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Book of Abstracts: Albany 2009

category image Albany 2009
Conversation 16
June 16-20 2009
© Adenine Press (2008)

Digestion of the λ cI Repressor with Various Serine Proteases and Correlation with its Three Dimensional Structure

Partial proteolysis of the λ cI repressor has been carried out systematically with trypsin, chymotrypsin, elastase, endoproteinase Glu-C, kallikrein and thrombin. The cleavage sites have been determined by (i) comparison of fragments produced and observed in SDS-polyacrylamide gel with known fragments and plots of distance migrated versus log (molecular weight of fragment), (ii) partial Edman sequencing of the stable C-terminal fragments to identify cleavage points, and (iii) electrospray mass spectrometry of fragments produced. Most cleavage points are found to occur in the region 86-137, saving some in the N-terminal domain observed for trypsin and Glu-C. Region 86-137 can be further subdivided into three regions 86-91, 114-121 and 128-137 prone to cleavage, with intermediate regions resistant to cleavage to all six proteases (1). These resistant regions show that much of the region 93-131 previously called a ?linker? is actually part of the C-domain as first proposed in all models from our laboratory (4). Region 92-114 includes the cleavage site Ala-Gly, which must be buried in the intact repressor. The observed cleavage points in region 114-137 can be used to judge the best among three previously proposed models (4) since they differ from each other in the structure of region 93-131. Model 1j5g is adjudged to be better than model 1lwq (which is based on 1kca, a crystal structure) as susceptible residues are more exposed in the former and lack of cleavages at six sites is better explained (1). Likewise, the models 1j5g and 1lwq are compared with a recent crystal structure of fragment 101-229 in 2ho0 (5) and another low resolution crystal structure in 3bdn (6).

References and Footnotes
  1. A. Pal, R. Chattopadhyaya, J. Biol. Str. Dyn. 26, 339-354 (2008).
  2. L. J. Beamer, C. O. Pabo, J. Mol. Biol. 227, 177-196 (1992).
  3. C. E. Bell, P. Frescura, A. Hochschild, M. Lewis. Cell 101, 801-811 (2000).
  4. R. Chattopadhyaya, K. Ghosh. J. Struct. Biol. 141, 103-114 (2003).
  5. D. Ndjonka, C. E. Bell. J. Mol. Biol. 362, 479-489 (2006).
  6. S. Staybrook et al. Nature 452, 1022-1026 (2008).

Atasi Pal
Rajagopal Chattopadhyaya

Department of Biochemistry
Bose Institute, P-1/12, C.I.T.
Scheme VII M, Calcutta 700054
India
raja@boseinst.ernet.in