Spider silk is one of the most fascinating materials found in nature. Besides its properties like biodegradability, low immunoreactivity, and biocompatibility, especially the mechanical properties outperforming today’s artificial high-tech materials like Kevlar® are of great interest in biomedicine or material science. Spider silk comprises highly repetitive amino acid sequence motives, whose structure is accepted to be responsible for the extraordinary properties of spider silk. Typically, hydrophilic sequence motives alternate with hydrophobic ones making spider silk proteins resemble block copolymers. Additionally, the simple amino acid sequence and the possibility to form fibrillar structures are common characteristics of spider silk proteins as well as intrinsically disordered proteins (IDP) or protein regions (IDR). Both are suspected of being involved in the development of certain neurodegenerative diseases like Alzheimer´s disease. These aspects open promising possibilities of the use of spider silk proteins in nanotechnology, but also as model systems for the fibrillization processes of IDPs and IDRs, which are still unresolved today. Currently, most of the research and application is focused on 1-dimensional spider silk protein fibrils and fibers or 0-dimensional spider silk particles. However, 2-dimensional spider silk protein films or porous 3-dimensional objects are highly relevant platforms with the potential for cell-supporting scaffolds, biodegradable electrolyte materials in transistors, or e.g., planar drug-eluting implant coatings. Generally, the effects of sequence-based and external influences on the self-assembly and folding of spider silk proteins have not yet been fully elucidated in all of these various dimensional spider silk materials, even concerning IDP and IDR models. Thus, basic research regarding assembly and folding processes is still needed, especially in films. Particularly, 2-dimensional films allow a broad spectrum of (surface) analytical techniques, from whose outcome general structure-property relations of spider silk materials across all material dimensions can be obtained. In this work, engineered spider silk proteins, which are based on the consensus sequence motives in the spider silk fibroin (spidroin) 3 and 4 of the European garden spider Araneus diadematus (eADF4(Cx), eADF3(AQ)x, eADF3(QAQ)x) as well as blends of two short peptides with the respective aa sequence of the hydrophobic (pep-c) and hydrophilic (pep-a) part of eADF4(Cx) proteins were used. Spider silk-related proteins and peptides were dissolved in 1,1,1,3,3,3-hexafluoroisopropanol or formic acid, processed as thin films, and post-treated with methanol vapor to induce β-sheet formation. Dichroic FTIR-spectroscopy was used, a powerful tool for studying protein secondary structure formation and orientation. Proteins reveal characteristic amide bands, which are highly sensitive to the conformation of the protein backbone. In the course of this work, a set of components for the line shape analysis (LSA) of the Amide I band was developed. Therby, each component was assigned to a typical secondary structure allowing a quantitative determination of the respective portions and their structural orientation. Quantitative secondary structure portions and their orientation could be determined on this basis. Furthermore, a comprehensive study of folding and self-assembly-influencing parameters like hydrophobic and hydrophilic sequences, molecular weight, the repeating sequence motive order, the film thickness, surface topography, and the surface chemistry in engineered spider silk protein and spider silk protein-based films was carried out. In general, methanol vapor post-treatment induced the formation of β-sheet structures in all films, causing phase separation and the formation of spherical and filamentous structures. The phase separation upon post-treatment was influenced by the covalent connectivity between hydrophobic and hydrophilic sequence parts as well as the repeating sequence motives. In thin films, the increased flexibility of shorter peptides enabled the formation of multipack filaments instead of spherical structures, which were formed by higher molecular weight proteins with several inter-connected repeating sequence motives. Stamping wrinkled structures using poly(dimethylsiloxane) substrates was possible. Filamentous structures were successfully assigned to β-sheet rich structures using infrared nanospectroscopy for the first time. Further, enhanced surface hydrophobicity led to the clustering of β-sheet filaments. The β-sheet content could be controlled by the amount of hydrophobic sequences in thin films. With a higher amount of hydrophobic sequences in the proteins or blends, the β-sheet content increased until a maximum β-sheet content of around 60% was reached. Additionally, β-sheet formation could be suppressed by increasing substrate hydrophobicity or by decreasing the number of repeating sequence motives by going from protein-like folding to peptide-like self-assembly. The backfolding of proteins with covalently linked repeating sequence motives further promoted the formation of more antiparallel β-sheets. Antiparallel β-sheet formation was also favored when the portion of the hydrophilic, amorphous phase was increased. Micrometer thick films did not reveal any preferred alignment of β-sheets, while a general out-of-plane orientation of β-sheets could be obtained in all thin protein, peptide, and blend films. Z-axial orientation in films was increased by using short pep-c and pep-a peptides, higher molecular weight proteins or the deposition of monolayered films instead of thin multilayered films. Also, increased hydrophilicity of the substrate promoted the alignment of β-sheets perpendicular to the substrate surface. The folding kinetics and final domain size were found to be directly correlated. The amount of hydrophobic phase, backfolding, and increased flexibility due to low chain lengths increased the folding kinetics and led to smaller domain sizes. Thus, competing effects of backfolding and flexibility of the protein/peptide backbone could be rationalized. The film integrity and water contact angle were directly related to the β-sheet content and the molecular weight. Beyond the classical protein conformation and orientation analysis, the possibilities and limits of orientation analysis using dichroic attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were elaborated on the seemingly ideal oriented polymer model system of end-grafted poly(N,N-dimethylaminoethylmethacrylate) chains. Such a system featured a polymer brush regime in the swollen state with z-axial orientation expected similarly high as thin spider silk films after ptm. Moreover, dichroic ATR-FTIR spectroscopy is a promising analytical method for closing gaps in the defined assignment of brush regimes. In summary, general models of the structure formation and self-assembly of spider silk protein in films depending on the parameters mentioned above could be developed and set in relation to IDP/IDR self-assembly by using dichroic FTIR spectroscopy as the basic analysis method. The herein postulated models on the molecular level contribute to the understanding and development of future industrial applications of spider silk protein-based materials and the clarification of unresolved questions regarding IDP and IDR systems.:Abstract V Kurzfassung IX List of Publications XIII Publications in Trade Journals XIII Presentations and Posters XIII Contribution to Joint Publications XV List of Abbrevations XVII List of Symbols XIX List of Figures XXV List of Tables XXXIII 1 Introduction and Motivation 1 2 Theory 5 2.1 Proteins and Peptides 5 2.1.1 General Definition of Proteins and Peptides 5 2.1.2 Structure of Globular Proteins 7 2.1.3 Protein Folding 10 2.1.4 Intrinsically Disordered Proteins and Protein Regions 11 2.2 Block Copolymers 14 2.3 Spiders and Spider Silks 17 2.3.1 Classification of Spiders 17 2.3.2 The Natural Spider Silk Spinning Process 18 2.3.3 Structure of Spider Silk and Spider Silk Proteins 19 2.3.4 Structure-Property Relationships of Spider Silk 21 2.4 Infrared Spectroscopy 23 2.4.1 Basic Principles of Infrared Spectroscopy 23 2.4.2 Basic Equipment and IR-Technologies 27 2.4.3 Orientation Analysis using Dichroic FTIR Spectroscopy 32 2.4.4 Infrared Spectroscopy of Proteins and Peptides 38 2.4.5 Quantitative Analysis of TRANS- and ATR-FTIR Protein Spectra 43 2.5 Electronic Circular Dichroism 46 2.5.1 Basics Principles of Circular Dichroism 46 2.5.2 Circular Dichroism of Proteins and Polypeptides 48 2.5.3 Spectra Analysis 50 2.6 Atomic Force Microscopy 51 2.6.1 Setup of Atomic Force Microscopes 51 2.6.2 Basic Principles of Atomic Force Microscopy 52 2.6.3 AFM Operation Modes 55 3 Experimental Section 57 3.1 Materials 57 3.1.1 Chemicals 57 3.1.2 Substrates 57 3.1.3 Film Preparation 58 3.2 Analytical Methods 60 3.2.1 Dichroic FTIR Spectroscopy 60 3.2.2 Atomic Force Microscopy 64 3.2.3 Electronic Circular Dichroism 64 3.2.4 Spectroscopic Ellipsometry 64 3.2.5 Infrared Nanospectroscopy 65 3.2.6 Grazing Incident Small Angle X-Ray Scattering 66 4 Results 67 4.1 Self-Assembly of eADF4(C16) Films 67 4.1.1 Motivation 67 4.1.2 Dichroic FTIR Spectroscopy Characterization of ß-sheet Orientation in Spider Silk Films on Silicon Substrates 68 4.2 Influence of the Hydrophilic and Hydrophobic Blocks on Peptide Self-Assembly 90 4.2.1 Motivation 90 4.2.2 β-Sheet Structure Formation within Binary Blends of Two Spider Silk Related Peptides 90 4.2.3 Influence of the Hydrophilic and Hydrophobic Blocks on the Inner Morphology in Spider Silk Protein Based Blend Films 122 4.3 Influence of the Sequence Motive Repeating Number on Spider Silk Protein Folding 123 4.3.1 Motivation 123 4.3.2 Influence of Sequence Motive Repeating Number on Protein Folding in Spider Silk Protein Films 124 4.4 Influence of the Module Order on Spider Silk Protein Self-Assembly 152 4.4.1 Motivation 152 4.4.2 Secondary Structure upon Post-treatment 153 4.4.3 β-Sheet Orientation after Post-treatment 157 4.4.4 Morphology and Surface Properties 158 4.4.5 Conclusion 160 4.5 Surface Induced Changes of Spider Silk Protein Self-Assembly 161 4.5.1 Motivation 161 4.5.2 Variation of the Substrate Surface Chemistry and Topography 161 4.5.3 Influence of the Surface Topography on Protein Self-Assembly 162 4.5.4 Influence of the Surface Chemistry on Protein Self-Assembly 164 4.5.5 Conclusion 169 4.6 Chances and Limits of Dichroic ATR-FTIR Spectroscopy 170 4.6.1 Motivation 170 4.6.2 Novel Insights into Swelling and Orientation of End-Grafted PDMAEMA Chains by In-Situ ATR-FTIR Complementing In-Situ Ellipsometry 171 5 Conclusion and Outlook 197 6 References 203 7 Appendix 219 8 Danksagung 227 9 Eidesstattliche Versicherung 229