DESIGN OF PROTEIN LINKERS FOR THE CONTROLLED ASSEMBLY OF NANOPARTICLES CHEN HAIBIN NATIONAL UNIVERSITY OF SINGAPORE 2009 DESIGN OF PROTEIN LINKERS FOR THE CONTROLLED ASSEMBLY OF NANOPARTICLES CHEN HAIBIN (B. ENG, XI’AN JIAOTONG UNIVERSITY, P. R. CHINA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS The pursuit of my doctoral study was full of painstaking effort, enormous care and constant encouragements from many people to whom I would like to sincerely express my greatest gratitude. Above all, I thank my supervisors, Dr. Choe Woo-Seok, Dr. Su Xiaodi and Prof. Neoh Koon-Gee, for their untiring guidance and inexhaustible patience throughout the course of my Ph.D. research work. Their rigorous research attitude and constructive criticism have helped me shape the research direction and attain the present achievement. The great experience to work with them will definitely benefit my future career. I am very grateful to all my colleagues and the staff in the Department of Chemical and Biomolecular Engineering. Special thanks are given to Mr. Nian Rui, Miss Tan Lihan, Mr. Ong Jeong Shing, Dr. Ang Ee Lui, Mr. Li Jianguo, Dr. Xiong Junying who have given direct help and support to my Ph.D. research work. And I also would like to express my sincere thanks to Miss Lee Chai Keng, Mr. Han Guangjun, Mr. Boey Kok Hong, Ms. Fam Hwee Koong, Ms. Li Xiang, and Ms. Li Fengmei for their professional technical services and laboratory management. My doctoral study would not have been accomplished without the encouragements and care from my friends in Singapore. They are too many to be listed here, but I deeply thank my girlfriend, Miss Ren Xinsheng, who brings so much light to my life. I must also appreciate the research scholarship provided by National University of Singapore and the opportunity to work in Dr. Su’s group in the Institute of Materials Research and Engineering. This thesis is dedicated to my parents, for their endless love! I TABLE OF CONTENTS ACKNOWLEDGEMENTS I TABLE OF CONTENTS II SUMMARY VII LIST OF ABBREVIATIONS . VIII LIST OF AMINO ACIDS, THEIR ABBREVIATIONS AND STRCTURES X LIST OF TABLES XI LIST OF FIGURES . XII CHAPTER INTRODUCTION 1.1 Background . 1.2 Objectives and scope . 1.3 Outline of the thesis CHAPTER LITERATURE REVIEW 2.1 Harnessing biomolecules for assembly of inorganic nanoparticles 11 2.2 Functionalization of nanoparticles using biomolecules 13 2.3 Combinatorial approaches in search of inorganic-binding peptides . 17 2.4 Mechanism of peptide binding to target inorganic materials 23 2.5 LacI-lacO conjugate as a logic switch 25 2.6 Exploring the interaction between biomolecules and inorganic surfaces II using QCM-D . 28 CHAPTER SELECTION OF PEPTIDES WITH SPECIFIC BINDING AFFINITY TO SIO2 AND TIO2 NANOPARTICLES, AND QCM-D ANALYSIS OF BINDING MECHANISM 3.1 Introduction . 31 3.2 Experimental Section 33 3.2.1 Isolation of inorganic-binding peptides 33 3.2.2 Phage binding assay 36 3.2.3 Zeta potential measurement of surface charge of TiO2 and SiO2 NPs 36 3.2.4 QCM-D measurements . 36 3.2.5 AFM characterization 38 3.3 Results and Discussion . 38 3.3.1 Peptide isolation 38 3.3.2 Deduction of binding mechanism based on pH-dependent surface charges of metal oxide NPs and STB1 41 3.3.3 QCM-D and AFM measurements show that binding of STB1-P to SiO2 and TiO2 is mediated by STB1 . 44 3.3.4 Further verification of binding mechanism at extreme pH and PZCs of SiO2 and TiO2 55 3.4 Summary . 59 CHAPTER PROBING THE INTERACTION BETWEEN PEPTIDES AND III METAL OXIDES USING POINT MUTANTS OF STB1 4.1 Introduction . 60 4.2 Experimental Section 63 4.2.1 Oligonucleotide-directed mutagenesis of M13 phage DNA . 63 4.2.2 QCM-D measurement . 64 4.2.3 Molecular dynamics simulation 65 4.3 Results and Discussion . 66 4.3.1 The contribution of each K residue . 72 4.3.2 QCM-D measurement of phage film 73 4.3.3 The collective effect of positively charged residues . 75 4.3.4 The influence of contextual residues 85 4.4 Summary . 88 CHAPTER ENGINEERING LACI WITH STB1 AND INVESTIGATING THE MECHANISM OF LACI BINDING TO SIO2 AND TIO2 5.1 Introduction . 89 5.2 Experimental Section 90 5.2.1 Protein expression and mutation . 90 5.2.2 Protein purification, characterization and proteolysis 92 5.2.3 QCM-D analysis of LacIs binding to planar SiO2 and TiO2 surface 94 5.3 Results and Discussion . 95 5.3.1 Protein characterization 95 IV 5.3.2 Qualitative study of LacIs binding to planar SiO2 and TiO2 . 97 5.3.3 Quantitative analysis of binding kinetics 104 5.4 Summary . 111 CHAPTER ASSEMBLY OF TIO2 NANOPARTICLES ON DNA SCAFFOLD USING ENGINEERED LACI 6.1 Introduction . 112 6.2 Experimental Section 113 6.2.1 SPR analysis of DNA/LacI-STB1/TiO2 NPs assembly process on Au surface . 113 6.2.2 TEM of DNA/LacI-STB1/TiO2 NPs assembly . 114 6.3 Results and Discussion . 115 6.4 Summary . 123 CHAPTER CONTEXT-DEPENDENT ADSORPTION BEHAVIOR OF CYCLIC AND LINEAR PEPTIDES ON METAL OXIDE SURFACES 7.1 Introduction . 124 7.2 Experimental Section 127 7.2.1 Site-directed mutagenesis . 127 7.2.2 QCM-D measurement . 127 7.2.3 Molecular dynamics simulation 128 7.3 Results and Discussion . 129 V 7.4 Summary . 144 CHAPTER CONCLUSIONS 8.1 Summary of major achievements 146 8.2 Suggestions for future work 150 REFERENCES . 153 APPENDIX I LIST OF PUBLICATIONS 162 VI SUMMARY Naturally occurring biomolecular machinery provides excellent platforms for assembling artificially synthesized inorganic materials into functional nanodevices as widely envisioned in the field of nanobiotechnology. Hybrid materials, coupling the unique physical properties of synthetic inorganic nanoparticles with the exquisite recognition and self-assembly abilities of biomolecules, are expected to revolutionize materials and devices of the next generation. In this study, a specific protein-DNA conjugate (LacI protein and lacO sequence) was successfully engineered as a biomolecular platform to assemble inorganic nanoparticles on DNA scaffold using the LacI molecule as a linker. Meanwhile, the interaction between peptides/proteins and inorganic surfaces was carefully investigated. The main achievements include 1) isolating a SiO2- and TiO2-binding peptide motif using combinatorial peptide libraries, 2) understanding the mechanism of peptide and LacI binding to SiO2 and TiO2, 3) genetically fusing the isolated peptide motif with LacI and assessing the binding behavior of wild-type LacI vs. engineered LacI, 4) assembling TiO2 nanoparticles on DNA scaffold using engineered LacI as a linker, and 5) revealing the interplay between local conformation and contextual milieu of displayed peptides with regard to their target recognition ability. This thesis not only provides a platform to assemble inorganic nanoparticles, given that the peptide sequence specifically binding to desired nanoparticles is available, but also sheds light on understanding the complicated interaction of proteins with solid surfaces. VII LIST OF ABBREVIATIONS AFM Atomic force microscope CD Circular dichroism spectroscopy cDNA Complementary deoxyribonucleic acid CSD Cell surface display technique D Dissipation factor DBD DNA binding domain DI deionized DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate DTT Dithiothreitol E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid f Resonance frequency FG Functional coupling group FPLC Fast protein liquid chromatography HRTEM High-resolution transmission electron microscopy IPTG Isopropyl-β-D-thiogalactopyranoside LacI Lactose repressor protein lacO lac operator DNA sequence LSTB1-P LSTB1-harboring phage mRNA Messenger ribonucleic acid NPs Nanoparticles NR Newton-Raphson NVT moles (N), volume (V) and temperature (T) PCR Polymerase chain reaction PDB Protein data bank PFU Plaque forming unit VIII Chapter engineer LacI protein as a linker to direct the assembly of inorganic NPs on DNA scaffold. In order to engineer LacI with the ability to recognize target NPs, a consensus peptide sequence STB1 (-CHKKPSKSC-) with specific binding affinity to both SiO2 and TiO2 nanoparticles was identified in Chapter using combinatorial peptide libraries displayed on the phage surface. The use of NPs as substrates and phage surface display technique provides high possibility to isolate consensus peptides with strong binding affinity to target materials. Subsequently, the underlying mechanism of STB1 binding to SiO2 and TiO2 was investigated. The binding behavior of the STB1-harboring phage particles (STB1-P) to SiO2 and TiO2 was extensively studied by QCM-D measurement at various pHs with the aid of AFM image analysis. The results proved that the binding of STB1-P to both metal oxides is mediated by the STB1 displayed on the phage surface, and the interaction between the STB1 and the metal oxides is electrostatic in a pH-dependent manner. A higher level of fundamental understanding of STB1 interaction with SiO2 and TiO2 was gained in Chapter 4. The binding affinity of 16 phage strains displaying various STB1 mutants to SiO2 and TiO2 was studied by means of QCM-D measurement and molecular dynamics simulations. Our results suggests that: 1) the three K residues of STB1 were essential and also sufficient to anchor phage particles on SiO2 and TiO2 surfaces; 2) in a defined peptide compositional and/or conformational context, there is an optimal number or distribution of charged residues to give the strongest electrostatic interaction between peptides and metal oxide 147 Chapter surfaces; 3) peptide geometries and contextual residues play important roles to modulate electrostatic interaction between basic residues and oxide surfaces. These findings may be further harnessed for fine tuning of the binding affinity and selectivity of peptides to the target material. Our systematic approaches for investigating peptide-inorganic interaction and the obtained results may shed light on understanding the complicated interaction between proteins or peptides with inorganic surfaces. Chapter elaborates on the engineering of LacI with STB1. Based on understanding the structure-function relationship of LacI, STB1 was genetically fused to the C-terminus of LacI to create LacI-STB1 in order not to disturb the N-terminal DNA-binding domain. The inserted STB1 peptides in the context of LacI-STB1 molecules were shown to actively interact with both SiO2 and TiO2 while LacI-STB1’s DNA-binding ability remained intact. Wild-type LacI was found to bind to the metal oxdies through its N-terminal DNA-binding domain. An interesting finding is that compared to wild-type LacI with one binding region (at N-terminus), two remote binding regions (at N-terminus and C-terminus) in LacI-STB1 did not lead to faster adsorption rates to the two metal oxides, but remarkably slowed down the desorption rates. The use of LacI-STB1 as a protein linker to assemble TiO2 NPs on DNA scaffolds is successfully demonstrated in Chapter 6. Moreover, it was found that the 148 Chapter specific interaction between LacI and lacO is affected by the buffer condition. By using pure DI water or optimized PBS buffer, the sandwich nanostructure of DNA/LacI-STB1/TiO2 NPs was assembled either through engineered LacI’s non-specific interaction with DNA scaffold or through engineered LacI’s specific binding to lacO. This achievement of our initial objective raises the potential to design nanostructures at a molecular level by harnessing engineered biomolecules with desired recognition capability toward various materials. Last but not least, there is a general concern that the inorganic-binding behavior of peptide molecules may vary with the conformation and the contextual environment surrounding the peptide moieties In light of this, the binding behavior of STB1 and its linear version LSTB1 on TiO2 and SiO2 surfaces was investigated in three different contexts including free, phage-hosted and LacI-fused peptide forms (Chapter 7). Our results suggest that the structural flexibility of peptides is an important factor to regulate their binding affinity and selectivity towards inorganic materials. Increasing the structural flexibility of inorganic-binding peptides may increase their binding affinity but would trade off their selectivity. Such interplay among peptides’ structural flexibility, binding affinity and selectivity should be considered in understanding the peptide-inorganic interaction as well as in tuning peptides’ inorganic-binding behavior. Overall the research presented in this thesis demonstrated a feasible route to 149 Chapter engineer desired protein molecules with specific binding ability to target inorganic NPs. In addition, QCM-D measurement, AFM imaging, site-directed mutagenesis and/or molecular dynamics simulations were systematically applied to investigate the interaction of peptides, proteins and/or phage particles with inorganic surfaces. Substantial understanding of such complicated interactions has been gained. 8.2 Suggestions for future work Following the above achievements in this thesis, future research can be conducted in several areas as follows to fully explore the potential impact: 1) The STB1 peptide identified in this thesis electrostatically binds to SiO2 and TiO2 with similar affinity. It is not able to differentiate between SiO2 and TiO2. In certain applications, it may be desirable to identify such a peptide that is able to specifically bind to SiO2 NPs in the presence of TiO2 NPs, or vice versa. The studies in Chap. and Chap. suggest that the contextual residues can modulate the binding affinity and the peptide geometry exerts great control over the accessibility of reactive amino acid side chains to reactive sites on target inorganic surfaces. It is therefore desirable to identify peptides with high specificity to either SiO2 or TiO2 through manipulating the contextual residues and peptide geometry and understanding the peptide-metal oxide interaction at atomic level. 150 Chapter 2) A process of assembling the sandwich nanostructure of DNA/LacI-STB1/TiO2 NPs was successfully monitored using SPR (Chap. 6). This opens the way to assemble nanostructures using biomolecules and inorganic NPs at a precision of molecular-level (e.g. to realize an assembly pattern where one engineered LacI molecule specifically binds to only one target nanoparticle). However, the stoichiometry of the DNA/LacI-STB1/TiO2 NPs assembly is not clear, which hinders its application as nanodevice components. In future, methods should be developed to precisely measure stoichiometry of the DNA/LacI-STB1/TiO2 NPs assembly, and subsequently factors to control the stoichiometry of assembly should be indentified. 3) LacI-lacO binding is reversible and can be modulated by incorporating inducer molecules (e.g. lactose, IPTG) to LacI’s core domain. The binding of inducer molecules to LacI triggers a conformational change of LacI which dramatically reduces its affinity to lacO. This feature makes LacI-lacO a potential logic switch for future nanodevices harnessing nanoelectronic particles. In future, it is worth investigating whether the LacI-STB1 molecule, once assembled in the DNA/LacI-STB1/TiO2 NPs sandwich nanostructure, can be released from DNA by incorporating inducer molecules. Secondly, it would also be interesting to design such a spacer region between STB1 and LacI that is sensitive to certain cleavage enzymes. In this way, the target NPs bound to engineered LacI 151 Chapter molecules can be released by enzymatic cleavage. These two investigations are aimed to render great flexibility to manipulate the DNA/LacI-STB1/TiO2 NPs nanostructure. Futhermore, functional nanodevices integrated with the engineered LacI-lacO machinery may be designed in the way that specific delivery of functional NPs is viable. This could facilitate the development of novel nanodevices of next generation which would find many useful applications ranging from molecular diagnostics to nanomedicine. 152 References REFERENCES Alivisatos, A. P. Nanocrystals: building blocks for modern materials design. Endeavour 1997, 21, 56-60. Baneyx, F.; Schwartz, D. T. Selection and analysis of solid-binding peptides. Curr. Opin. Biotechnol. 2007, 18, 312-317. Brown, S. Engineering iron oxide-adhesion mutants of the Escherichia-coli phage-lambda receptor. Proc.Natl. Acad. Sci. USA 1992, 89, 8651-8655. Brown, S. Metal-recognition by repeating polypeptides. Nat. Biotechnol. 1997, 15, 269-272. Astier, Y.; Bayley, H.; Howorka, S. Protein components for nanodevices. Curr. Opin. Chem. Biol. 2005, 9, 576-584. Bäuerlein, E. Biomineralization, Wiley-VCH, Weinheim, 2004. Calabretta, M. K.; Matthews, K. S.; Colvin, V. L. DNA binding to protein-gold nanocrystal conjugates. Bioconjugate Chem. 2006, 17, 1156-1161. Cerofolini, G. F.; Arena, G.; Camalleri, C. M.; Galati, C.; Reina, S.; Renna, L.; Mascolo, D. A hybrid approach to nanoelectronics. Nanotechnology 2005a, 16, 1040-1047. Cerofolini, G. F.; Arena, G.; Camalleri, M.; Galati, C.; Reina, S.; Renna, L.; Mascolo, D.; Nosik, V. Strategies for nanoelectronics. Microelectron. Eng. 2005b, 81, 405-419. Chen, H.; Su, X.; Neoh, K.-G.; Choe, W.-S. QCM-D analysis of binding mechanism of phage particles displaying a constrained heptapeptide with specific affinity to SiO2 and TiO2. Anal. Chem. 2006, 78, 4872-4879. Choe, W.-S.; Sastry, M. S. R.; Thai, C. K.; Dai, H.; Schwartz, D. T.; Baneyx, F. Conformational control of inorganic adhesion in a designer protein engineered for cuprous oxide binding. Langmuir 2007, 23, 11347-11350. Connolly, S.; Fitzmaurice, D. Programmed assembly of gold nanocrystals in aqueous solution. Adv. Mater. 1999, 11, 1202-1205. Dai, H.; Choe, W.-S.; Thai, C. K.; Sarikaya, M.; Traxler, B. A.; Baneyx, F.; Schwartz, 153 References D. T. Nonequilibrium synthesis and assembly of hybrid inorganic-protein nanostructures using an engineered DNA-binding protein. J. Am. Chem. Soc. 2005, 127, 15637-15643. Doi, N.; Yanagawa, H.; Genotype-phenotype linkage for directed evolution and screening of combinatorial protein libraries. Comb. Chem. High Throughput Screen 2001, 4, 497-509. Eteshola, E.; Brillson, L. J.; Lee, S. C. Selection and characteristics of. peptides that bind thermally grown silicon dioxide films. Biomol. Eng. 2005, 22, 201-204. Fairman, R.; Åkerfeldt, K. S. Peptides as novel smart materials. Curr. Opin. Struct. Biol. 2005, 15, 453-463. Falcon, C. M.; Matthews, K. S. Operator DNA Sequence Variation Enhances High Affinity Binding by Hinge Helix Mutants of Lactose Repressor Protein. Biochemistry 2000, 39, 11074-11083. Feynman, R. P. in Miniaturization (Ed.: H. D. Gilbert), Reinhold, New York, 1961, 282-296. Flynn, C. E.; Mao, C.; Hayhurst, A.; Williams, J. L.; Georgiou, G.; Iverson, B.; Belcher, A. M. Synthesis and organization of nanoscale II-VI semiconductor materials using evolved peptide specificity and viral capsid assembly. J. Mater. Chem. 2003, 13, 2414-2421. Geisler, N.; Weber, K. Isolation of the amino-terminal fragment of lactose repressor necessary for DNA binding. Biochemistry 1977, 16, 938-943. Goede, K.; Grundmann, M.; Holland-Nell, K.; Beck-Sickinger, A. G. Cluster properties of peptides on (100) semiconductor surfaces. Langmuir 2006, 22, 8104-8108. Goodsell, D. S. Bionanotechnology, Lessons from Nature. Wiley-Liss, Hoboken, 2004. Granéli, A.; Edvardsson, M.; Höök, F. DNA-based formation of a supported, three-dimensional lipid vesicle matrix probed by QCM-D and SPR. ChemPhysChem 2004, 5, 729-733. Gray, J. J. The interaction of proteins with solid surfaces. Curr. Opin. Struct. Biol. 2004, 14, 110-115. Gyorvary, E.; Schroedter, A.; Talapin, D. V.; Weller H.; Pum, D.; Sleytr, U. B. 154 References Formation of Nanoparticle Arrays on S-Layer Protein Lattices. J. Nanosci. Nanotechnol. 2004, 4, 115-120. Hayashi, T.; Sano, K. I.; Shiba, K.; Kumashiro, Y.; Iwahori, K.; Yamashita, I.; Hara, M. Mechanism Underlying Specificity of Proteins Targeting Inorganic Materials. Nano Lett. 2006, 6, 515-519. Hoffman, M. R.; Martin, S. T.; Choi, W. Y.; Bahnemann. D. W. Environmental applications of semiconductor photocatalysis. Chem. Rev. 1995, 95, 69-96. Höök, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Energy dissipation kinetics for protein and antibody-antigen adsorption under shear oscillation on a quartz crystal microbalance. Langmuir 1998, 14, 729-734. Hunter, R. J. Foundations of Colloid Science. Oxford University Press, New York, 1989. Imamura, K.; Kawasaki, Y.; Awadzu, T.; Sakiyama, T.; Nakanishi, K. Contribution of acidic amino residues to the adsorption of peptides onto a stainless steel surface. J. Colloid Interface Sci. 2003, 267, 294-301. Janshoff, A.; Galla, H. J.; Steinem, C. Piezoelectric mass-sensing devices as biosensors - An alternative to optical biosensors. Angew. Chem. Int. Ed. 2000, 39, 4004-4032. Kalodimos, C. G.; Biris, N.; Bonvin, A. M. J. J.; Levandoski, M. M.; Guennuegues, M.; Boelens, R.; Kaptein, R. Structure and flexibility adaptation in nonspecific and specific protein-DNA complexes. Science 2004, 305, 386-389. Karlsson, R.; Michaelsson, A.; Mattsson, L. Kinetic analysis of monoclonal antigen-antibody interactions with a new biosensor based analytical system. J. Immunol. Methods 1991, 145, 229-240. Kase, D.; Kulp, J. L. III; Yudasaka, M.; Evans, J. S.; Iijima, S.; Shiba, K. Affinity selection of peptide phage libraries against single-wall carbon nanohorns identifies a peptide aptamer with conformational variability. Langmuir 2004, 20, 8939-8941. Kay, B. K.; Winter, J.; McCafferty, J. Phage display of peptides and proteins: a laboratory manual. Academic Press, San Diego, 1996. Keren, K.; Krueger, M.; Gilad, R.; Ben-Yoseph, G.; Sivan, U.; Braun, E. Sequence-specific molecular lithography on single DNA molecules. Science 2002, 297, 72-75. 155 References Kjaergaard, K.; Sorensen, J. K.; Schembri, M. A.; Klemm, P. Sequestration of zinc oxide by fimbrial designer chelators. Appl. Environ. Microbiol. 2000, 66, 10-14. Kosmulski, M. pH-dependent surface charging and points of zero charge II. Update. J. Colloid Interface Sci. 2004, 275, 214-224. Krauland, E. M.; Peelle, B. R.; Wittrup, K. D.; Belcher, A. M. Peptide tags for enhanced cellular and protein adhesion to single-crystalline sapphire. Biotechnol. Bioeng. 2007, 97, 1009-1020. Kriplani, U.; Kay, B. Selecting peptides for use in nanoscale materials using phage-displayed combinatorial peptide libraries. Curr. Opin. Biotechnol. 2005, 16, 470-475. Kröger, N.; Deutzmann, R.; Sumper, M. Polycationic Peptides from diatom biosilica that direct silica nanosphere formation. Science 1999, 286, 1129-1132. Kumar, A.; Galaev, I. Y.; Mattiasson, B. Purification of lac repressor protein using polymer displacement and immobilization of the protein. Bioseparation 1999, 8, 307-316. Lagier, C. M.; Efimov, I.; Hillman, A. R. Film resonance on acoustic wave devices: the roles of frequency and contacting fluid. Anal. Chem. 2005, 77, 335-343. Lee, S. Y., Choi, J. H., Xu, Z. H. Microbial cell-surface display. Trends Biotechnol. 2003, 21, 45-52. Lee, W. S.; Mao, C.; Flynn, C. E.; Belcher, A. M. Ordering of Quantum Dots Using Genetically Engineered Viruses. Science 2002, 296, 892-895. Lewis, M.; Chang, G.; Horton, N. C.; Kercher, M. A.; Pace, H. C.; Schumacher, M. A.; Brennan, R. G.; Lu, P. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 1996, 271, 1247-1254. Li, C. M.; Botsaris, G. D.; Kaplan, D. L. Selective in vitro effect of peptides on calcium carbonate crystallization. Cryst. Growth Des. 2002, 2, 387-393. Li, H.; LaBean, T. H.; Kenan, D. J. Single-chain antibodies against DNA aptamers for use as adapter molecules on DNA tile arrays in nanoscale materials organization. Org. Biomol. Chem. 2006, 18, 3420-3426. Li, H.; Park, S. H.; Reif, J. H.; LaBean, T. H.; Yan, H. DNA-templated self-assembly of protein and nanoparticle linear arrays. J. Am. Chem. Soc. 2004, 126, 418-419. 156 References Linsebigler, A. L.; Lu G. Q.; Yates, J. T. Jr. Photocatalysis on TiO2 surfaces: principles, mechanisms, and selected results. Chem. Rev. 1995, 95, 735-758. Lipovsek, D.; Pluckthun, A. In-vitro protein evolution by ribosome display and mRNA display. J. Immun. Meth. 2004, 290, 51-67. Lowe, C. R. Nanobiotechnology: the fabrication and applications of chemical and biological nanostructures. Curr. Opin. Struct. Biol. 2000, 10, 428-434. Lowenstam, H. A.; Weiner, S. On Biomineralization, Oxford University Press, New York, 1989. Mann, S. Biomineralization, Oxford University Press, Oxford, New York, 2001. Martin, S. J.; Bandey, H. L.; Cernosek, R. W. Equivalent-circuit model for the thickness-shear mode resonator with a viscoelastic film near film resonance. Anal. Chem. 2000, 72, 141-149. Marx, K. A. Quartz crystal microbalance: A useful tool for studying thin polymer films and complex biomolecular systems at the solution-surface interface Biomacromolecules. 2003, 4, 1099-1120. Matthews, K. S. Tryptic core protein of lactose repressor binds operator DNA. J. Biol. Chem. 1979, 254, 3348-3353. Matthews, K. S.; Nichols, J. C. Lactose repressor protein: functional properties and structure. Prog. Nuc. Acid Res. Mol. Biol. 1998, 58, 127–164. Mirkin, C. A.; Taton, T. A. Semiconductors Meet Biology. Nature 2000, 405, 626-627. Mirkin, C. A. Programming the Assembly of Two- and Three-Dimensional Architectures with DNA and Nanoscale Inorganic Building Blocks. Inorg. Chem. 2000, 39, 2258-2272. Muller-Hill, B.; Beyreuther, K.; Gilbert, W. Lac repressor from Escherichiu coli. Methods Enzymol. 1971, 21, 483-487. Naik, R. R.; Brott, L.; Carlson, S. J.; Stone, M. O. Silica-precipitating peptides isolated from a combinatorial phage display peptide library. J. Nanosci. Nanotechnol. 2002a, 2, 95-100. Naik, R. R.; Stringer, S. J.; Agarwal, G.; Jones, S. E.; Stone, M. O. Biomimetic synthesis and patterning of silver nanoparticles. Nat. Mater. 2002b, 1, 169–172. 157 References Nakanishi, K.; Sakiyama, T.; Imamura, K. On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon. J. Biosci. Bioeng. 2001, 91, 233-244. Niemeyer, C. M. Nanoparticles, proteins, and nucleic acids: Biotechnology meets materials science. Angew. Chem. Int. Ed. 2001, 40, 4128-4158. Niemeyer, C. M.; Mirkin, C. A. (Eds.) Nanobiotechnology: Concepts, Applications and Perspectives. Wiley-VCH, Weinheim, 2004. Nygaard, S.; Wendelbo, R.; Brown, S. Surface-specific zeolite-binding proteins. Adv. Mater. 2002, 14, 1853-1856. Olsen, J. V.; Ong, S. E.; Mann, M. Trypsin cleaves exclusively C-terminal to arginine and lysine Residues. Mol. Cell. Proteomics 2004, 3, 608-614. Patwardhan, S. V.; Patwardhan, G.; Perry, C. C. Interactions of biomolecules with inorganic materials: principles, applications and future prospects. J. Mater. Chem. 2007, 17, 2875-2884. Peelle, B. R.; Krauland, E. M.; Wittrup, K. D.; Belcher, A. M. Design criteria for engineering inorganic material-specific peptides. Langmuir 2005, 21, 6929-6933. Peh, W. Y. X.; Reimhult, E.; Teh, H. F.; Thomsen, J. S.; Su, X. Understanding ligand binding effects on conformation of Estrogen Receptor-DNA complex: A combinational QCM-D and SPR study. Biophys. J. 2007, 92, 4415-4423. Peters, E. A.; Schatz, P. J.; Johnson, S. S.; Dower, W. J. Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors. J. Bacteriol. 1994, 176, 4296-4305. Rappocciolo, E. Antimicrobial peptides as carriers of drugs. Drug Discovery Today 9, 2004, 470. Record, M. T. Jr.; Ha, J.-H.; Fisher, M. A. Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA. Methods Enzymol. 1991, 208, 291-343. Regan. O. B.; Gratzel, M. A low-cost, high-efficiency solar cell based on dye-sensitized colloidal TiO2 films. Nature 1991, 353, 737. Ringler, P.; Schulz, G. E. Self-assembly of proteins into designed networks. Science 2003, 302, 106-109. 158 References Rodahl, M.; Höök, F.; Fredriksson, C.; Keller, C. A.; Krozer, A.; Brzezinski, P.; Voinova, M.; Kasemo, B. Simultaneous frequency and dissipation factor QCM measurements of biomolecular adsorption and cell adhesion. Faraday Discuss. 1997, 107, 229-246. Rodahl, M.; Höök, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. Quartz crystal microbalance setup for frequency and Q-factor measurements in gaseous and liquid environments. Rev. Sci. Instrum. 1995, 66, 3924-3930. Sambrook, J.; Russell, D. W. Molecular Cloning: A Laboratory Manual (3rd Ed.) Cold Spring Harbor Laboratory Press, New York, 2001. Vol. 2: Chap. 13. Samuelson, P.; Gunneriusson, E.; Nygren, P.; Stahl, S. Display of proteins on bacteria. J. Biotechnol. 2002, 96, 129-154. Sano, K. I.; Ajima, K.; Iwahori, K.; Yudasaka, M.; Iijima, S.; Yamashita, I.; Shiba, K. Endowing a ferritin-like cage protein with high affinity and selectivity for certain inorganic materials. Small 2005, 1, 826-832. Sano, K. I.; Sasaki, H.; Shiba, K. Specificity and Biomineralization Activities of Ti-Binding Peptide-1 (TBP-1). Langmuir 2005, 21, 3090-3095. Sano, K. I.; Sasaki, H.; Shiba, K. Utilization of the pleiotropy of a peptidic aptamer to fabricate heterogeneous nanodot-containing multilayer nanostructures. J. Am. Chem. Soc. 2006, 128, 1717-1722. Sano, K. I.; Shiba, K. A hexapeptide motif that electrostatically binds to the surface of titanium. J. Am. Chem. Soc. 2003, 125, 14234-14235. Santore, M. M.; Wertz, C. F. Protein spreading kinetics at liquid-solid interfaces via an adsorption probe method. Langmuir 2005, 21, 10172-10178. Sarikaya, M.; Tamerler, C.; Jen, A. K. Y.; Schulten, K.; Baneyx, F. Molecular biomimetics: Nanotechnology Through Biology. Nat. Mater. 2003, 2. 577-585. Sarikaya, M.; Tamerler, C.; Schwartz, D. T.; Baneyx, F. O. Materials assembly and formation using engineered polypeptides. Annu. Rev. Mater. Res. 2004, 34, 373-408. Sauerbrey, G. Z. Use of quartz crystal vibrator for weighting thin films on a microbalance. Z. Phys. 1959, 155, 206-222. Schembri, M. A.; Kjaergaard, K.; Klemm, P. Bioaccumulation of heavy metals by fimbrial designer adhesions. FEMS, Microbial. Lett. 1999, 170, 363-371. 159 References Seker, U. O. S.; Wilson, B.; Dincer, S.; Kim, I. W.; Oren, E. E.; Evans, J. S.; Tamerler, C.; Sarikaya, M. Adsoprtion behavior of genetically engineered linear and cyclic Pt-binding peptides. Langmuir 2007, 23, 7895-7900. Shenton, W.; Pum, D.; Sleytr, U. B.; Mann, S. Synthesis of cadmium sulphide superlattices using self-assembled bacterial S-layers. Nature 1997, 389, 585-587. Shimizu, K.; Cha, J.; Stuck, G. D.; Morse, D. E. Silicatein α: Cathepsin L-like protein in sponge biosilica. Proc. Natl. Acad. Sci. USA 1998, 95, 6234-6238. Shipway, A. N.; Katz, E.; Willner, I. Nanoparticle arrays on surfaces for electronic, optical, and sensor applications. Chemphyschem 2002, 1, 18-52. Smith, G. P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228, 1315-1317. Smothers, J. F.; Henikoff, S.; Carter, P. Affinity selection from biological libraries. Science 2002, 298, 5593. Steinem, C.; Janshoff, A. (Eds.) Piezoelectric sensors, Springer-Verlag, Berlin, 2007. Su, X.; Wu, Y.-J.; Robelek, R.; Knoll, W. Surface plasmon resonance spectroscopy and quartz crystal microbalance study of streptavidin film structure effects on biotinylated DNA assembly and target DNA hybridization. Langmuir 2005, 21, 348-353. Su, X.; Lin, C.-Y.; O’Shea, S. J.; Teh, H. F.; Peh, W. Y. X.; Thomsen, J. S. Combinational application of SPR and QCM for studying nuclear hormone receptor-response element interactions. Anal. Chem. 2006, 78, 5552-5558. Thai, C. K.; Dai, H.; Sastry, M. S. R.; Sarikaya, M.; Schwartz, D. T.; Baneyx, F. Identification and characterization of Cu2O and ZnO binding polypeptides by Escherichia coli cell surface display: towards an understanding of metal oxide binding. Biotechnol. Bioeng. 2004, 87, 129-137. Tie, Y.; Calonder, C.; Van Tassel P. R. Protein adsorption: Kinetics and history dependence. J. Colloid Interface Sci. 2003, 268, 1-11. Wang, S. Q.; Humphreys, E. S.; Chung, S. Y.; Delduco, D. F.; Lustig, S. R.; Wang H., Parker, K. N.; Rizzo, N. W.; Subramoney, S.; Chiang, Y. M.; Jagota, A. Peptides with selective affinity for carbon nanotubes. Nat. Mater. 2003, 2, 196-200. Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Selection of 160 References peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature 2000, 405, 665-668. Willett, R. L.; Baldwin, K. W.; West, K. W.; Pfeiffer, L. N. Differential adhesion of amino acids to inorganic surfaces. Proc. Natl. Acad. Sci. USA 2005, 102, 7817-7822. Yun, C. S.; Khitrov, G. A.; Vergona, D. E.; Reich, N. O.; Strouse, G. F. Enzymatic manipulation of DNA-nanomaterial constructs. J. Am. Chem. Soc. 2002, 124, 7644-7645. Zhang, S. Fabrication of novel biomaterials through molecular self-assembly. Nat. Biotechnol. 2003, 21, 1171-1178. Zhou, C.; Friedt, J. M.; Angelova, A.; Choi, K. H.; Laureyn, W.; Frederix, F.; Francis, L. A.; Campitelli, A.; Engelborghs, Y.; Borghs, G. Human immunoglobin adsorption investigated by means of quartz crystal microbalance dissipation, atomic force microscopy and surface acoustic wave, and surface plasmon resonance techniques. Langmuir 2004, 20, 5870-5878. 161 Appendix I APPENDIX I LIST OF PUBLICATIONS Parts of this thesis have been presented elsewhere as listed below: Journal publications: Chen, H.; Su, X.; Neoh, K.-G.; Choe, W.-S. QCM-D analysis of binding mechanism of phage particles displaying a constrained heptapeptide with specific affinity to SiO2 and TiO2. Anal. Chem. 2006, 78, 4872-4879. Chen, H.; Su, X.; Neoh, K.-G.; Choe, W.-S. Probing the interaction between peptides and metal oxides using point mutants of a TiO2-binding peptide. Langmuir 2008, 24, 6852- 6857. Chen, H.; Su, X.; Neoh, K.-G.; Choe, W.-S. Context-dependent adsorption behavior of cyclic and linear peptides on metal oxide surfaces. Langmuir 2009, 25, 1588-1593. Chen, H.; Su, X.; Neoh, K.-G.; Choe, W.-S. Engineering LacI for self-assembly of inorganic nanoparticles on DNA scaffold through the understanding of LacI binding to solid surfaces. Adv. Funct. Mater. 2009, 19, 1186-1192. Presentation on International Conference: Chen, H.; Su, X.; Neoh, K.-G.; Choe, W.-S. Identification of heptapeptide motifs specifically binding to TiO2 and SiO2 nanoparticles. NanoBio-Europe’06, 12-14 Jun, 2006. Grenoble, France. Poster presentation. 162 [...]... films In the case of phage particles binding to SiO2 as illustrated in Scheme 4.1c, the viscoelasticity of the phage film would depend only on the amount of the phage particles bound on SiO2: the more phage particles bind to SiO2, the more viscoelastic is the phage film and thus the larger ΔD is, which is well justified from the continuous increase of ΔD during the 30 min binding process of STB1-P... mediated by the STB1 moiety displayed on the phage surface in a pH dependant manner, indicating that the binding is largely governed by electrostatic interaction Furthermore, the interpretation of QCM-D signals (i.e frequency shift and dissipation shift), with the aid of AFM image analysis of the phage particles bound on the surface 6 Chapter 1 of the two metal oxides, elucidated whether the nature of phage... Investigation of contextual influence of peptides on their target binding behavior Assembly of the nanostructure of DNA-Engineered LacI-NPs Assessment of the binding behavior of wild-type vs engineered LacI 10 Chapter 2 CHAPTER 2 LITERATURE REVIEW 2.1 Harnessing biomolecules for assembly of inorganic nanoparticles With unique molecular recognition abilities, biomolecules are employed as cross -linkers in the. .. any creature on the earth The structure of DNA or proteins could easily be tailored by biochemical methods or genetic engineering, which enables us to design DNA or proteins with desired recognition properties Therefore, hybrid materials, coupling the unique physical properties of synthetic 2 Chapter 1 inorganic nanoparticles with the exquisite recognition and self -assembly abilities of biomolecules,... 2007) This opens the way to engineer protein linkers to assemble inorganic nanoparticles based on protein- containing biomolecular machinery 3 Chapter 1 1.2 Objectives and scope With the aim to explore the potential of engineering protein linkers for assembling inorganic nanoparticles, we chose a protein- DNA conjugate, i.e lac repressor protein (LacI) and its DNA operator sequence (lacO), as the biomolecular... concentrations The dotted line marks the slope of the first binding phase Throughout the first binding phase, ΔD - Δf plots at various concentrations for either LacI-C7AC or LacI-STB1 exhibit the same slope The ΔD - Δf plots for LacI-C7AC or LacI-STB1 binding to TiO2 surface (not shown) look similar to those for SiO2 surface 108 Figure 6.1 (a) Real-time monitoring of the assembly of DNA/LacI-STB1/TiO2... first 5 min was used to calculate the binding kinetics parameters 138 Figure 7.5 Snapshots of the simulated conformations of (a) STB1 and (b) LSTB1 (c) shows the RMSD, i.e root mean square deviation, of the backbone of the simulated conformations (produced during the 200 ps dynamics XIX production) of STB1 and LSTB1 Inside each snapshot, the left image shows the surface electrostatic potential... couple with the modified surfaces of nanoparticles In another word, the interface between biomolecules and inorganic nanoparticles consists of a linker attached to the surface of the nanoparticles and a functional coupling group (FG) tagged to biomolecules There is a specific and strong interaction 13 Chapter 2 (usually covalent bond) between the linker and FG FGs in various DNA /protein- nanoparticles. .. SiO2 and TiO2 The consensus peptides sequence for either SiO2 or TiO2 nanoparticles were then identified and used to engineer LacI 2) To investigate the mechanism of the identified peptides binding to SiO2 or TiO2 The chemistry of the identified peptides and SiO2 or TiO2 surface was 4 Chapter 1 studied, and their interaction was measured using QCM-D technique (see the review in Chap 2) The binding mechanism... and fusion protein forms, was investigated 5 Chapter 1 1.3 Outline of the thesis This thesis is composed of eight chapters Chapter 1 describes the motivation and defines the objectives and scope of the work The current literatures relevant to this study are reviewed in Chapter 2 Experimental works and main findings are presented and discussed from Chapters 3 to 7 Chapter 8 concludes the thesis and . DESIGN OF PROTEIN LINKERS FOR THE CONTROLLED ASSEMBLY OF NANOPARTICLES CHEN HAIBIN NATIONAL UNIVERSITY OF SINGAPORE 2009 DESIGN OF PROTEIN LINKERS FOR THE CONTROLLED. deviation, of the backbone of the simulated conformations (produced during the 200 ps dynamics production) of free STB1 and H 1 /K|P 4 /K|S 5 S 7 /A peptides. RMSD measures the flexibility of the. contrast of circular DNA strand against the background demonstrates the efficient assembly of TiO 2 NPs onto the DNA strand. (b) TEM image of the same plasmid DNA molecule in the presence of wild-type