Browsing by Subject "Protein conformation"
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Item Novel insights into macromolecularly imprinted polymers for the specific recognition of protein biomarkers(2011-08) Kryscio, David Richard; Peppas, Nicholas A., 1948-; Hilt, James Z.; Roy, Krishnendu; Maynard, Jennifer A.; Ellison, Christopher J.Bulk imprinted polymers were synthesized using traditional small molecular weight imprinting techniques for the recognition of bovine serum albumin (BSA). Reproducibility and capacity concerns prompted the use of circular dichroism to investigate the potential effects that conditions commonly employed have on the structure of the protein prior to polymerization. These studies clearly showed a substantial change in the secondary structure of three common model protein templates when in the presence of various monomers and crosslinkers. Molecular docking was used to further examine the interactions taking place at the molecular level. Docking simulations revealed that significant amounts of non-covalent interactions are occurring between the amino acid side chains and ligands; although, the interactions taking place amongst the analyte and polypeptide backbone are responsible for the experimentally observed conformational change. The computational studies also showed that several of the ligands preferentially ‘docked’ to the same amino acids in the protein, indicating that if multiple monomers are employed, this competition for similar binding sites will potentially result in non-specific recognition. These findings are important as they offer insight into the fundamental reasons why recognition of macromolecular templates has proven difficult as well as provide guidance for future success in the field. Using this information, novel surface imprinted polymers were synthesized via a facile technique for the specific recognition of BSA. Thin films based on 2-(dimethylamino)ethyl methacrylate (DMAEMA) as the functional monomer and varying amounts of either N,N’ methylenebisacrylamide (MBA) or poly(ethylene glycol) (400) dimethacrylate (PEG400DMA) as crosslinker were synthesized via UV free-radical polymerization. A clear and reproducible increase in recognition of the template was demonstrated for these systems as 1.6-2.5 times more BSA was recognized by the MIP sample relative to the control polymers. Additionally, these polymers exhibited specific recognition of the template relative to similar competitor proteins with up to 2.9 times more BSA adsorbed than either glucose oxidase or bovine hemoglobin. These synthetic antibody mimics hold significant promise as the next generation of robust recognition elements in a wide range of bioassay and biosensor applications.Item The RING finger binding protein is a nuclear membrane protein that interacts with RUSH transcription factors(Texas Tech University, 2001-05) Mansharamani, Malini; Chilton, Beverly S.; Lee, Vaughan H.; Pfarr, Curt M.; Pressley, Thomas A.; Webster, Daniel R.; Whelley, Sandra M.Molecular regulation of Uteroglobin gene expression by progesterone and prolactin is mediated by RUSH transcription factors. The RUSH family of proteins, which includes rabbit RUSH-la and P and the human, mouse and plant homologs of RUSH lα, are SWI/SNF related chromatin remodeling proteins. These proteins have a novel C3HC4 RING finger, at their -COOH terminus that has been implicated in mediating protein-protein interactions. When this motif was identified in RUSH proteins, it was used to screen an expression library to isolate cDNAs for proteins that complex with it. A single phage clone (~1.6kb insert) was identified. Sequence analysis of this RING Finger Binding Protein (RFBP) clone, revealed a partial cDNA that lacked an initiator codon but contained a stop codon. RACE PCR was then used to extend the 5' and 3' ends of the cDNA. The predicted amino acid sequence from the composite cDNA sequence (4286-bp) is that of a putative Type IV P-type ATPase. P-type ATPases are membrane transporters that use the energy of ATP hydrolysis to transport substrate across the membrane. Genomic cloning and ClustalW alignment indicate that RFBP is an atypical P-type ATPase that has only seven of eight core regions and nine of ten transmembrane domains typical of this family of proteins. Core region D that contains transmembrane domain four is absent from this protein. Western blot analysis, coupled with immunoelectron microscopy data, indicates that RFBP is present in the inner nuclear membrane. Coimmunoprecipitation and GST pulldown experiments showed a direct interaction between RUSH and RFBP. The RUSH binding site lies within aa 612-804 of the RFBP protein. Competitive quantitative RT-PCR indicates that RFBP is ubiquitous in its expression, with the expression pattern correlating with that of RUSH in these same tissues. In addition, expression of RFBP is hormonally regulated in the endometrium, suggesting that RFBP function and expression may be closely linked with the function of the RUSH proteins in regulating gene expression in the reproductive system. Current studies provide important information about RFBP as a RUSH binding interaction between the proteins to shed light on the mechanism of hormone regulation of uteroglobin gene expression.