Internal mobility of reactive-site-hydrolyzed recombinant Cucurbita maxima trypsin inhibitor-V characterized by NMR spectroscopy: evidence for differential stabilization of newly formed C- and N-termini.
Internal mobility of reactive-site-hydrolyzed recombinant Cucurbita maxima trypsin inhibitor-V characterized by NMR spectroscopy: evidence for differential stabilization of newly formed C- and N-termini.
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通过 NMR 光谱表征反应位点水解重组南瓜胰蛋白酶抑制剂-V 的内部迁移率：新形成的 C 端和 N 端差异稳定的证据。
DOI：
10.1021/bi9609329
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Krishnamoorthi,R
中科院分区：
文献类型：
--
作者：
Liu,J;Prakash,O;Huang,Y;Wen,L;Wen,JJ;Huang,JK;Krishnamoorthi,R
The solution structure and internal dynamics of the reactive-site (Lys44−Asp45peptide bond) hydrolyzed form of recombinantCucurbita maximatrypsin inhibitor-V (rCMTI-V*) were characterized by the application of two-dimensional1H−15N NMR methods to the uniformly15N-labeled protein. The1H−15N chemical shift correlation spectra of rCMTI-V* were assigned, and the chemical shift data were compared with those available for rCMTI-V [Liu, J., Prakash, O., Cai, M., Gong, Y., Huang, Y., Wen, L., Wen, J. J., Huang, J.-K., & Krishnamoorthi, R. (1996)Biochemistry 35, 1516−1524] and CMTI-V* [Cai, M., Gong, Y., Prakash, O., & Krishnamoorthi, R. (1995)Biochemistry 34, 12087−12094] for which three-dimensional solution structures have been determined. It was deduced that the solution structure of rCMTI-V* was almost the same as that of CMTI-V*.15N spin−lattice and spin−spin relaxation rate constants (R1andR2, respectively) and {1H}−15N steady-state heteronuclear Overhauser effects were measured for the peptide NH units and arginine and tryptophan NεH groups in rCMTI-V*, and the model-free parameters [Lipari, G., & Szabo, A. (1982)J. Am. Chem. Soc. 104, 4546−4559, 4559−4570] were computed. Most of the backbone of rCMTI-V* is found to be highly constrained (⟨S2⟩ = 0.85), including the N-terminal residues 3−6 (⟨S2⟩ = 0.77). Residues 39−44, forming the C-terminal fragment of the binding loop, exhibit increased mobility (⟨S2⟩ = 0.51); however, the N-terminal segment (residues 46−48) retains rigidity as in the intact form (⟨S2⟩ = 0.83). TheS2values, 0.78 and 0.59, respectively, of Arg50and Arg52side chain NHs provide evidence not only for the conservation of the Arg hydrogen-bonds with the binding loop segments but also for the difference in strength between them. This is consistent with the earlier observation made from a study of rCMTI-V at two different pHs and its R50 and R52 mutants [Cai, M., Huang, Y., Prakash, O., Wen, L., Dunkelbarger, S. P., Huang, J.-K., Liu, J., & Krishnamoorthi, R. (1996)Biochemistry35, 4784−4794]. The dynamical results suggest the main-chain oxygen atom of Asp45as the hydrogen bond acceptor of Arg50. Residues Trp9and Trp54, which interact with many others in the protein scaffold and the binding loop region, respectively, remain rigid in the cleaved inhibitor with theS2values of 0.84 and 0.71 determined for their respective NεHs. The internal dynamics of rCMTI-V* was compared with that of the noncovalent complex formed between the two fragments of reactive-site-hydrolyzed chymotrypsin inhibitor-2 from barley seeds [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995)Biochemistry 34, 2225−2233], another potato I family inhibitor that lacks the Cys3−Cys48disulfide present in rCMTI-V*.
