Harikrishna Pillai, Brijesh Singh Yadav, Navaneet Chaturvedi, Arif Tasleem Jan, Girish Kumar Gupta, Mohammad Hassan Baig and Sanjeev Kumar Bhure Pages 186 - 192 ( 7 )
Background: Regucalcin (RGN), a calcium regulating protein having anti-prolific, antiapoptotic functions, plays important part in the biosynthesis of ascorbic acid. It is a highly conserved protein that has been reported from many tissue types of various vertebrate species. Employing its effect of regulating enzyme activities through reaction with sulfhydryl group (-SH) and calcium, structural level study believed to offer a better understanding of binding properties and regulatory mechanisms of RGN, was performed.
Material and method: Using sample from testis of Bubalus bubalis, amplification of regucalcin (RGN) gene was subjected to characterization by performing digestion using different restriction endonucleases (RE). Alongside, cDNA was cloned into pPICZαC vector and transformed in DH5α host for custom sequencing. To get a better insight of its structural characteristics, three dimensional (3D) structure of protein sequence was generated using in silico molecular modelling approach. The full trajectory analysis of structure was achieved by the Molecular Dynamics (MD) that explains the stability, flexibility and robustness of protein during simulation in a time of 50ns. Molecular docking against 1,5-anhydrosorbitol was performed for functional characterization of RGN.
Results: Preliminary screening of amplified products on Agarose gel showed expected size of ~893 bp of PCR product corresponding to RGN. Following sequencing, BLASTp search of the target sequence revealed that it shares 91% similarity score with human senescence marker protein-30 (pdb id: 3G4E). Molecular docking of 1,5-anhydrosorbitol reveals information regarding important binding site residues of RGN. 1,5-anhydrosorbitol was found to interact with binding free energy of - 6.01 Kcal/mol. RMSD calculation of subunits A, B and D-F might be responsible for functional and conserved regions of modeled protein.
Conclusion: Three dimensional structure of RGN was generated and its interactions with 1,5- anhydrosorbitol, demonstrates the role of key binding residues. Until now, no structural details were available for buffalo RGN proteins, hence this study will broaden the horizon towards understanding the structural and functional aspects of different proteins in cattle.
PCR amplification, cloning, Regucalcin (RGN) Protein, Molecular Dynamics (MD), simulation, Bubalus bubalis.
Division of Biochemistry, Indian Veterinary Research Institute, Izatnaga, Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Telaviv-Yafo, Department of Biochemistry & Molecular Biology, Tel Aviv University, Telaviv-Yafo, Department of Medical Biotechnology, Yeungnam University, Gyeongsan, Gyeongsangbukdo, Department of Pharmaceutical Chemistry, Maharishi Markandeshwar College of Pharmacy, Maharishi Markandeshwar University, Mullana, Ambala-133207, Haryana, Department of Medical Biotechnology, Yeungnam University, Gyeongsan, Gyeongsangbukdo, Republic of, Division of Biochemistry, Indian Veterinary Research Institute, Izatnaga