. If this assumption cannot be made, as for example for proteins which undergo domain motions or for unfolded or partially folded proteins, one needs a method to establish for each residue whether it undergoes fast (ps) or slow (ns) internal motion. Even then it is impossible to determine the correct overall tumbling time, &tgr;_m⁰, via the usual method from the ratio of the longitudinal and transverse relaxation times, if the majority of residues do not undergo fast, small amplitude internal motions. The latter problem is solved when &tgr;_m⁰ can be determined independent of the time scale, &tgr;_i, or the amplitude, S², of the internal motion. We propose a new protocol, called PINATA, for analyzing ¹⁵N relaxation data acquired at minimally two field strengths, where no a priori assumption about time scales or amplitude of internal motions needs to be made, and overall tumbling can either be isotropic or anisotropic. The protocol involves four steps. First, for each residue, it is detected whether it undergoes ps- or ns-internal motion, via the combination of the ratio of the longitudinal relaxation time at two fields and the hetero-nuclear NOE. Second, for each residue &tgr;_m⁰ and the exchange broadening, Rex, are iteratively determined. The accuracy of the determination of &tgr;_m⁰ is ca. ±0.5 ns and of Rex ca ± 0.7 s⁻¹, when the relaxation data are of good quality and &tgr;_m⁰>5 ns, S²>0.3, and &tgr;_i<≈3 ns. Third, given &tgr;_m⁰ and Rex, step 1 is repeated to iteratively improve on the internal motion and obtain better estimates of the internal parameter values. Fourth, final time scales and amplitudes for internal motions are determined via grid search based fitting and &khgr;²-analysis. The protocol was successfully tested on synthetic and experimental data sets. The synthetic dataset mimics internal motions on either fast or slow time scales, or a combination of both, of either small- or large amplitude, superimposed onto anisotropic overall motion. The procedures are incorporated into MATLAB scripts, which are available on request.