5hir: Difference between revisions

Revision as of 20:15, 21 February 2008

SOLUTION STRUCTURE OF RECOMBINANT HIRUDIN AND THE LYS-47 (RIGHT ARROW) GLU MUTANT. A NUCLEAR MAGNETIC RESONANCE AND HYBRID DISTANCE GEOMETRY-DYNAMICAL SIMULATED ANNEALING STUDY

OverviewOverview

The solution structure of recombinant wild-type hirudin and of the putative active site mutant Lys-47----Glu has been investigated by nuclear magnetic resonance (NMR) spectroscopy at 600 MHz. The 1H NMR spectra of the two hirudin variants are assigned in a sequential manner with a combination of two-dimensional NMR techniques. Some assignments made in our previous paper [Sukumaran, D. K., Clore, G. M., Preuss, A., Zarbock, J., & Gronenborn, A. M. (1987) Biochemistry 26, 333-338] were found to be incorrect and are now corrected. Analysis of the NOE data indicates that hirudin consists of an N-terminal compact domain (residues 1-49) held together by three disulfide linkages and a disordered C-terminal tail (residues 50-65) which does not fold back on the rest of the protein. This last observation corrects conclusions drawn by us previously on hirudin extracted from its natural source, the leech Hirudo medicinalis. The improved sensitivity of the 600-MHz spectrometer relative to that of our old 500-MHz spectrometer, the availability of two variants with slightly different chemical shifts, and the additional information arising from stereospecific assignments of methylene beta-protons and methyl protons of valine have permitted the determination of the solution structure of hirudin with much greater precision than before. Structure calculations on the N-terminal domain using the hybrid distance geometry-dynamical simulated annealing method were based on 685 and 661 approximate interproton distance restraints derived from nuclear Overhauser enhancement (NOE) data for the wild-type and mutant hirudin, respectively, together with 16 distance restraints for 8 backbone hydrogen bonds identified on the basis of NOE and amide NH exchange data and 26 phi backbone and 18 chi 1 side-chain torsion angle restraints derived from NOE and three-bond coupling constant data. A total of 32 structures were computed for both the wild-type and mutant hirudin. The structure of residues 2-30 and 37-48 which form the core of the N-terminal domain is well determined in both cases with an average atomic rms difference between the individual structures and the respective mean structures of approximately 0.7 A for the backbone atoms and approximately 1 A for all atoms. As found previously, the orientation of the exposed finger of antiparallel beta-sheet (residues 31-36) with respect to the core could not be determined on the basis of the present data due to the absence of any long-range NOEs between the exposed finger and the core.(ABSTRACT TRUNCATED AT 250 WORDS)

About this StructureAbout this Structure

5HIR is a Single protein structure of sequence from Hirudo medicinalis. Full crystallographic information is available from OCA.

ReferenceReference

Solution structure of recombinant hirudin and the Lys-47----Glu mutant: a nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study., Folkers PJ, Clore GM, Driscoll PC, Dodt J, Kohler S, Gronenborn AM, Biochemistry. 1989 Mar 21;28(6):2601-17. PMID:2567183

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-[[Image:5hir.gif|left|200px]]<br /><applet load="5hir" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:5hir.gif|left|200px]]<br /><applet load="5hir" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="5hir" />
 '''SOLUTION STRUCTURE OF RECOMBINANT HIRUDIN AND THE LYS-47 (RIGHT ARROW) GLU MUTANT. A NUCLEAR MAGNETIC RESONANCE AND HYBRID DISTANCE GEOMETRY-DYNAMICAL SIMULATED ANNEALING STUDY'''<br />
 ==Overview==
-The solution structure of recombinant wild-type hirudin and of the, putative active site mutant Lys-47----Glu has been investigated by nuclear, magnetic resonance (NMR) spectroscopy at 600 MHz. The 1H NMR spectra of, the two hirudin variants are assigned in a sequential manner with a, combination of two-dimensional NMR techniques. Some assignments made in, our previous paper [Sukumaran, D. K., Clore, G. M., Preuss, A., Zarbock, J., &amp; Gronenborn, A. M. (1987) Biochemistry 26, 333-338] were found to be, incorrect and are now corrected. Analysis of the NOE data indicates that, hirudin consists of an N-terminal compact domain (residues 1-49) held, together by three disulfide linkages and a disordered C-terminal tail, (residues 50-65) which does not fold back on the rest of the protein. This, last observation corrects conclusions drawn by us previously on hirudin, extracted from its natural source, the leech Hirudo medicinalis. The, improved sensitivity of the 600-MHz spectrometer relative to that of our, old 500-MHz spectrometer, the availability of two variants with slightly, different chemical shifts, and the additional information arising from, stereospecific assignments of methylene beta-protons and methyl protons of, valine have permitted the determination of the solution structure of, hirudin with much greater precision than before. Structure calculations on, the N-terminal domain using the hybrid distance geometry-dynamical, simulated annealing method were based on 685 and 661 approximate, interproton distance restraints derived from nuclear Overhauser, enhancement (NOE) data for the wild-type and mutant hirudin, respectively, together with 16 distance restraints for 8 backbone hydrogen bonds, identified on the basis of NOE and amide NH exchange data and 26 phi, backbone and 18 chi 1 side-chain torsion angle restraints derived from NOE, and three-bond coupling constant data. A total of 32 structures were, computed for both the wild-type and mutant hirudin. The structure of, residues 2-30 and 37-48 which form the core of the N-terminal domain is, well determined in both cases with an average atomic rms difference, between the individual structures and the respective mean structures of, approximately 0.7 A for the backbone atoms and approximately 1 A for all, atoms. As found previously, the orientation of the exposed finger of, antiparallel beta-sheet (residues 31-36) with respect to the core could, not be determined on the basis of the present data due to the absence of, any long-range NOEs between the exposed finger and the core.(ABSTRACT, TRUNCATED AT 250 WORDS)
+The solution structure of recombinant wild-type hirudin and of the putative active site mutant Lys-47----Glu has been investigated by nuclear magnetic resonance (NMR) spectroscopy at 600 MHz. The 1H NMR spectra of the two hirudin variants are assigned in a sequential manner with a combination of two-dimensional NMR techniques. Some assignments made in our previous paper [Sukumaran, D. K., Clore, G. M., Preuss, A., Zarbock, J., &amp; Gronenborn, A. M. (1987) Biochemistry 26, 333-338] were found to be incorrect and are now corrected. Analysis of the NOE data indicates that hirudin consists of an N-terminal compact domain (residues 1-49) held together by three disulfide linkages and a disordered C-terminal tail (residues 50-65) which does not fold back on the rest of the protein. This last observation corrects conclusions drawn by us previously on hirudin extracted from its natural source, the leech Hirudo medicinalis. The improved sensitivity of the 600-MHz spectrometer relative to that of our old 500-MHz spectrometer, the availability of two variants with slightly different chemical shifts, and the additional information arising from stereospecific assignments of methylene beta-protons and methyl protons of valine have permitted the determination of the solution structure of hirudin with much greater precision than before. Structure calculations on the N-terminal domain using the hybrid distance geometry-dynamical simulated annealing method were based on 685 and 661 approximate interproton distance restraints derived from nuclear Overhauser enhancement (NOE) data for the wild-type and mutant hirudin, respectively, together with 16 distance restraints for 8 backbone hydrogen bonds identified on the basis of NOE and amide NH exchange data and 26 phi backbone and 18 chi 1 side-chain torsion angle restraints derived from NOE and three-bond coupling constant data. A total of 32 structures were computed for both the wild-type and mutant hirudin. The structure of residues 2-30 and 37-48 which form the core of the N-terminal domain is well determined in both cases with an average atomic rms difference between the individual structures and the respective mean structures of approximately 0.7 A for the backbone atoms and approximately 1 A for all atoms. As found previously, the orientation of the exposed finger of antiparallel beta-sheet (residues 31-36) with respect to the core could not be determined on the basis of the present data due to the absence of any long-range NOEs between the exposed finger and the core.(ABSTRACT TRUNCATED AT 250 WORDS)
 ==About this Structure==
-HIR is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Hirudo_medicinalis Hirudo medicinalis]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=5HIR OCA].
+HIR is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Hirudo_medicinalis Hirudo medicinalis]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5HIR OCA].
 ==Reference==
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 [[Category: Hirudo medicinalis]]
 [[Category: Single protein]]
-[[Category: Clore, G.M.]]
+[[Category: Clore, G M.]]
-[[Category: Gronenborn, A.M.]]
+[[Category: Gronenborn, A M.]]
 [[Category: coagulation inhibitor]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 19:33:29 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 19:15:21 2008''