The interaction of proteins with solid surfaces is a fundamental phenomenon with implications on nanotechnology, biomaterials and biotechnological processes. Although "most proteins interact with most surfaces", the particular strength, mechanism, and kinetics of each interaction has significant consequences in the final conformation of the adsorbed protein. The basis for the adsorption is generally provided by some combination of hydrophobic and electrostatic interactions. While generally recognized as the major contributor to the favorable free energy change driving the binding, hydrophobic interactions induce significant rearrangements and losses in biological activity in the adsorbed protein. Electrostatic interactions, though typically too weak to provide long-term stability, enabe preserving the protein structure and can be controlled by a variety of ways. Hence, we propose to 1) exploit electrostatic forces to control the adsorption process of proteins to electrode surfaces and 2) manipulate the activity of the adsorbed proteins by adjusting the potential applied to the surface. The hypothesis of this project is that by controlling the potential applied to the surface (electrode), it will be possible to affect the adsorption process (affinity, mechanism, and kinetics) and most importantly, the biological activity of the adsorbed proteins. Current evidence, though only sparsely reported and mostly qualitatively expressed, supports this hypothesis. Thus, the main goal of this project is to systematically demonstrate that (and understand how, why, and how fast) changes in electrode potential can affect the adsorption, orientation, conformation, activity, and stability of adsorbed proteins. For these studies, we have selected a group of proteins called ankyrins. These proteins display an unusually high stability and fully reversible spring-like behavior. Besides the fact that there are no reports related to adsorption behavior of these proteins, this proposal will determine the fundamental mechanism of surface potential in the adsorption and final conformation, which is crucial to bolster the rational development and application of sensors and nanodevices. Furthermore, understanding how ankyrins and other membrane proteins, interact with nanomaterials will form the basis for the development of a high-throughput model to study a broad range of pathologies linked to defective protein-protein interactions, such as hereditary spherocytosis, spinocerebellar ataxia, cardiac arrhythmias, and a variety of channelopathies. The proposed project will also develop a novel surface-based method to study real-time binding of proteins in the presence of pharmaceutical compounds that would otherwise interfere with the association of other proteins in cells.
This project propose to generate a unique pool of proteins (ankyrins) with similar structure but different characteristics and use them to investigate the rol of surface potential on the structure and activity of such adsorbed proteins. It is expected that the findings of this project will enable the rational design of biomedical devices, in which the structure of the adsorbed protein defines the clinical condition of a patient.
|Bhakta, Samir A; Benavidez, Tomas E; Garcia, Carlos D (2014) Immobilization of glucose oxidase to nanostructured films of polystyrene-block-poly(2-vinylpyridine). J Colloid Interface Sci 430:351-6|
|Benavidez, Tomás E; Torrente, Daniel; Marucho, Marcelo et al. (2014) Adsorption and catalytic activity of glucose oxidase accumulated on OTCE upon the application of external potential. J Colloid Interface Sci 435:164-70|
|Evans, Elizabeth; Gabriel, Ellen Flávia Moreira; Benavidez, Tomás E et al. (2014) Modification of microfluidic paper-based devices with silica nanoparticles. Analyst 139:5560-7|
|Moreira Gabriel, Ellen Flávia; Tomazelli Coltro, Wendell Karlos; Garcia, Carlos D (2014) Fast and versatile fabrication of PMMA microchip electrophoretic devices by laser engraving. Electrophoresis 35:2325-32|
|Benavidez, Tomas E; Garcia, Carlos D (2013) Potential-assisted adsorption of bovine serum albumin onto optically transparent carbon electrodes. Langmuir 29:14154-62|
|Felhofer, Jessica L; Scida, Karen; Penick, Mark et al. (2013) Simultaneous solid phase extraction and derivatization of aliphatic primary amines prior to separation and UV-absorbance detection. Talanta 115:688-93|
|Segato, Thiago P; Bhakta, Samir A; Gordon, Matthew et al. (2013) Microfab-less Microfluidic Capillary Electrophoresis Devices. Anal Methods 5:1652-1657|
|Benavidez, Tomas E; Garcia, Carlos D (2013) Spectroscopic and electrochemical characterization of nanostructured optically transparent carbon electrodes. Electrophoresis 34:1998-2006|
|Alharthi, Sarah A; Benavidez, Tomas E; Garcia, Carlos D (2013) Ultrathin optically transparent carbon electrodes produced from layers of adsorbed proteins. Langmuir 29:3320-7|
|Scida, Karen; Stege, Patricia W; Haby, Gabrielle et al. (2011) Recent applications of carbon-based nanomaterials in analytical chemistry: critical review. Anal Chim Acta 691:6-17|
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