Interrogation and engineering of RAD51:BRC repeat interactions

The maintenance of genetic information is a fundamental function of every organism. The DNA in every human cell endures thousands of lesions per day, and eukaryotes have developed a sophisticated response to tackle this damage. Double strand breaks (DSBs) are the most toxic type of DNA damage, potentially leading to chromosomal rearrangements and tumorigenesis. Homologous recombination (HR) is a repair pathway that uses a homologous template to faithfully repair the DSBs. Central to HR is a recombinase protein RAD51, which forms a nucleoprotein filament with the broken ends, and allows efficient homology search. RAD51 is a promising therapeutic target in oncology. BRCA2 is a crucial mediator of RAD51 function and interacts with RAD51 through a set of 8 BRC repeats. A BRC repeat consists of two modules, an FxxA and an LFDE module, each containing hot-spot residues that mediate binding. In this work, I set out to utilise these for the pharmacological targeting of human RAD51. First, using biophysical methods, I investigated a novel, high-affinity chimeric repeat BRC8-2 composed of FxxA and LFDE modules from different natural repeats. I determined the X-ray crystallographic structure of its complex, which allowed me to uncover the determinants of high affinity binding. I applied these findings to the design of novel macrocyclic peptide inhibitors of RAD51. A method for cysteine stapling of recombinantly produced peptides was optimised to yield the correct product of high purity. The optimised stapling methodology provides a promising general approach for recombinant production and evaluation of cysteine-stapled peptides. Peptides were rationally designed in a structure-guided manner, and a variety of stapling architectures were evaluated for binding. The resulting purified molecules exhibit high potency and are stable in serum. Further crystallographic studies shed light on how the stapling moiety affects the peptide binding mode. The derived peptides serve as novel modalities for targeting Rad51 protein-protein interactions and can inform subsequent development of therapeutic drugs. In addition to the human proteins, I investigated the BRC:RAD51 interactions in orthologs from Leishmania infantum, the causative agent of leishmaniasis. Using crystallography and biophysical methods, I uncovered novel features of BRC repeat binding. The presented work expands our understanding of the structural determinants of homologous recombination.MRC DT

RosettaRemodel: A Generalized Framework for Flexible Backbone Protein Design

We describe RosettaRemodel, a generalized framework for flexible protein design that provides a versatile and convenient interface to the Rosetta modeling suite. RosettaRemodel employs a unified interface, called a blueprint, which allows detailed control over many aspects of flexible backbone protein design calculations. RosettaRemodel allows the construction and elaboration of customized protocols for a wide range of design problems ranging from loop insertion and deletion, disulfide engineering, domain assembly, loop remodeling, motif grafting, symmetrical units, to de novo structure modeling

Navigating the Extremes of Biological Datasets for Reliable Structural Inference and Design

Structural biologists currently confront serious challenges in the effective interpretation of experimental data due to two contradictory situations: a severe lack of structural data for certain classes of proteins, and an incredible abundance of data for other classes. The challenge with small data sets is how to extract sufficient information to draw meaningful conclusions, while the challenge with large data sets is how to curate, categorize, and search the data to allow for its meaningful interpretation and application to scientific problems. Here, we develop computational strategies to address both sparse and abundant data sets. In the category of sparse data sets, we focus our attention on the problem of transmembrane (TM) protein structure determination. As X-ray crystallography and NMR data is notoriously difficult to obtain for TM proteins, we develop a novel algorithm which uses low-resolution data from protein cross-linking or scanning mutagenesis studies to produce models of TM helix oligomers and show that our method produces models with an accuracy on par with X-ray crystallography or NMR for a test set of known TM proteins. Turning to instances of data abundance, we examine how to mine the vast stores of protein structural data in the Protein Data Bank (PDB) to aid in the design of proteins with novel binding properties. We show how the identification of an anion binding motif in an antibody structure allowed us to develop a phosphate binding module that can be used to produce novel antibodies to phosphorylated peptides - creating antibodies to 7 novel phospho-peptides to illustrate the utility of our approach. We then describe a general strategy for designing binders to a target protein epitope based upon recapitulating protein interaction geometries which are over-represented in the PDB. We follow this by using data describing the transition probabilities of amino acids to develop a novel set of degenerate codons to create more efficient gene libraries. We conclude by describing a novel, real-time, all-atom structural search engine, giving researchers the ability to quickly search known protein structures for a motif of interest and providing a new interactive paradigm of protein design

Pulling apart the intermolecular interactions of the Parkinson’s disease linked protein alpha synuclein

Amyloidoses are a group of protein misfolding diseases that are characterised by the abnormal accumulation of highly ordered filamentous assemblies known as amyloid. This phenomenon is associated with more than 50 human diseases, some of which are the most debilitating disorders that threaten human health today. Many of these disorders have age as the main contributing risk factor and, therefore, pose an ever-increasing risk in the developed world with aging societies. Despite intense research, much remains unknown about the fundamental processes driving protein aggregation in these diseases and there are few disease modifying treatments available. A protein that undergoes amyloid formation and causes disease is the intrinsically disordered neuronal protein α-synuclein (αSyn), the aggregation of which leads to several diseases including Parkinson’s disease (PD) which is the second-most common neurodegenerative disorder that affects 2–3% of the population ≥65 years of age. Importantly, the toxic species on the aggregation pathway are difficult to identify and determine in molecular detail. This thesis was motivated by this fact and aimed to study the initial intermolecular events in αSyn self-assembly (dimerisation) on a single molecule scale. Single molecule force spectroscopy (SMFS) methodologies were therefore utilised in order to study these early protein-protein interaction events. A display system was firstly designed and validated in which small regions of highly aggregation-prone sequences can be presented in a protein scaffold in a robust and reproducible manner for SMFS studies. It was demonstrated that intermolecular interactions of these sequences could be analysed by implementing this system. A novel heterodimeric interaction between the central aggregation-prone regions of αSyn (residues 71-82) and the same region of its human homologue γSynuclein (γSyn), were revealed by using this system. Further study led to the finding that this interaction played a role in the inhibiting the aggregation of αSyn. The dimerisation interaction of full length αSyn has also been analysed in this thesis and several important findings have been demonstrated. The SMFS experiments show that force-resistant structure forms in the dimeric species of αSyn and that this structure is dependent on the environmental conditions. SMFS utilising different immobilisation regimes of αSyn have also allowed the location of a novel interaction interface involving the N-terminal region of the protein. Further SMFS experiments investigating the effects of salt and hydrophobicity have on dimerisation, alongside bioinformatics analyses of the protein sequence led to the hypotheses that the dimeric interaction is driven by hydrophobic stretches in the N-terminal region, but modulated by local electrostatics. In vitro aggregation assays and SMFS on non-aggregation-prone synuclein homologues (β- and γSyn) indicated that that this interaction is protective against aggregation, considering these finding with existing literature prompted speculation that the interactions observed in SMFS may indeed be physiologically relevant. This may therefore be an important finding in regards to targeting the aggregation process with disease modifying agents

Kinetic And Thermodynamic Analysis Of Genetically Engineered Inorganic Binding Peptides For Bionanotechnology

Tez (Doktora) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2009Thesis (PhD) -- İstanbul Technical University, Institute of Science and Technology, 2009Bu çalışma kapsamında genetik mühendisliği kullanılarak oluşturulmuş anorganiklere seçici olarak bağlanan peptidlerin (GEPI) kinetik ve termodinamik araçlarla moleküler tanıma özellikleri incelenmiştir. Faj gösterim ya da hücre yüzey gösterim teknikleri kullanılarak seçilen GEPI moleküllerinin karakterizasyonu yapılmıştır. GEPI’lerin moleküler karakterizasyonu, adsorpsiyon kinetikleri ve termodinamikleri, ikincil yapı çalışmaları ile bir bütünlük içerisinde gerçekleştirilmiştir. GEPI’lerin bağlanma kinetik ve termodinamikleri yüzey plazmon rezonans spektrometresi ve kuvartz kristal mikroterazi kullanılarak gerçekleştirilmiştir. Altın , silika ve platin yüzeylere bağlanan peptidlerin sadece afiniteleri değil, aynı zamanda malzeme seçicilikleri de incelenmiştir. Bir durum çalışması olarak, altın yüzeye bağlanan peptid için yapı-aktivite ilişkisi de, termodinamik bir bakış açısı kullanılarak gerçekleştirilmiştir. GEPI’lerin, moleküler karakterizayonu takiben, moleküler bağlayıcı ve malzeme sentezleyicisi olarak kullanımları araştırılmıştır. Bu bağlamda, GEPI’ler, moleküler bağlayıcı olarak, alkali fosfataz enziminin altın, silika ve platin yüzeylerine immobilizasyonu ve yarı iletken nanotaneciklerin LED aygıtlarının yüzeylerine tutuklanması çalışmalarında, moleküler tutucu olarak ise, Huntington hastalık etmen proteininin fibrilasyonunun gerçek zamanlı olarak incelenmesinde kullanılmıştır. Ayrıca, morfoloji kontrollü silika sentezi için GEPI’lerin malzeme yapıcı olarak kullanımları gösterilmiştir. GEPI’lerin birçok değişik biyo-nano teknolojik uygulamalarda moleküler bağlayıcı olarak kullanımları, nano- biyo- arakesitindeki zorlukları aşabilmek için uygun moleküler araçlar olduğunu ortaya koyacak şekilde sunulmuştur.Molecular recognition properties of genetically engineered inorganic binding peptides (GEPI), were investigated with respect to their binding kinetics and thermodynamics. Phage display or cell surface display selected GEPIs for different materials were characterized. In the molecular characterization of GEPIs, adsorption kinetics and thermodynamics of GEPIs were realized in conjugation with secondary structural analysis of the peptides. To characterize binding kinetics and thermodynamics of GEPIs, surface plasmon resonance sensor, quartz crystal microbalance were employed. Both the affinity and materials selectivity of gold, platinum and silica binding peptides were investigated. The effect of conformational constraints and multiple repeats on the affinity and material selectivity of GEPIs was tested. As a case study the structureaffinity relationship in gold binding peptides was analyzed, using a thermodynamic approach. Following the molecular characterization of GEPIs, proof of demonstration studies were carried out, in which GEPIs were utilized as molecular linkers and material synthesizer. In this perspective, we utilized GEPIs as molecular linkers, for the immobilization of alkaline phosphatase on gold, platinum and silica; for the targeted assembly of semiconductor nanoparticles on LED device; as molecular erector for the real time monitoring of fibrillation in Huntington’s disease; and finally as synthesizer in the morphology controlled synthesis of silica. In the scope of the thesis, GEPIs have been exploited to create new generation of biomimetic molecular linkers. Various practical bio-nanotechnological examples in the usage of GEPIs as molecular linkers were demonstrated as representative towards their wide range of applications to overcome the challenges at the nano- bio- interface.DoktoraPh

Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy

This book is a compendium of scientific articles submitted to a Special Issue of International Journal of Molecular Sciences, fostered by MDPI and curated by Dr. Annamaria Sandomenico and Dr. Menotti Ruvo from the Institute of Biostructure and Bioimaging of the National Research Council. All articles underwent a rigorous peer review and were selected to highlight the properties that make monoclonal antibodies and their functional fragments some of the most useful and versatile assets in therapy and diagnosis

Design, expression, and characterization of FimH antigen as single recombinant protein or exposed on nanoparticles

Uropathogenic Escherichia coli (UPEC) accounts for approximately 85% of all urinary tract infections (UTIs), causing a global economic burden. E. coli is one of the pathogens mentioned in the ESKAPEE list drafted by OMS, meaning that the increasing antibiotic resistance acquired by UPEC is and will be a serious health problem in the future. Amongst the immunogenic antigens exposed on the surface of UPEC, FimH represent a potential target for vaccine development, since it is involved in the early stages of infection. As already demonstrated, immunizations with FimH elicit functional antibodies that prevent UPEC infections even though the number of doses required to elicit a strong immune response is not optimal. In this work, we aimed to stabilize FimH as a soluble recombinant antigen exploiting the donor strand complementation mechanism by generating different chimeric constructs constituted by FimH and FimG donor strand. To explore the potential of self-assembling nanoparticles to display FimH through genetic fusion, different constructs have been computationally designed and produced. In this work a structure-based design, using available crystal structures of FimH and three different NPs was performed to generate different constructs with optimized properties. Despite the different conditions tested, all the constructs designed (single antigen or chimeric NPs), resulted to be un-soluble proteins in E. coli. To overcome this issue a mammalian expression system has been tested. Soluble antigen expression was achieved for all constructs tested in the culture supernatants. Three novel chimeric NPs have been characterized by transmission electron microscopy (TEM) confirming the presence of correctly assembled NPs displaying UPEC antigen. In vivo study has shown a higher immunogenicity of the E. coli antigen when displayed on NPs surface compared to the single recombinant antigen. The antibodies elicited by chimeric NPs showed a higher functionality in the inhibition of bacterial adhesion

Doctor of Philosophy

dissertationMolecular biorecognition is at the heart of all biological processes and has come to the center stage in designing new biologics. Coiled-coils result from molecular biorecognition of multiple protein α-helices. We have designed a series of coiled-coil motifs that self-assemble into supercoils, which in turn function as physical cross-linkers in the construction of hybrid biomaterials. This dissertation describes our endeavors following the hypothesis below: the selfassembly of coiled-coil forming peptides into coiled-coils can be used as a cross-linking mechanism in the construction of hybrid hydrogels and the development of a drug-free macromolecular therapeutic. In the first part, a macromonomer free radical copolymerization strategy was developed and HPMA graft copolymers containing coiled-coil motifs of different chain lengths were synthesized. Results indicated that the primary structure of these motifs greatly influenced gel formation. At least four heptads were needed to mediate effective gelation. The gelation process was highly dependent on the environmental temperature and copolymer concentration. In the second part, a drug-free macromolecular therapeutic was designed to take full advantage of the facts that CD20 is one of the most reliable biomarkers for B-cell non- Hodgkin's lymphoma (NHL), and cross-linking of CD20 antigens induces apoptosis of B cells. The drug-free therapeutic was composed of two components: Fab' fragment of 1F5 iv anti-CD20 antibody conjugated with one coiled-coil motif (CCE) and polyHPMA copolymer grafted with multiple copies of the complementary coiled-coil motif (CCK). In vitro studies showed the conjugates could colocalize on Raji cell surfaces and a clinically relevant magnitude of apoptosis was achieved. A systemic NHL murine model was used to evaluate in vivo efficacy. Significant delay of hind-limb paralysis onset was observed after treatment. In groups receiving multiple-dose treatment, the surviving mice were disease-free and no presence of Raji B cell in bone marrow could be detected after 100 days. In summary, the possibility of integrating molecular biorecognition of short nonnatural coiled-coil motifs into new smart biomaterials is presented. This specific biorecognition can be used with or without adjuvant pharmacology to mediate a biofunctional process in combating diseases

Computational design with flexible backbone sampling for protein remodeling and scaffolding of complex binding sites

Dissertation presented to obtain the Doutoramento (Ph.D.) degree in Biochemistry at the Instituto de Tecnologia Qu mica e Biol ogica da Universidade Nova de LisboaComputational protein design has achieved several milestones, including the design of a new protein fold, the design of enzymes for reactions that lack natural catalysts, and the re-engineering of protein-protein and protein-DNA binding speci city. These achievements have spurred demand to apply protein design methods to a wider array of research problems. However, the existing computational methods have largely relied on xed-backbone approaches that may limit the scope of problems that can be tackled. Here, we describe four computational protocols - side chain grafting, exible backbone remodeling, backbone grafting, and de novo sca old design - that expand the methodological protein design repertoire, three of which incorporate backbone exibility. Brie y, in the side chain grafting method, side chains of a structural motif are transplanted to a protein with a similar backbone conformation; in exible backbone remodeling, de novo segments of backbone are built and designed; in backbone grafting, structural motifs are explicitly grafted onto other proteins; and in de novo sca olding, a protein is folded and designed around a structural motif. We developed these new methods for the design of epitope-sca old vaccines in which viral neutralization epitopes of known three-dimensional structure were transplanted onto nonviral sca old proteins for conformational stabilization and immune presentation.(...