Design of protein linkers for the controlled assembly of nanoparticles

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. 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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