Adsorption-Induced Conformational Changes in Fibronectin Due to Interactions with Well-Defined Surface Chemistries Kristin E. Michael, †,‡ Varadraj N. Vernekar, § Benjamin G. Keselowsky, ‡,| J. Carson Meredith, ⊥ Robert A. Latour, § and Andre ´s J. Garcı ´a* ,†,‡ Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, Department of Bioengineering, Clemson University, Clemson, South Carolina 29634, Coulter School of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, and School of Chemical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332 Received May 12, 2003. In Final Form: June 30, 2003 Protein adsorption onto synthetic materials influences cell adhesion and signaling events that direct cell function in numerous biomedical applications. Adsorption of fibronectin (FN) to different surfaces alters protein structure and modulates R51 integrin binding, cell adhesion, cell spreading, and cell migration. In the present study, self-assembled monolayers of alkanethiols on Au were used to analyze the effects of surface chemistry (CH 3, OH, NH2, and COOH) on the adsorption of a recombinant fragment of FN, FNIII7-10, that incorporates both the synergy and RGD cell binding motifs. Surface chemistry potentiated differential FNIII7-10 adsorption kinetics and adsorbed structure as determined by surface plasmon resonance spectroscopy and antibody binding assays. FNIII7-10 functional activity, determined by cell adhesion strength, was modulated in a fashion consistent with these structural changes (OH ) NH2 > COOH > CH3). However, these changes in protein parameters did not correlate simply to differences in surface hydrophobicity, indicating that additional surface parameters influence protein adsorption. These results demonstrate that surface chemistry modulates adsorbed protein structure and activity and establish a relationship between surface-dependent changes in structural domains of FNIII 7-10 and functional activity. Introduction Protein adsorption plays a critical role in numerous biomedical and biotechnological applications. Adsorption of proteins onto synthetic surfaces is a thermodynamically driven process. 1 Due to the diverse circumstances in which proteins and surfaces come in contact, an understanding of protein adsorption is fundamental to fields as varied as bioseparation, development of biosensors, food process- ing, and implant technology. 1,2 In addition to activating blood clotting and inflammatory responses, adsorbed proteins mediate cell adhesion to synthetic surfaces. Cell adhesion to adsorbed proteins is particularly important in cell function, host responses to implants, and design of tissue engineering substrates. 3-5 Protein adsorption is a complex, dynamic process involving noncovalent interactions, including hydrophobic interactions, electrostatic forces, hydrogen bonding, and van der Waals forces. 1 Protein parameters including primary structure, size, and structural stability as well as surface properties such as surface energy, roughness, and chemistry have been identified as key factors influ- encing the adsorption process. 6-9 In particular, surface chemistry influences adsorbed protein type, quantity, and conformation. 10-12 For example, adsorption of the extra- cellular matrix protein fibronectin (FN) on different surfaces alters protein structure and modulates cell adhesion, spreading, and migration. 13-16 Although these adsorption studies provide insights into the relationship * Corresponding author. Address: Woodruff School of Mechanical Engineering, 315 Ferst Drive, Room 2314 IBB, Atlanta, GA 30332- 0363. E-mail: andres.garcia@me.gatech.edu. Phone: 404-894-9384. Fax: 404-385-1397. † Woodruff School of Mechanical Engineering, Georgia Institute of Technology. ‡ Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology. § Department of Bioengineering, Clemson University. | Coulter School of Biomedical Engineering, Georgia Institute of Technology. ⊥ School of Chemical Engineering, Georgia Institute of Technol- ogy. (1) Andrade, J. D.; Hlady, V. Adv. Polym. Sci. 1986, 79,1-63. (2) Brash, J. L.; Horbett, T. A. In Proteins at Interfaces II: Funda- mentals and Applications; Horbett, T. A., Brash, J. L., Eds.; ACS Symposium Series No. 602; American Chemical Society: Washington, DC, 1995; pp 1-25. (3) Park, P. K.; Jarrell, B. E.; Williams, S. K.; Carter, T. L.; Rose, D. G.; Martinez-Hernandez, A.; Carabasi, R. A., III J. Vasc. Surg. 1990, 11, 468-475. 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