Theoretical Investigations of the Interactions Between Peptides and Inorganic Surfaces Zur Erlangung des akademischen Grades eines DOKTORS DER NATURWISSENSCHAFTEN (Dr. rer. nat.) von der Fakultät für Chemie und Biowissenschaften des Karlsruher Instituts für Technologie (KIT) genehmigte DISSERTATION von Magister Monika Borkowska-Panek aus Zielona Góra, Polen KIT-Dekan: Prof. Dr. W. Klopper Referent: PD Dr. K. Fink Korreferent: Prof. Dr. W. Klopper Tag der mündlichen Prüfung: 10. Februar 2017 Contents 1 Introduction 1 1.1 Peptides in biotechnology – applications . .2 1.2 Experimental techniques to investigate peptide-surface interactions . .2 1.3 Computational methods for the investigation of peptide-surface interactions .3 2 Background 5 2.1 Binding mechanisms of single amino acids to inorganic surfaces . .5 2.1.1 Covalent bonding . .5 2.1.2 Noncovalent bonding . .5 2.1.2.1 Electrostatic interaction . .6 2.1.2.2 Hydrophobic effects . .7 2.1.2.3 Van der Waals interactions . .8 2.2 Iron-oxide surface and nanoparticles . .8 2.2.1 Structure and magnetic properties of magnetite . .8 2.2.2 Compounds containing iron and other metal centers – ferrites . 12 2.2.3 Iron oxide magnetic nanoparticles . 12 2.2.4 Surface of magnetite and magnetic nanoparticles . 14 2.3 Experimental methods providing large sets of peptide sequences . 16 2.3.1 Peptide array screening . 16 3 Methods 19 3.1 Molecular dynamics . 19 3.2 Metropolis Monte Carlo . 20 3.3 Metadynamics . 21 3.4 Effective Implicit Surface Model (EISM) . 22 3.4.1 Energy of the peptide in vacuum, EINT ................. 23 3.4.2 The SLIM energy term . 26 3.4.3 The Solvent Accessible Surface Area (SASA) term . 27 3.4.4 The surface Lennard-Jones term, ESLJ ................. 29 3.4.5 The pit potential, EPIT .......................... 29 3.4.6 Limitations of the model . 29 4 Tests of the EISM model 31 4.1 Calibration of the EISM model . 31 4.1.1 Inconsistency of the reference data . 31 4.2 Gold-binding peptides . 32 4.2.1 The first calibration set . 32 4.2.2 The second calibration set . 37 4.2.3 Optimization of the EISM parameters . 39 4.3 Silver-binding peptides . 41 4.4 Summary . 41 5 Interactions of peptides with magentic nanoparticles (MNPs) 45 5.1 Experimental conditions and observations . 45 5.1.1 Foundations of the model . 47 5.1.2 Reactions of oppositely charged species . 50 5.1.3 Reactions of the uniformly charged species . 53 i 5.1.4 Summary . 57 5.2 Calibration of the EISM against experimental data . 57 5.2.1 Method of calibration . 57 5.2.2 Validation of the calibration . 59 5.2.2.1 D homopeptides . 59 5.2.2.2 Effects of mutations . 62 5.2.3 Summary . 65 5.3 Towards a theoretical peptide design . 67 5.3.1 Strong binders . 67 5.3.2 Mutations with strong binders . 68 5.3.3 Mutations with weak binders . 69 5.3.4 Summary . 74 6 Electronic structure of magnetite and its derivatives 75 6.1 Test calculations on model clusters . 75 6.1.1 Inverse and normal spinel systems . 78 6.2 Point Charge Field Embedding . 78 6.2.1 Preparation of the system . 81 6.2.2 Geometry optimization of different spinel structures . 83 6.3 Population analysis of small clusters compared to the full system . 86 6.4 Summary . 89 7 Summary 91 Bibliography 95 Acknowledgments 107 ii 1 Introduction Investigating the interactions between peptides and inorganic surfaces is essential to un- derstand the more complex phenomenon of interactions in biologically relevant systems of a mixed organic-inorganic nature. This knowledge is invaluable in various fields, like, biotechnology, nanotechnology, and materials science for designing new biomimetic hybrid materials[1–3]. In medicine, in turn, such materials can be used as transporters delivering drugs into the cells[4–12]. Gaining a detailed understanding of the surface-peptide interac- tions is essential for the selection of appropriate surfaces and peptides. This was the aim of a joint experimental and theoretical project entitled “Rational design of peptide-surface interactions”, funded by the Federal Ministry of Education and Research (BMBF) in the Biotechnology 2020+ initiative. The goal was to find and to design new peptide sequences which are selective direct binders to nanoparticle surfaces of magnetic iron oxides. Iron- oxide nanoparticles are preferred in various applications due to their magnetic properties, and the low cost of production. The experimental investigations were performed at the Technical University of Munich (TUM) in the group of Prof. Sonja Berensmeier, the the- oretical investigations at the Institute of Nanotechnology (INT) of Karlsruhe Institute of Technology (KIT). The experimental part was devoted to characterization of the surface properties, the structures of the interacting peptides, as well as to the verification of the interactions. The aim of the thesis was the development of protocols for the description of the peptide- surface interactions under various experimental conditions. Next, to provide and test the methodology for the efficient theoretical assessment of the binding affinity between pep- tides and surfaces. The setup was parametrized with literature data and partially from the experimental results. It allows to reproduce the experimental results, as well as pre- dict sequences of well-binding peptides. Finally, to employ quantum-chemical methods to investigate electronic properties of iron oxide and derivative mixed-center systems. This thesis is organized as follows. In the remaining parts of the introduction, applica- tions, experimental and theoretical methods for characterization of peptide-surface inter- actions are described. First, various applications of inorganic surfaces coated by organic molecules (mostly proteins) are presented in Chapter 1.1. Next, experimental and theoret- ical methods used to investigate peptide-surface interactions are presented in Chapters 1.2, and 1.3, respectively. In Chapter 2.1, the interactions occurring between amino acids and inorganic surfaces are introduced. In Chapter 2.2, the electronic and magnetic properties of iron oxide (magnetite) are considered, including spinel structures and its derivatives with manganese and zinc. Furthermore, the properties of magnetic nanoparticles (MNPs) are discussed in particular. In Chapter 2.3, the main experimental method, namely, pep- tide array screening, used by the experimental collaborators, to measure peptide-surface affinity is described. In Chapter 3, the main computational protocols used in this thesis to predict peptide-surface affinity are described. These rely on Metropolis Monte Carlo simulations supported with the metadynamics approach and the Effective Implicit Surface Model (EISM). In Chapter 4, the EISM calibrated against theoretical reference data was 1 Chapter 1. Introduction used to reproduce reference peptide binding affinities to gold (111) and silver (111) sur- faces. Chapter 5 is divided into two parts directly connected to the results provided by the experimental group from TU Munich. In Chapter 5.1, the affinity of magnetic nanoparti- cles to a set of peptides is measured under different pH and solvent conditions. In order to explain and reproduce these results, an analytical model, based on possible equilibrium reactions occurring during the experimental procedure, is introduced. In Chapter 5.2, the EISM model is calibrated against the experimentally obtained binding affinities of amino acids and used to reproduce and predict binding affinities of various peptide sequences. The EISM results are then directly compared with the experimental results. Further analysis in Chapter 5.3 shows an influence of the composition and length of the peptide chain on the changes of the Gibbs free energy of binding. Based on these results, peptide sequences, which bind selectively to the magnetic nanoparticles, can be designed. Finally, in Chap- ter 6 the electronic properties of magnetite and its derivatives with manganese and zinc are investigated by means of the density-functional theory (DFT) methods employing Point Charge Field Embedding model. In Chapter 7, the summary of this thesis is given. 1.1 Peptides in biotechnology – applications Peptides find various application in biotechnology and bioengineering. A combination of the protein collagen and the mineral hydroxyapatite was found to build bones and other tissues with different flexibility[13–15] and became important in bone and dentin tis- sue engineering[16]. Implants covered with proteins can be protected from an immune response[17]. A deeper insight into bacterial resistance to antibiotics showed that it is due to the biofilm which is made by bacteria[18,19], interruption in the process of bind- ing the protein to the substrate could solve this important problem. In nanotechnology the interactions of peptides and inorganic materials are essential for crystal growth regu- lation[20–26], surface biocompatibility and synthesis of nanoparticles[23,25,27–33]. New, func- tionalized nanoparticles are characterized by various and unique properties[34–37]. Mussels Adhesive Proteins (MAPs) produced by marine mussels[38] are containing a specific amino acid, 3,4-dihydroxyphenylalanine (DOPA), which generates the adhesion to plenty of inor- ganic surfaces such as metals, oxides and polymers. Functionalized nanoparticles could be used to produce, e.g., biodegradable glues, characterized by high mechanical strength, able to connect components of different type[39–44]. Combination of nanoparticles with peptides can also have influence on the optical properties of the system. The size and the shape of nanoparticles, as well as occurrence of molecular recognition groups (peptides, surfactants) determine the plasmon resonance frequency in such a way that it can induce adequate shifts in the UV/vis absorption spectrum[45–47]. 1.2 Experimental techniques to investigate peptide-surface interactions There are many experimental tools, which can be used to investigate the nature of inter- actions between peptides and inorganic surfaces. In the following section some of the most prominent experimental methods are introduced. Phage display is one of the most impor- tant techniques for verifying the presence of binding to the surface[2,22,24–26,48,49].