http://192.168.1.40:8080/xmlui/handle/123456789/1129 Sun, 31 May 2026 10:19:40 GMT 2026-05-31T10:19:40Z http://192.168.1.40:80/xmlui/bitstream/id/de9b3436-9f0f-4968-80f8-8c514e2fa9b6/raja.jpg http://192.168.1.40:8080/xmlui/handle/123456789/1129 http://192.168.1.40:8080/xmlui/handle/123456789/2322 Improved Model of a LexA Repressor Dimer Bound to recA Operator Chattopadhyaya, Rajagopal; Pal, A. complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Delta(1-67) S119A, S119A, G85D and Delta(1-67) quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the 'cleavable' cleavage site region found in the crystal structures is actually the more relevant form of the region in wildtype LexA since it agrees with the value of the pre-exponential Arrhenius factor for its autocleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a 'conformational switch' inferred from the crystal structures is meaningless. DOI:10.1080/07391102.2004.10506959 Thu, 01 Apr 2004 00:00:00 GMT http://192.168.1.40:8080/xmlui/handle/123456789/2322 2004-04-01T00:00:00Z http://192.168.1.40:8080/xmlui/handle/123456789/2319 pH-dependent autocleavage of lambda repressor occurs in the operator-bound form: characterization of lambda repressor autocleavage Ghosh, K.; Pal, A.; Chattopadhyay, Rajagopal The first-order rate constants for the RecA-independent, spontaneous, pH-dependent autocleavage of the lambda cl repressor was measured in the present study at pH 10.6 at 27, 37 and 42 degreesC respectively. Autocleavage of the repressor Occurs also at pH 9 and 8, although at progressively slower rates. We demonstrate that the spontaneous autocleavage occurs also in the operator-bound state, at a rate either higher than or equal to the rate in solution, depending on the pH value. Owing to the near equality of the rate constant in both operator-free and operator-bound repressors, it can be inferred that the cleavage site has a similar structure and dynamics with respect to the catalytic site in both forms at neutral pH. Covalent modification using PMSF, brought about by a large molar excess of the reagent, inhibits autocleavage of the; repressor. The difficulty in obtaining this covalent modification is rationalized using our recent lambda repressor models. Bimolecular type II trans-cleavage was observed previously for mutant LexA repressors lacking a crucial catalytic serine or lysine residue [Kim and Little (1993) Cell (Cambridge, Mass.) 73, 1165-1173], but it could still be cleaved by an 85-202 'enzyme' fragment possessing an improved or hypercleavable character lacking its own cleavage site. Such a type II trans-cleavage was not observed with the covalently modified intact lambda repressor used as substrate and the purified wild-type lambda repressor 112-236 fragment used as the 'enzyme'. All these results show that for the wild-type lambda repressor, the catalytic site is close to the cleavage site in both operator-free and -bound states. In the lytic pathway, the repressor is mainly cleaved via RecA-mediated cleavage, which occurs much faster than the spontaneous auntocleavage; the possible biological significance of this slow, spontaneous, but constant, autocleavage is related to the lysogenic state, when RecA-mediated cleavage is absent. DOI: 10.1042/BJ20031834 Thu, 15 Apr 2004 00:00:00 GMT http://192.168.1.40:8080/xmlui/handle/123456789/2319 2004-04-15T00:00:00Z http://192.168.1.40:8080/xmlui/handle/123456789/2074 Three-dimensional models of NB-ARC domains of disease resistance proteins in tomato, arabidopsis, and flax Chattopadhyaya, Rajagopal; Pal, Amita Three dimensional models of NB-ARC domains in five different proteins were constructed based on the recently published crystal structure of the apoptotic protease activating factor 1, of which two are for tomato species, one each for flax, Arabidopsis, and nematode. Standard multiple sequence alignment was performed for chosen members of the NB-ARC domains, very divergent from each other in protein sequence, followed by homology model building and structure refinement. In this alignment, amino acid insertions and deletions between members generally fall in loop regions or at ends of alpha helices. Despite the presence of sequence divergence between the species, it is argued that the NB-ARC domains carry out the similar biological functions in the various species, highlighting the ATP binding and ATPase activity. By our comparative study of these models, it is predicted that NB-ARC domains should bind ADP/ATP rather than GDP/GTP. Both natural and induced mutants of Arabidopsis within the RPS2 locus and their phenotypes for disease reaction against Pseudomonas syringae are rationalized from the protein model. Apaf-1 Thr263 and Arg265 positions conserved totally within the NB-ARC domains are predicted to take active part in the catalytic activity of kinase-3 motif, the arginine known as the sensor I motif in AAA+ proteins. This was later verified for the Ced-4 crystal structure in complex with Ced-9. Our model of Ced-4 based on Apaf-1 was also compared with its crystal structure in the Ced-4-Ced-9 complex; the 3 layered alpha/beta domain superposes quite well, helical domain I is shifted by about 5 A but the winged helix domain is rotated away to a new position. Since Apaf-1 was crystallized with ADP and Ced-4-Ced9 with magnesium-ATP, this rotation signifies a change in structure of these NB-ARC domains between the two forms. Further, we hypothesize that certain mutants in the plant R proteins called 'constitutive gain-of-function' or 'autocatalytic' dispose their winged helix domains permanently like the magnesium-ATP form as observed for Ced-4, avoiding the closed ADP conformation. The models are also validated with mutagenesis data for a related tomato protein I-2, tomato prf and flax, including loss of function, wild type and autocatalytic phenotypes, and compared with similar data for potato and tobacco proteins, for which models were not built. These three dimensional models would help us to understand the spatial arrangement, function of R proteins and their conserved motifs. DOI:10.1080/07391102.2008.10507184 Fri, 01 Feb 2008 00:00:00 GMT http://192.168.1.40:8080/xmlui/handle/123456789/2074 2008-02-01T00:00:00Z http://192.168.1.40:8080/xmlui/handle/123456789/1928 Digestion of the lambda cI Repressor with Various Serine Proteases and Correlation with Its Three Dimensional Structure Pal, Atasi; Chattopadhyaya, Rajagopal Partial proteolysis of the lambda cl repressor has been carried Out systematically with trypsin. chymortrypsin, elastase, endoproteinase Glu-C, kallikrein, and thrombin. The cleavage sites have been determined by (i) comparison of fragments produced and observed in SDS-poly-acrylamide 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. These resistant regions show that Much of the region 93-131 previously called a 'linker' is actually part of the C-domain its first proposed in all models from our laboratory. 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 call be used to judge the best among three previously proposed models since they differ front each other in the structure of region 93-131. Model lj5g, is adjudged to be better than model 11wq (which is based oil 1kca. a crystal structure) as Susceptible residues are more exposed in the former and lack of cleavages at six sites is better explained. Likewise, the models lj5j and 11wq are compared with a recent crystal Structure of fragment 101-229 in 2ho0 and another low resolution crystal structure in 3bdn. DOI:10.1080/07391102.2008.10507249 Mon, 01 Dec 2008 00:00:00 GMT http://192.168.1.40:8080/xmlui/handle/123456789/1928 2008-12-01T00:00:00Z