1mit: Difference between revisions

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RECOMBINANT CUCURBITA MAXIMA TRYPSIN INHIBITOR V (RCMTI-V) (NMR, MINIMIZED AVERAGE STRUCTURE)

OverviewOverview

The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I family--eglin c [Peng, J. W., & Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225-2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a beta-sheet, appears to be more rigid.

About this StructureAbout this Structure

1MIT is a Single protein structure of sequence from Cucurbita maxima. Full crystallographic information is available from OCA.

ReferenceReference

Solution structure and backbone dynamics of recombinant Cucurbita maxima trypsin inhibitor-V determined by NMR spectroscopy., Liu J, Prakash O, Cai M, Gong Y, Huang Y, Wen L, Wen JJ, Huang JK, Krishnamoorthi R, Biochemistry. 1996 Feb 6;35(5):1516-24. PMID:8634282

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-[[Image:1mit.gif|left|200px]]<br /><applet load="1mit" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1mit.gif|left|200px]]<br /><applet load="1mit" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1mit" />
 '''RECOMBINANT CUCURBITA MAXIMA TRYPSIN INHIBITOR V (RCMTI-V) (NMR, MINIMIZED AVERAGE STRUCTURE)'''<br />
 ==Overview==
-The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V, (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine, residue, was determined by means of two-dimensional (2D) homo and 3D, hetero NMR experiments in combination with a distance geometry and, simulated annealing algorithm. A total of 927 interproton distances and, 123 torsion angle constraints were utilized to generate 18 structures. The, root mean squared deviation (RMSD) of the mean structure is 0.53 A for, main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues, 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the, same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., &amp;, Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone, dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N, NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1, and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser, effect enhancements were measured for the peptide NH units and, using the, model-free formalism [Lipari, G., &amp; Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559, 4559-4570], the following parameters were determined:, overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms, to account for motions on a slower time scale (second) due to chemical, exchange and/or conformational averaging (R(ex)). Most of the backbone NH, groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with, the exception of those in the binding loop (residues 41-48, (S2) = 0.71), and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions, show large RMSD values in the average NMR structure. Residues involved in, turns also appear to have more mobility ((S2) = 0.80). Dynamical, properties of rCMTI-V were compared with those of two other inhibitors of, the potato I family--eglin c [Peng, J. W., &amp; Wagner, G. (1992), Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2;, Shaw, G. L., Davis, B., Keeler, J., &amp; Fersht, A. R. (1995) Biochemistry, 34, 2225-2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to, somewhat reduce the N-terminal flexibility; likewise, the C-terminal of, rCMTI-V, being part of a beta-sheet, appears to be more rigid.
+The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., &amp; Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., &amp; Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I family--eglin c [Peng, J. W., &amp; Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., &amp; Fersht, A. R. (1995) Biochemistry 34, 2225-2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a beta-sheet, appears to be more rigid.
 ==About this Structure==
-MIT is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Cucurbita_maxima Cucurbita maxima]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1MIT OCA].
+MIT is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Cucurbita_maxima Cucurbita maxima]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1MIT OCA].
 ==Reference==
@@ Line 15: / Line 15: @@
 [[Category: Cai, M.]]
 [[Category: Gong, Y.]]
-[[Category: Huang, J.K.]]
+[[Category: Huang, J K.]]
 [[Category: Huang, Y.]]
 [[Category: Krishnamoorthi, R.]]
 [[Category: Liu, J.]]
 [[Category: Prakash, O.]]
-[[Category: Wen, J.J.]]
+[[Category: Wen, J J.]]
 [[Category: Wen, L.]]
 [[Category: serine protease inhibitor (rcmti-v)]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 21:26:28 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:55:38 2008''