Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2011 Fluoro-Silane as a Functional Monomer for Protein Conformational Imprinting Yun Peng Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Peng, Yun, "Fluoro-Silane as a Functional Monomer for Protein Conformational Imprinting" (2011). All Graduate Theses and Dissertations. 906. https://digitalcommons.usu.edu/etd/906 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. FLUORO-SILANE AS A FUNCTIONAL MONOMER FOR PROTEIN CONFORMATIONAL IMPRINTING by Yun Peng A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Engineering Approved: David W. Britt, Ph.D. Timothy E. Doyle, Ph.D. Major Professor Committee Member Marie K. Walsh, Ph.D. Soonjo Kwon, Ph.D. Committee Member Committee Member Jixun Zhan, Ph.D. Byron Burnham Committee Member Dean of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2011 ii Copyright © Yun Peng 2011 All Rights Reserved iii ABSTRACT Fluoro-silane as a Functional Monomer for Protein Conformational Imprinting by Yun Peng, Doctor of Philosophy Utah State University, 2011 Major Professor: Dr. David W. Britt Department: Biological Engineering By using the technology of molecularly imprinted polymer (MIP), we propose to synthesize a protein conformational imprint that also acts as a plastic enzyme, inducing protein structural transitions. The imprint aims at MIP-induced stabilization and / or formation of bound protein secondary structure and the applications associated with analysis and correction of misfolded proteins. The screening of polymeric functional monomers being able to induce the conformational transitions in proteins is investigated in this report. The fluoro-silanes (3-heptafluoroisopropoxy)propalethoxysilane (7F) and 3,3,3-trifluoropropylmethoxysilane (3F) were employed as functional monomers for synthesis of this catalytic protein conformational imprint via sol-gel reactions. 3F was demonstrated superior to 7F for fluoro-modification of tetraethylorthosilicate (TEOS) gel in terms of retaining gel transparency and increasing hydrophobicity while maintaining a uniform distribution of encapsulated protein. Both hydrolyzed 3F and polymerized 3F exhibited strong influences on structure transitions of three template proteins: bovine iv serum albumin (BSA), beta-lactoglobulin (BLG), and bovine carbonic anhydrase (BCA). The formation of molten globule intermediates that stabilized by increased alpha-helices was induced by the trifluoro-silane in BLG and BCA. Additionally, 3F was effective at a lower concentration than the benchmark fluoro-alcohol 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), validating the application of 3F as a functional monomer for protein conformational imprinting. (213 pages) v – 以此献给我的母亲及爱我的家人 – vi ACKNOWLEDGMENTS I would like to offer my sincerest gratitude to Dr. David W. Britt, who gave me invaluable guidance and advice through my entire graduate research period. I would also like to thank all of the members of my dissertation committee, Dr. Timothy E. Doyle, Dr. Marie K. Walsh, Dr. Soonjo Kwon, and Dr. Jixun Zhan, for their professional opinions and lab support. Special thanks are given to Dr. Kwon for his long-term support in the lab and to Dr. Walsh for her kindly help on the enzyme activity assays. Great thanks are given to Dr. Timothy A. Taylor for his constantly standing by me and willingness to help whenever needed. Also, I would like to acknowledge Russell and Hui Apple for giving me helpful comments. You are thoughtful friends. Finally, I would like to dedicate this work to my family. To my husband Ang Shen, I thank you for always being there for me. To my lovely daughters Li and Zi, I thank you for bringing me happiness every day; you are my angels. To my sister Tao Peng and my brother Zhang hui Peng, I thank you for your understanding and encouragement every step of the way. Most importantly, to my mother, Yue ying Wang, I thank you for your endless love. This work could not have been accomplished without your unconditional and unending help through all these years. Yun Peng vii CONTENTS Page ABSTRACT ....................................................................................................................... iii DEDICATION .....................................................................................................................v ACKNOWLEDGMENTS ................................................................................................. vi LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES .......................................................................................................... xii LIST OF SCHEMES ........................................................................................................ xvi ABBREVIATIONS ......................................................................................................... xvii CHAPTER 1. INTRODUCTION ................................................................................................1 PROTEIN CONFORMATIONAL IMPRINTING ...............................................1 HYPOTHESIS AND OBJECTIVES ....................................................................5 DISSERTATION OUTLINE .................................................................................7 REFERENCES .....................................................................................................9 2. LITERATURE REVIEW .................................................................................... 11 DEVELOPMENT OF MOLECULARLY IMPRINTED POLYMERS ............................................................................................... 11 MOLECULARLY IMPRINTED POLYMERS SYNTHESIS STRATEGIES ............................................................................................12 PROTEIN IMPRINTING: TOWARD ARTIFICIAL ANTIBODIES / ENZYMES ......................................................................15 POLYMERS FOR PROTEIN SELECTIVE MOLECULAR IMPRINTING ............................................................................................16 Acrylamide-based MIPs.......................................................................16 Stimuli-sensitive hydrogel ...................................................................19 Sol-gel ..................................................................................................22 3-Aminophenylboronic acid (APBA) based MIPs ..............................25 FUNCTIONAL MONOMERS FOR NON-COVALENT MIP ..........................27 Functional monomer-template interaction ...........................................27 Commonly used functional monomers ................................................28 Hydrophobic interaction in monomer-template complex ....................30 Optimization of pre-polymerization complex: ratios of functional monomer/cross-linker and of functional viii monomer/template ........................................................................31 Functional monomer selection strategy ...............................................33 PROTEIN SELECTIVE IMPRINTING APPLICATIONS ................................35 Analytical separation ...........................................................................35 Biosensors ............................................................................................38 FLUORO-ALCOHOL-PROTEIN INTERACTION: MOTIVATION FOR APPLYING FLUORO-SILANE AS FUNCTIONAL MONOMER ....................................................................41 Fluoro-alcohol-induced protein structure transition ............................41 Mechanisms for fluoro-alcohol-induced protein structural transitions .....................................................................46 From fluoro-alcohol to fluoro-silane ...................................................50 REFERENCES ...................................................................................................50 3. BOVINE SERUM ALBUMIN ENCAPSULATION AND CONFORMATION IN FLUORO-MODIFIED SILICA GELS .........................68 ABSTRACT ........................................................................................................68 INTRODUCTION ..............................................................................................69 EXPERIMENTAL ..............................................................................................71 Materials ..............................................................................................71 Gel preparation and optimization .........................................................72 Fluorescence microscopy .....................................................................73 Fluorescence spectroscopy ...................................................................74 Gel surface hydrophobicity ..................................................................75 RESULTS AND DISCUSSION ..........................................................................76 TEOS sol-gel synthesis ........................................................................76