Background: Foamy viruses (FVs) have developed a unique budding strategy within the retrovirus family. FV release requires co-expression and a highly specific interaction between capsid (Gag) and glycoprotein (Env), which cannot be complemented by heterologous Env proteins. The interaction domain in FV Env has been mapped in greater detail and resides mainly in the N-terminal tip of the cytoplasmic domain of the Env leader peptide subunit. In contrast, the corresponding domain within Gag is less well defined. Previous investigations suggest that it is located within the N-terminal part of the protein. Results: Here we characterized additional Gag interaction determinants of the prototype FV (PFV) isolate using a combination of particle release, GST pull-down and single cycle infectivity analysis assays. Our results demonstrate that a minimal PFV Gag protein comprising the N-terminal 129 aa was released into the supernatant, whereas proteins lacking this domain failed to do so. Fine mapping of domains within the N-terminus of PFV Gag revealed that the N-terminal 10 aa of PFV Gag were dispensable for viral replication. In contrast, larger deletions or structurally deleterious point mutations in C-terminally adjacent sequences predicted to harbor a helical region abolished particle egress and Gag – Env protein interaction. Pull-down assays, using proteins of mammalian and prokaryotic origin, support the previous hypothesis of a direct interaction of both PFV proteins without requirement for cellular cofactors and suggest a potential direct contact of Env through this N-terminal Gag domain. Furthermore, analysis of point mutants within this domain in context of PFV vector particles indicates additional particle release-independent functions for this structure in viral replication by directly affecting virion infectivity. Conclusions: Thus, our results demonstrate not only a critical function of an N-terminal PFV Gag motif for the essential capsid - glycoprotein interaction required for virus budding but also point out additional functions that affect virion infectivity.

Cytochromes P450 represent a large group of proteins metabolizing variety of substrates. Many of them are responsible for metabolism of xenobiotics including drugs and chemical carcinogens. Heme-protein cytochrome b5 is a single-electron donor cooperating with a NADPH:cytochrome P450 reductase and NADH:cytochrome b5 reductase 3 enzyme. Cytochrome b5 can affect the xenobiotic metabolism via modulation of the cytochromes P450 activity. One of the goals of the Ph.D. thesis was to utilize site directed mutagenesis of cytochromes P450 family 1 to elucidate the mechanism of their nitroreductase activity. Another aim was to study the interaction between cytochrome b5 and cytochromes P450 of the 1A subfamily using site directed mutagenesis on presumed protein-protein contact interface. Another goal was to utilize the combination of theoretical and experimental approaches to explain variance in the reduction state of several human cytochromes P450 heterologously expressed in intact bacterial cells. The results found in the thesis show that nitroreductase activity of CYP1A1, CYP1A2 and CYP1B1 is mediated by the presence of a particular hydroxyl group in their active centre. Single mutation introducing a hydroxyl group to the specific part of CYP1B1 active site to the active site turned on its artificial...

Background: Foamy viruses (FVs) have developed a unique budding strategy within the retrovirus family. FV release requires co-expression and a highly specific interaction between capsid (Gag) and glycoprotein (Env), which cannot be complemented by heterologous Env proteins. The interaction domain in FV Env has been mapped in greater detail and resides mainly in the N-terminal tip of the cytoplasmic domain of the Env leader peptide subunit. In contrast, the corresponding domain within Gag is less well defined. Previous investigations suggest that it is located within the N-terminal part of the protein. Results: Here we characterized additional Gag interaction determinants of the prototype FV (PFV) isolate using a combination of particle release, GST pull-down and single cycle infectivity analysis assays. Our results demonstrate that a minimal PFV Gag protein comprising the N-terminal 129 aa was released into the supernatant, whereas proteins lacking this domain failed to do so. Fine mapping of domains within the N-terminus of PFV Gag revealed that the N-terminal 10 aa of PFV Gag were dispensable for viral replication. In contrast, larger deletions or structurally deleterious point mutations in C-terminally adjacent sequences predicted to harbor a helical region abolished particle egress and Gag – Env protein interaction. Pull-down assays, using proteins of mammalian and prokaryotic origin, support the previous hypothesis of a direct interaction of both PFV proteins without requirement for cellular cofactors and suggest a potential direct contact of Env through this N-terminal Gag domain. Furthermore, analysis of point mutants within this domain in context of PFV vector particles indicates additional particle release-independent functions for this structure in viral replication by directly affecting virion infectivity. Conclusions: Thus, our results demonstrate not only a critical function of an N-terminal PFV Gag motif for the essential capsid - glycoprotein interaction required for virus budding but also point out additional functions that affect virion infectivity.

5 Abstract Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) belongs to the serine/ threonine protein kinase family, which is involved in the calcium signaling pathway. The increase of intracellular calcium concentration induces the activation of calmodulin (CaM), which then activates its binding partners including CaMKII, CaMKIII, CaMKK1 and CaMKK2. CaMKK2 activates CaMKI, CaMKIV and AMP-dependent kinase, AMPK, by phosphorylation. CaMKK2 is naturally present in cells in an autoinhibited state, which is caused by the steric hindrance of the active site by the autoinhibitory domain. When calmodulin binds to the calmodulin-binding domain, the autoinhibitory domain is removed and the active site becomes accessible. Upon activation, CaMKK2 undergoes autophosphorylation, which increases its enzyme activity. Negative regulation of CaMKK2 is mediated by cAMP-dependent protein kinase A (PKA)- and GSK3-dependent phosphorylation. Sites phosphorylated by PKA have been identified for both CaMKK1 and CaMKK2. Two of them are also motifs recognized by scaffolding 14-3-3 proteins. Previous studies have shown that the 14-3-3 protein binding maintains phosphorylated CaMKK2 in an inhibited state by blocking the dephosphorylation of S495, which prevents the binding to calmodulin. However, it is unclear if it is the...

The SCP-RICM assay employs the measurable surface energy (or adhesive work W_adh) of a micrometer-sized polymeric sphere (soft colloidal probe, SCP) interacting with a glass chip using reflection interfer-ence contrast microscopy (RICM). Depending on those two interacting surfaces' nature and functional-ization, the SCP will deform, creating a contact area with the hard glass chip. This contact area is clearly distinguishable from the sphere’s interference ring pattern and can be measured. The adhesive surface energy W_adh can be calculated from the size of the contact area. An immobilization can be overcome by choosing a two-component analyte-dependent interaction, here presented for the copper (Cu) detection. The detection of Cu was chosen as a proof-of-concept system. However, detecting metal ions is an essential endeavor because, in excessive amounts, they present a severe threat to health and the environment. The copper-dependent interaction of the yeast chaperones yCox17 (also Cox17) and ySco1 (also Sco1) were chosen as the two-component analyte-dependent interaction. The chaperones partic-ipate in vivo in the formation of the electron transport chain of S. cerevisiae and interact in the mito-chondrial inner membrane to transfer one Cu(I) ion from Cox17 to Sco1. It was necessary to immobilize one protein to the SCPs and one to the chip surface, to transfer the copper chaperones' interaction into the SCP-RICM assay core detection components. The unique self-assembling characteristics of the class I hydrophobin Ccg-2 from N. crassa were used to immobilize one interaction partner to the chip surface. Class I hydrophobins are known for the formation of re-sistant and uniform layers at hydrophilic/hydrophobic interfaces. Initial SCP-RICM assay measurements with Sco1Δ95_a-SCPs and the Cox17_c-chips indicate that copper detection using the proposed mechanism is possible (Figure 39-3). Measurements can be differentiated between 0 and 0.1 mM Cu(I) concentration in solution. Further screening of concentrations be-low 0.1 mM is still necessary. The presented proof-of-principle system for the indirect detection of copper shows copper-dependent behavior. These positive results give rise to many more options to use the SCP-RICM assay as an indirect detection system. The application range of the SCP-RICM assay could be enlarged for different analytes such as other heavy metals, bacteriophages, biomarkers, et cetera, and is relevant for fields from medicine to environmental monitoring.:TABLE OF CONTENT Table of Content I List of Figures VII List of Tables IX List of Abbreviations XI 1 Introduction 1 1.1 Biosensors 1 1.2 Analytical Detection Methods: Copper 2 1.3 SCP-RICM Assay 3 1.3.1 Sensor Chip Surface 4 1.3.2 Soft Colloidal Probes 5 1.3.3 Reflection Interference Contrast Microscopy 6 1.4 Hydrophobins 9 1.4.1 Structure and Functions of Hydrophobins 9 1.4.2 Ex vivo Applications of Hydrophobins 11 1.4.3 Class I Hydrophobin: Ccg-2 12 1.5 Mitochondrial Respiratory Chain 14 1.5.1 Copper Transport in Yeast 14 1.5.2 S. cerevisiae Sco1 protein 18 1.5.3 S. cerevisiae Cox17 protein 21 1.6 SCP-RICM Assay for Copper Detection 23 1.7 Aim of the Study 24 2 Materials and Methods 25 2.1 Laboratory Equipment 25 2.1.1 Devices 25 2.1.2 Chemicals 26 2.1.3 Consumables 28 2.1.4 Antibodies 29 2.1.5 Enzymes 30 2.1.6 Molecular Weight Standards 30 2.1.7 DNA Oligonucleotides 31 2.1.8 Plasmids and Vectors 32 2.2 Microorganisms 33 2.2.1 Strains 33 2.2.2 Cultivation of Microorganisms 34 2.2.3 Preparation of Electrocompetent E. coli Cells 36 2.2.4 Preparation of E. coli Glycerol Stocks 36 2.3 Protein Design 37 2.4 Molecular Cloning Methods 38 2.4.1 Vector Template Preparation 38 2.4.2 Agarose Gel Electrophoresis 40 2.4.3 DNA Extraction from Agarose Gels 41 2.4.4 Polymerase Chain Reaction 41 2.4.5 DNA Restriction Digest 42 2.4.6 DNA Dialysis 43 2.4.7 Ligation of DNA Fragments 43 2.4.8 Isolation of DNA from E. coli 44 2.4.9 DNA Sequencing 45 2.4.10 Transformation of E. coli via Electroporation 45 2.5 Protein Detection and Quantification 46 2.5.1 SDS PAGE 46 2.5.2 Coomassie Staining 50 2.5.3 Western Blot Analysis 51 2.5.4 Immunological Detection 51 2.5.5 Protein Quantification: Lowry Assay 52 2.5.6 Protein Quantification: Bradford Assay 53 2.5.7 Protein Quantification: NanoDrop Measurement 53 2.6 Protein Purification and Storage 54 2.6.1 Expression Analysis of Recombinant Proteins 54 2.6.2 Solubility Analysis 54 2.6.3 Protein Purification by Ni2+ Affinity Chromatography 55 2.6.4 Quantification of Purified Proteins 64 2.6.5 Dialysis of Purified Proteins 65 2.7 Glass Surface Functionalization 65 2.7.1 Glass Surface Preparation 66 2.7.2 Hydrophobin and Fusion Protein-Based Coating 66 2.7.3 Contact Angle Measurement 67 2.7.4 DRoPS Test 67 2.7.5 Atomic Force Microscopy 67 2.8 SCP Functionalization 68 2.8.1 Functionalization of SCPs with Proteins 68 2.8.2 Validation of SCP Functionalization with FITC Staining 69 2.9 SCP-RICM Assay and Its Analysis 69 3 Results 73 3.1 Generation of Recombinant Fusion Proteins 73 3.1.1 Sco1 and Sco1∆95 73 3.1.2 Cox17 84 3.1.3 Ccg-2 88 3.1.4 Overview: Optimization of Expression and Purification of Recombinant Proteins 90 3.2 His-Tag Cleavage 92 3.3 Chip Surface Functionalization 94 3.3.1 Optimization of the Glass Chip Preparation 94 3.3.2 Macroscopic Properties of the Functionalized Chip Surface 95 3.3.3 AFM Measurements 102 3.3.4 Theoretical Package of Hydrophobin Ccg-2 on the Chip Surface 103 3.4 SCP Functionalization 104 3.4.1 SCP Functionalization and FITC Staining 104 3.4.2 Theoretical Package of Proteins on SCPs 106 3.5 SCP-RICM Assay 107 4 Discussion and Further Prospectives 113 4.1 Discussion: SCP-RICM Assay and Protein-Protein Interaction 113 4.2 Outlook and Further Prospects 119 4.2.1 Heterologous Protein Expression and Purification: Methods, Cleavage and Refolding 119 4.2.2 Further Analysis of Chip Surface Functionalization 124 4.2.3 Alternative Chip Surface Functionalization Methods 126 4.2.4 SCP-RICM Assay: Data Acquisition and Evaluation 128 4.2.5 SCP-RICM Assay: Copper Detection 130 4.2.6 Exploiting the SCP-RICM Assay using Protein-Protein Interactions 131 4.2.7 Exploiting the SCP-RICM Assay with Alternative Interactions 133 5 Summary 137 6 Bibliography 141 7 Appendix 165 7.1 Sequences of Protein Constructs 165 7.1.1 Sequences of the Protein Construct Cox17_a 165 7.1.2 Sequences of the Hydrophobin-Cox17 Fusion Protein Cox17_b 165 7.1.3 Sequences of the Hydrophobin-Cox17 Fusion Protein Construct Cox17_c 166 7.1.4 Sequences of the Protein Construct Sco1_a and Sco1Δ95_a 167 7.1.5 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_b and Sco1Δ95_b 169 7.1.6 Sequences of the Hydrophobin-Sco1 Fusion Protein Constructs Sco1_c and Sco1Δ95_c 171 7.1.7 Sequences of the Hydrophobin Ccg-2 173 7.2 pET-28b(+): Plasmid Map 173 7.3 Nickel Removal During Dialysis 175 7.4 DGR Assay 176 7.5 SCP diameter 179 Acknowledgements 181 Declaration of Authorship 183

Indiana University-Purdue University Indianapolis (IUPUI) / Protein-protein interactions play a crucial role in a wide range of biological processes. Research on the design and synthesis of small molecules to modulate these proteinprotein interactions can lead to new targets and drugs to modulate their function. In Chapter one, we discuss the design and synthesis of small molecules to probe a proteinprotein interaction in a voltage-gated Ca2+ channel. Virtual screening identified a compound (BTT-3) that contained a 3,4-dihydro-3,4’-pyrazole core. This compound had modest biological activity when tested in a fluorescence polarization (FP) assay. The synthetic route to BTT-3 consisted of six steps. In addition, analogs of BTT-3 were made for a structure-activity study to establish the importance of a carboxylate moiety. We also synthesized a biotinylated benzophenone photo-affinity probe and linked it to BTT-3 to identify additional protein targets of the compound. In Chapter two, small-molecule antagonists targeting uPA-uPAR protein-protein interaction are presented. A total of 500 commercially-available compounds were previously identified by virtual screening and tested by a FP assay. Three classes of compounds were found with biological activity. The first class of compounds contains pyrrolidone core structures represented by IPR- 1110, the second class has a novel pyrrolo[3,4-c]pyrazole ring system, represented by xv IPR-1283 and the last series had compounds with a 1,2-disubstituted 1,2- dihydropyrrolo[3,4-b]indol-3(4H)-one core structure, represented by IPR-540. Each of these three compounds were synthesized and assessed by FP and ELISA assays. A binding mode of IPR-1110 with uPA was subsequently proposed. Based on this binding mode, another 61 IPR-1110 derivatives were synthesized by us to illustrate the SAR activity. Analogs of the other two series were also synthesized.

BACKGROUND: A long-standing challenge in the post-genomic era of Bioinformatics is the prediction of protein-protein interactions, and ultimately the prediction of protein functions. The problem is intrinsically harder, when only amino acid sequences are available, but a solution is more universally applicable. So far, the problem of uncovering protein-protein interactions has been addressed in a variety of ways, both experimentally and computationally. MOTIVATION: The central problem is: How can protein complexes with solved threedimensional structure be utilized to identify and classify protein binding sites and how can knowledge be inferred from this classification such that protein interactions can be predicted for proteins without solved structure? The underlying hypothesis is that protein binding sites are often restricted to a small number of residues, which additionally often are well-conserved in order to maintain an interaction. Therefore, the signal-to-noise ratio in binding sites is expected to be higher than in other parts of the surface. This enables binding site detection in unknown proteins, when homology based annotation transfer fails. APPROACH: The problem is addressed by first investigating how geometrical aspects of domain-domain associations can lead to a rigorous structural classification of the multitude of protein interface types. The interface types are explored with respect to two aspects: First, how do interface types with one-sided homology reveal convergently evolved motifs? Second, how can sequential descriptors for local structural features be derived from the interface type classification? Then, the use of sequential representations for binding sites in order to predict protein interactions is investigated. The underlying algorithms are based on machine learning techniques, in particular Hidden Markov Models. RESULTS: This work includes a novel approach to a comprehensive geometrical classification of domain interfaces. Alternative structural domain associations are found for 40% of all family-family interactions. Evaluation of the classification algorithm on a hand-curated set of interfaces yielded a precision of 83% and a recall of 95%. For the first time, a systematic screen of convergently evolved motifs in 102.000 protein-protein interactions with structural information is derived. With respect to this dataset, all cases related to viral mimicry of human interface bindings are identified. Finally, a library of 740 motif descriptors for binding site recognition - encoded as Hidden Markov Models - is generated and cross-validated. Tests for the significance of motifs are provided. The usefulness of descriptors for protein-ligand binding sites is demonstrated for the case of "ATP-binding", where a precision of 89% is achieved, thus outperforming comparable motifs from PROSITE. In particular, a novel descriptor for a P-loop variant has been used to identify ATP-binding sites in 60 protein sequences that have not been annotated before by existing motif databases.

Indiana University-Purdue University Indianapolis (IUPUI) / The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.

The concept of 3D proteomics is a technique that couples chemical cross-linking with mass spectrometry and has emerged as a tool to study protein conformations and protein-protein interactions. In this thesis I present my work on improving the analytical workflow and developing applications for 3D proteomics in the structural analysis of proteins and protein complexes through four major tasks. I. As part of the technical development of an analytical workflow for 3D proteomics, a cross-linked peptide library was created by cross-linking a mixture of synthetic peptides. Analysis of this library generated a large dataset of cross-linked peptides. Characterizing the general features of cross-linked peptides using this dataset allowed me to optimize the settings for mass spectrometric analysis and to establish a charge based enrichment strategy for cross-linked peptides. In addition to this, 1185 manually validated high resolution fragmentation spectra gave an insight into general fragmentation behaviours of cross-linked peptides and facilitated the development of a cross-linked peptide search algorithm. II. The advanced 3D proteomics workflow was applied to study the architecture of the 670 kDa 15-subunit Pol II-TFIIF complex. This work established 3D proteomics as a structure analysis tool for large multi-protein complexes. The methodology was validated by comparing 3D proteomics analysis results and the X-ray crystallographic data on the 12-subunit Pol II core complex. Cross-links observed from the Pol II–TFIIF complex revealed interactions between the Pol II and TFIIF at the peptide level, which also reflected the dynamic nature of Pol II-TFIIF structure and implied possible Pol II conformational changes induced by TFIIF binding. III. Conformational changes of flexible protein molecules are often associated with specific functions of proteins or protein complexes. To quantitatively measure the differences between protein conformations, I developed a quantitative 3D proteomics strategy which combines isotope labelling and cross-linking with mass spectrometry and database searching. I applied this approach to detect in solution the conformational differences between complement component C3 and its active form C3b in solution. The quantitative cross-link data confirmed the previous observation made by X-ray crystallography. Moreover, this analysis detected the spontaneous hydrolysis of C3 in both C3 and C3b samples. The architecture of hydrolyzed C3-C3(H2O) was proposed based on the quantified cross-links and crystal structure of C3 and C3b, which revealed that C3(H2O) adopted the functional domain arrangement of C3b. This work demonstrated that quantitative 3D proteomics is a valuable tool for conformational analysis of proteins and protein complexes. IV. Encouraged by the achievements in the above applications with relatively large amounts of highly purified material, I explored the application of 3D proteomics on affinity purified tagged endogenous protein complexes. Using an on-beads process which connected cross-linking and an affinity purification step directly, provided increased sensitivity through minimized sample handling. A charge-based enrichment step was carried out to improve the detection of cross-linked peptides. The occurrence of cross-links between complexes was monitored by a SILAC based control. Cross-links observed from low micro-gram amounts of single-step purified endogenous protein complexes provided insights into the structural organization of the S. cerevisiae Mad1-Mad2 complex and revealed a conserved coiled-coil interruption in the S. cerevisiae Ndc80 complex. With this endeavour I have demonstrated that 3D proteomics has become a valuable tool for studying structure of proteins and protein complexes.

BACKGROUND:A hub protein is one that interacts with many functional partners. The annotation of hub proteins, or more generally the protein-protein interaction "degree" of each gene, requires quality genome-wide data. Data obtained using yeast two-hybrid methods contain many false positive interactions between proteins that rarely encounter each other in living cells, and such data have fallen out of favor.RESULTS:We find that protein "stickiness", measured as network degree in ostensibly low quality yeast two-hybrid data, is a more predictive genomic metric than the number of functional protein-protein interactions, as assessed by supposedly higher quality high throughput affinity capture mass spectrometry data. In the yeast Saccharomyces cerevisiae, a protein's high stickiness, but not its high number of functional interactions, predicts low stochastic noise in gene expression, low plasticity of gene expression across different environments, and high probability of forming a homo-oligomer. Our results are robust to a multiple regression analysis correcting for other known predictors including protein abundance, presence of a TATA box and whether a gene is essential. Once the higher stickiness of homo-oligomers is controlled for, we find that homo-oligomers have noisier and more plastic gene expression than other proteins, consistent with a role for homo-oligomerization in mediating robustness.CONCLUSIONS:Our work validates use of the number of yeast two-hybrid interactions as a metric for protein stickiness. Sticky proteins exhibit low stochastic noise in gene expression, and low plasticity in expression across different environments.