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Molecular recognition of a protein by an antibody involves a relatively small area of the protein. It is a -local- recognition event. We propose to develop a - global- protein sensor through molecular imprinting and apply the imprinted membranes as protein - conformation sensors- as well as test their ability to act as artificial chaperones and direct protein conformational changes, such as inducing a beta-sheet to alpha-helix conformational transition using imprints formed against the alpha-helix conformer (and vice versa). The central hypothesis of this work is that a global protein recognition element (i.e. an imprint that is complementary to a given protein in terms of size, shape, and chemistry) will bind this protein with higher affinity than its conformationally altered counterpart or a different protein (native or denatured). Corollary to this is that an imprint formed against a native protein may catalyze refolding of its denatured counterpart, thus acting as an artificial chaperonin.
NON-TECHNICAL SUMMARY: This research is directly relevant to the food safety and agricultural and food security agricultural NRI programs. This work aims to construct protein conformational sensors through molecular imprinting approaches. <P>APPROACH: We propose to use bulk sol-gel imprinting and novel surface templating methods to form -plastic global antibodies- , in respective monolith and membrane geometries, with activities against several model proteins, including: beta-lactoglobulin A, lysozyme, carbonic anhydrase B (CAB), and albumin. We will characterize protein / imprint interactions and imprint distribution, stability, and specificity using an array of surface sensitive techniques, namely: scanning probe microscopy, total internal reflection fluorescence spectroscopy / microscopy, Fourier transform infrared spectroscopy, far UV circular dichroism, and near field scanning optical microscopy. In addition to the spectroscopy methods sensitive to protein conformation, the enzymatic activity of lysozyme and CAB will be used as conformation indicators. Molecular modeling will be employed to aid in the design and evaluation of the proposed global recognition elements.
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PROGRESS: 2005/07 TO 2008/12<BR>
OUTPUTS: The main goal of demonstrating recognition of protein conformation through molecular imprinting has been demonstrated for the model protein, beta-lactoglobulin. Molecular imprints of three isoforms of BLG have been formed in polymers of amino-phenyl boronic acid (APBA). Rebinding studies of the proteins to thin APBA films coupled to quartz crystal microbalance sensors indicate enhanced rebinding of the imprinted protein vs. conformationally altered challenger protein. This is highly significant as the isoforms have the same molecular weight and differ only in shape. Prior imprinting of biomacromolecules has generally been limited to discriminating among proteins having distinct molecular weights and/or isoelectric points. This collaborative work between Utah State University, The University of Utah, and Cranfield University, UK, has been reported at the Institute of Food Technologists annual conference, Orlando, FL, June 24-28, 2006,the 234th National American Chemical Society Meeting, Boston, MA, August 19-24, 2007, The Tenth World Congress on Biosensors, Shanghai, China, May 14-16, 2008. In addition to employing APBA as a protein imprinting platform, we have developed fluorosilane / TEOS sol-gel platforms, imprinted against BLG and BSA. Three fluorosilanes were employed: 3,3,3-trifluoropropoxymethoxysilane (3F), (3-heptafluoroisopropoxy)propalethoxysilane (7F), and pentafluorophenyltriethoxysilane (5F). A protocol for premixing protein with hydrolyzed silane monomers was developed. BLG was mixed with fluorosilane monomers in pH 2.5, 20mM HCl. These conditions slowed condensation allowing hydrolyzed fluorosilane monomers to bind to hydrophobic sites on BLG. The solution was mixed with TEOS at pH 6.5 to induce gelation. BLG conformational transitions were measured pre- and post gelation via shifts in BLG intrinsic fluorescence and through BLG interaction with 1-anilino-8-naphhalene sulfonic acid, a probe used to detect the molten-globule state of proteins. Intrinsic BLG fluorescence in presence of silane, fluoroalcohol. Hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) were used as benchmarks with which to compare fluorosilane induced conformational changes. 3F hydrolyzed monomers induced conformational changes at concentrations below HFIP and TFE. Once integrated into the silica gel, 3F doped at 20% (relative to TEOS) as well as 7F doped at 10%, showed a similar influence on protein structure. Above 20% both fluorosilanes led to significant loss of gel transparency and promoted protein aggregation. 5F was restricted as a functional monomer due to limited solubility in buffer. Our results demonstrate that 3F fluorosilane is more active than fluoroalcohols in inducing conformational changes in solution. Gelation of the 3F-BLG complex into a TEOS matrix preserves the induced conformation. This work has been presented at the 237th ACS National Meeting, Salt Lake City, Utah, March 22-26, 2009. <BR>PARTICIPANTS: Utah State University/University of Utah/Cranfield University, UK <BR>TARGET AUDIENCES: cattle industry, dairy industry, biotechnology industry
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IMPACT: 2005/07 TO 2008/12<BR>
The ability to detect misfolded isoforms of proteins is important as a diagnostic tool for -protein conformational diseases- such as scrapie, bovine spongiform encephalitis, and chronic wasting disease. Fundamental studies of protein imprinting in polymer matrices provides a means to form so called -plastic antibodies- that are robust, low-cost alternative to monoclonal antibodies.