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Biophysical and Biochemical Characterization of Protein D/E: A Putative Glycoprotein Involved in Sperm/Egg Binding and Fusion
Author(s) -
Copeland Clinton L.,
Socolovsky Eduardo A,
Ramesh Vani,
Ramesh Govindarajan T,
Hall Joseph C
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1001.6
Subject(s) - fusion protein , circular dichroism , protein sequencing , in silico , sperm , protein purification , biology , biochemistry , computational biology , peptide sequence , chemistry , biophysics , genetics , gene , recombinant dna
The purpose of the present investigation was to develop a more efficient and rapid method for purifying protein D/E, also known as Cysteine‐rich secretory protein‐1 (CRISP‐1) from rat epididymal tissue. While there is ample experimental evidence in the literature which demonstrate the involvement of protein D/E in sperm/egg binding and/or membrane‐mediated fusion events, biophysical characterization and x‐ray crystallographic analysis of protein D/E has not been reported. Protein D/E was purified to apparent homogeneity, as assessed by one‐and‐two dimensional gel electrophoresis, in three conventional chromatographic steps, with a percent recovery of 0.5%. Preliminary biophysical characterization of protein D/E using circular dichroism spectroscopy suggests the presence of significant α‐helical content, suggesting that there may be functional binding motifs in the protein structure. Computer‐assisted drug design modeling of protein D/E structure was initiated using computational tools to identify potential structural binding motifs. Using the protein D/E amino acid sequence that was deduced from the gene sequence and computational model tools (Fugue and Biopolymer), potential protein binding motifs were identified on the protein surface. These data will be used to optimize egg/ligand interactions, synthesize and test ligands as potential inhibitors of sperm/egg binding and membrane‐mediated fusion events. [ This study was supported, in part, by an NIH grant (P20 MS001822‐02) awarded to Dr. Joseph C. Hall, Director of the Center for Biotechnology and Biomedical Sciences and Vice President for Research and Economic Development, Norfolk State University, 700 Park Avenue, Norfolk, VA 23504 ]