Submitted to the faculty of the Bioinformatics Graduate Program in partial fulfillment of the requirements for the degree Master of Science in the School of Informatics, Indiana University December 2005 / The last decade has witnessed numerous proteomic studies which have predicted and successfully confirmed the existence of extended structurally flexible regions in protein molecules. Parallel to these advancements, the last five years of structural bioinformatics has also experienced an explosion of results on molecular recognition and its importance in protein-protein interactions. This work provides an extension to past and ongoing research efforts by looking specifically at the “flexibility and disorder†found in protein sequences involved in molecular recognition processes and known as, Molecular Recognition Elements or Molecular Recognition Features (MoREs or MoRFs, as we call them). MoRFs are relatively short in length (10 – 70 residues length); loosely structured protein regions within longer sequences that are largely disordered in nature. Interestingly, upon binding to other proteins, these MoRFs are able to undergo disorder-to-order transition. Thus, in our interpretation, MoRFs could serve as potential binding sites, and that this binding to another protein lends a functional advantage to the whole protein complex by enabling interaction with their physiological partner. There are at least three basic types of MoRFs: those that form α-helical structures upon binding, those that form β-strands (in which the peptide forms a β-sheet with additional β-strands provided by the protein partner), and those that form irregular structures when bound. Our proposed names for these structures are α-MoRF (also known as α-MoRE, alpha helical molecular recognition feature/element), β-MoRF (beta sheet molecular recognition feature/element), and I-MoRF (Irregular molecular recognition feature/element), respectively. The results presented in this work suggest that functionally significant residual structure can exist in MoRF regions prior to the actual binding event. We also demonstrate profound conformational preferences within MoRF regions for α-helices. We believe that the results from this study would subsequently improve our understanding of protein-protein interactions especially those related to the molecular recognition, and may pave way for future work on the development of protein binding site predictions. We hope that via the conclusions of this work, we would have demonstrated that within only a few of years of its conception, intrinsic protein disorder has gained wide-scale importance in the field of protein-protein interactions and can be strongly associated with molecular recognition.

Exploiting the functional diversity of proteins for fundamental research and biotechnological applications requires their functional organization into micro- and nanostructures while preserving their functional integrity to the highest possible level. My PhD research aimed to establish generic techniques based on photolithography which could be used to control the spatial as well as temporal organization of recombinantly expressed proteins on surfaces. My thesis describes in detail four strategies that I developed for achieving this goal. In the first approach a photo-induced Fenton reaction was used to selectively destroy tris(nitrilotriacetic acid) (tris-NTA) moieties on a surface. UV-irradiation through a photomask allowed localized photo-destruction and targeting of His-tagged proteins to non-irradiated regions. Photo-destruction could also be achieved by scanning selected regions with the UV laser of a confocal laser scanning microscope (CLSM) thus allowing flexible creation and modification of protein patterns. The second strategy was based on the photosensitive nitroveratryloxycarbonyl (NVOC) protection group, which was used to cage amine groups on a surface. Sequential uncaging by UV-irradiation through a photomask followed by reactions with biotin and coenzyme A was used to pattern streptavidin and ybbR-tagged proteins into microstructures. In the third approach a photo-fragmentable Histidine peptide was used to block tris-NTA surfaces against binding of His-tagged proteins. UV-irradiation through a photomask or by using a UV laser in a CLSM cleaved the peptide into short fragments which quickly dissociated from the surface due to loss in multivalency. His-tagged proteins could be efficiently targeted into irradiated regions even from a complex cell lysate. Sequential uncaging and immobilization allowed the construction of multiplexed protein patterns with a high degree of temporal control. The fourth strategy used combined peptide tags comprising of a His-tag as well as a Halo- or ybbR-tag to achieve rapid covalent immobilization of recombinant fusion proteins on surfaces functionalized with specific ligands. In combination with a photo-fragmentable histidine peptide as described above, stable spatio-temporal organization of proteins carrying these combined tags was possible. The techniques developed in this thesis enabled the photolithographical micropatterning of recombinant proteins carrying specific peptide or protein tags on surfaces in a functional manner. Owing to the generic nature of immobilization strategies, coupled with the ease of patterning, highly versatile applications of these methods both in fundamental research as well as bio-technological and analytical applications can be envisioned.

Dans la cellule, les protéines évoluent dans un environnement très dense et interagissent ainsi avec un grand nombre de partenaires spécifiques et non-spécifiques qui entrent en compétition. L’objectif de ma thèse est de caractériser les propriétés physiques et évolutives des surfaces protéiques pour comprendre comment la pression de sélection s’exerce sur les protéines, façonnant leurs interactions et régulant ainsi cette sévère compétition.Pour cela, j’ai développé une méthodologie permettant de caractériser la propension des protéines à interagir avec les protéines de leur environnement, par des approches de docking. La cartographie moléculaire permettant la visualisation et la comparaison des propriétés de la surface des protéines, j’ai donc mis en place un nouveau cadre théorique basé sur une représentation des paysages énergétiques d'interaction par des cartes d'énergies. Ces cartes (en deux dimensions) reflètent de manière synthétique la propension des surfaces protéiques à engager des interactions avec d’autres protéines. Elles sont donc d’un grand intérêt pratique pour déterminer les régions des surfaces protéiques les plus enclines à engager des interactions avec d’autres molécules.Ce nouveau cadre théorique a permis de montrer que les surfaces des protéines comprennent des régions de différents niveaux d'énergies de liaison (régions chaudes, intermédiaires et froides pour les régions d'interaction favorables, intermédiaires et défavorables respectivement).Une partie importante de la thèse a consisté à caractériser les propriétés physico-chimiques et évolutives de ces différentes régions. L'autre partie a consisté à appliquer cette méthode sur plusieurs systèmes : complexes homomériques, protéines du cytosol de S. cerevisiae, familles d'interologues. Ce travail ouvre la voie à un grand nombre d'applications en bioinformatique structurale, telles que la prédiction de sites de liaison, l’annotation fonctionnelle ou encore le design de nouvelles interactions.En conclusion, la stratégie mise en place lors de ma thèse permet d’explorer la propension d’une protéine à interagir avec des centaines de partenaires d'intérêts, et donc d'investiguer le comportement d’une protéine dans un environnement cellulaire spécifique. Cela va donc au-delà de l'utilisation classique du docking "binaire" puisque notre stratégie fournit une vision systémique des interactions protéiques à l’échelle des "résidus". / In the crowded cell, proteins interact with their functional partners, but also with a large number of non-functional partners that compete with the functional ones. The goal of this thesis is to characterize the physical properties and the evolution of protein surfaces in order to understand how selection pressure exerts on proteins, shaping their interactions and regulating this severe competition.To do this I developed a framework based on docking calculations to characterize the propensity of protein surfaces to interact with other proteins. Molecular cartography enables the visualization and the comparison of surface properties of proteins. I implemented a new theoretical framework based on the representation of interaction energy landscapes by 2-D energy maps. These maps reflect in a synthetic manner the propensity of the surface of proteins to interact with other proteins. These maps are useful from a practical point view for determining the regions of protein’s surface that are more prone to interact with other proteins. Our new theoretical framework enabled to show that the surface of proteins harbor regions with different levels of propensity to interact with other proteins (hot regions, intermediate and cold regions to favorable, intermediate and unfavorable regions respectively).A large part of this thesis work consisted in characterizing the physico-chemical properties and the evolution of these regions. The other part of this thesis work consisted in applying this methodology on several study systems: homomeric complexes, cytosolic proteins from S. cerevisiae, families of interologs. This work opens the way to numerous practical applications in structural bioinformatics, such as binding site prediction, functional annotation and the design of new interactions.To conclude, the strategy implemented in this work enable the exploration of the propensity of a protein to interact with hundred of protein partners. It thus enables the investigation of the behavior of a protein in a crowded environment. This application goes beyond the classical use of protein docking as a, because our strategy provides a systemic point of view of protein interactions at an atomic resolution.

Indiana University-Purdue University Indianapolis (IUPUI) / In an effort to advance the methodology in analyzing RNAPII protein-protein interaction network and to determine the role of the CTD in controlling RNAPII transcription, we devised a method to specifically isolate the CTD-associated and CTD-less RNAPII to identify the proteins that interact with both the CTD and the globular core of RNAPII using novel purification scheme coupled to quantitative proteomics.

Indiana University-Purdue University Indianapolis (IUPUI) / Thousands of missense mutations have been found to be associated with human diseases, ~60% of which have been predicted to affect protein stability and/or protein-protein interactions (PPIs). Current proteomic methods for studying the effects of mutations on the cell focus on measures of protein abundance or post-translational modifications (PTMs), which cannot directly be used for PPI analysis. High-throughput methodology to evaluate how mutations in a single protein affect PPI networks would help streamline the characterization of global effects caused by mutant proteins and aid in the prediction of phenotypic outcomes resulting from genomic mutations. Temperature sensitive Mutant Proteome Profiling (TeMPP) is a novel application of a mass spectrometry (MS) based thermal proteome profiling (TPP) approach that measures changes in missense mutant containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. This study measures the impact of temperature sensitivity-inducing missense mutations of proteins in the ubiquitin proteasome system and the transcription termination machinery on the thermal stability of the proteome at large. Results reveal distinct mechanistic details that were not obtained using only steady-state transcriptome and proteome analyses. Furthermore, my data suggests that TeMPP is highly specific to proteins functionally related to the mutated protein of interest and capable of differentiating effects between two proteins in the same complex. Overall, TeMPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, non-biased fashion. Use of this method along with other complementary -omics approaches will help to characterize how missense mutations affect cellular protein homeostasis and thus enable deeper insight into disease phenotypes. / 2022-08-10

The helix-loop-helix (HLH) family of transcription regulatory proteins are fundamental regulators in the processes of cell proliferation and differentiation, cell lineage determination, myogenesis, neurogenesis, and sex determination in a wide range of multicellular organisms. A gene encoding a novel class II HLH protein has recently been identified from a human B-cell eDNA library using a yeast two-hybrid screen. The predicted human ABF -1 polypeptide sequence was used to search the Caenorhabditis elegans genome database for a C. elegans ABF-1 homolog. This bHLH protein, called C. elegans ABF -1 (CeABF -1 ), has a bHLH domain that shares 72% amino acid similarity with its human ABF-1 relative. The expression of the CeABF-1 mRNA has been detected in larval stages L2, L3, L4, and adult, however the mRNA is most highly expressed at the L3 and L4 stages. CeABF -1 protein is capable of heterodimerizing with the human E2A gene product, E4 7. Like human ABF -1, CeABF -1 expression in the presence of the E4 7 protein results in a reduction in E2A mediated gene activation. It has therefore been concluded that CeABF -1 , like human ABF -1 , also acts as a transcriptional repressor. Because C. elegans shares many conserved genes with higher eukaryotic organisms it has become a model organism for in depth genetic studies. It has therefore become increasingly desirable to investigate the possibility of alternative protein-protein interactions that can potentially occur within C. elegans, so it was necessary to construct a C. elegans eDNA library along with the appropriate bait vector expressing the CeABF- 1 protein. The titer ofthe primary library was calculated to be 9.7 x I06 clones, 10-fold greater than minimum titer requirement of I x I 06 clones for a good representational library. Sequencing of the CeABF -I insert confirmed successful construction of a mutation-free bait construct suitable for use in yeast two-hybrid screening. Yeast-two hybrid analysis revealed two new interactors, one of which was identified as an aldose reductase homolog, while the other remains uncharacterized.

The ability of intracellular pathogens to invade and spread from non-phagocytic cell to another is an imperative mechanism broadly investigated in cellular biology. Listeria monocytogenes (Lm) –one example of intracellular pathogens, invades specifically human epithelial cells using its surface proteins Internalin A (InlA) and InlB, respectively. InlA alone is sufficient to internalise the pathogen into the host cells by interacting with human E-cadherin –specifically the N-terminal domain 1 (hEC1). The InlA variant (InlAm) that was previously made to increase the binding affinity to hEC1 was successfully engineered in this study. This variant was found to interact with N-terminal domain 1 of murine E-cadherin (mEC1) by isothermal titration calorimetry (ITC). Previously, the InlAm was reported to allow Lm invasion into M villous cells that express murine N-cadherin –possibly via the N-terminal domain 1 (mNC1). In this study, InlAm did not have affinity for mNC1 or N-terminal domain 1 of human N-cadherin (hNC1) when analysed by ITC –possibly due to amino acid sequences variation from both mEC1 and hEC1. However, by structurally engineering the complexes (InlAm/mNC1 and InlAm/hNC1) and studying their interaction interfaces, it was revealed that mNC1 and hNC1 can be recognised by InlAm just like hEC1. This was supported by the distances between interacting amino acid residues in InlAm/hEC1 crystal structure complex, which were also conserved in the engineered complexes. These observations related to the fact that the N-terminal domains of E- and N-cadherin are structurally conserved, therefore that could have attributed to similarities observed in the engineered complexes. Therefore, future studies would aim at using alternative methods that could support or disprove one of the two findings, that is whether InlAm and any of the N-terminal domains of N-cadherin interact or not. / Dissertation (MSc)--University of Pretoria, 2016. / National Research Foundation (NRF) / The Allan Gray Orbis Foundation / The Mandela Rhodes Foundation / Biochemistry / MSc / Unrestricted

Huntington’s disease (HD) is a neurodegenerative disease caused by a CAG expansion in the HTT gene, which causes an expansion in the polyglutamine tract of the huntingtin protein. In 2018, the cryo-EM structure of the 350 kDa protein huntingtin (Htt) in complex with huntingtin associated protein of 40 kDa (HAP40) was solved, which demonstrated that huntingtin had to be co-translated and complexed with HAP40 to retain structure. However, little is known about HAP40 and thus the biological relevance of this structure. In this project, we transduced cells with fluorescently labelled recombinant apo-Htt or Htt-HAP40 to determine if HAP40 must be complexed with huntingtin in order for huntingtin to have biological activity. This method has not been implemented in HD research and may also improve current fluorescent microscopy models for huntingtin, as it has the advantage of looking at full-length protein rather than small fragments. We also found that with the huntingtin lowering drug branaplam, there is a linear correlation between huntingtin and HAP40 levels, where HAP40 levels will decrease when huntingtin levels are directly decreased as detected by western blot analysis. Furthermore, we found that this lowering effect by branaplam ameliorates DNA repair deficits in HD. With the potential for branaplam to become a treatment for HD, we should continue to test its effect on other HD-associated phenotypes to determine the effect of huntingtin and downstream HAP40 lowering. / Thesis / Master of Health Sciences (MSc)

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