ISSN:
1573-5001
Schlagwort(e):
Collagenase
;
Assignments
;
Structure
;
MMP
;
3D NMR
;
Matrix metalloproteinase
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
,
Chemie und Pharmazie
Notizen:
Abstract We report here the backbone 1HN, 15N, 13Cα, 13CO, and 1Hα NMR assignmentsfor the catalytic domain of human fibroblast collagenase (HFC). Three independentassignment pathways (matching 1H, 13Cα, and 13CO resonances) were used to establishsequential connections. The connections using 13Cα resonances were obtained fromHNCOCA and HNCA experiments; 13CO connections were obtained from HNCO andHNCACO experiments. The sequential proton assignment pathway was established from a 3D(1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement withrecent X-ray structures of human fibroblast collagenase and human neutrophil collagenase aswell as similar to recent NMR structures of a highly homologous protein, stromelysin. Allthree helices were located; a five-stranded β-sheet (four parallel strands, one antiparallelstrand) was also determined. β-Sheet regions were identified by cross-stranddαN and dNN connections and by strong intraresidue dαN correlations, and were corroborated byobserving slow amide proton exchange. Chemical shift changes in a selectively 15N-labeledsample suggest that substantial structural changes occur in the active site cleft on the bindingof an inhibitor.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1023/A:1018615400129
Permalink