Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection. / QC 20100729

The interaction between two 6 kDa proteins has been investigated. The studied complex of micromolar affinity (Kd) consists of the Z domain derived from staphylococcal protein A and the related protein ZSPA-1, belonging to a group of binding proteins denoted affibody molecules generated via combinatorial engineering of the Z domain. Affibody-target protein complexes are good model systems for structural and thermodynamic studies of protein-protein interactions. With the Z:ZSPA-1 pair as a starting point, we determined the solution structure of the complex and carried out a preliminary characterization of ZSPA-1. We found that the complex contains a rather large (ca. 1600 Å2) interaction interface with tight steric and polar/nonpolar complementarity. The structure of ZSPA-1 in the complex is well-ordered in a conformation that is very similar to that of the Z domain. However, the conformation of the free ZSPA-1 is best characterized by comparisons with protein molten globules. It shows a reduced secondary structure content, aggregation propensity, poor thermal stability, and binds the hydrophobic dye ANS. This molten globule state of ZSPA-1 is the native state in the absence of the Z domain, and the ordered state is only adopted following a stabilization that occurs upon binding. A more extensive characterization of ZSPA-1 suggested that the average topology of the Z domain is retained in the molten globule state but that it is represented by a multitude of conformations. Furthermore, the molten globule state is only marginally stable, and a significant fraction of ZSPA-1 exists in a completely unfolded state at room temperature. A complete thermodynamic characterization of the Z:ZSPA-1 pair suggests that the stabilization of the molten globule state to an ordered three helix structure in the complex is associated with a significant conformational entropy penalty that might influence the binding affinity negatively and result in an intermediate-affinity (µM) binding protein. This can be compared to a dissociation constant of 20-70 nM for the complex Z:Fc of IgG where Z uses the same binding surface as in Z:ZSPA-1. Structure analyses of Z in the free and bound state reveal an induced fit response upon complex formation with ZSPA-1 where a conformational change of several side chains in the binding surface increases the accessible surface area with almost 400 Å2 i.e. almost half of the total interaction surface in the complex. Two cysteine residues were introduced at specific positions in ZSPA-1 for five mutants in order to stabilize the conformation of ZSPA-1 by disulfide bridge formation. The mutants were thermodynamically characterized and the binding affinity of one mutant showed an improvement by more than a factor of ten. The improvement of the introduced cysteine bridge correlates with an increase in binding enthalpy rather than with entropy. Further analysis of the binding entropy suggests that the conformational entropy change in fact is reduced but its favorable contribution is opposed by a less favorable desolvation enthalpy change. These studies illustrate the structural and thermodynamic complexity of protein-protein interactions, but also that this complexity can be dissected and understood. In this study, a comprehensive characterization of the ZSPA-1 affibody has gained insight into the intricate mechanisms involved in complex formation. These theories were supported by the design of a ZSPA-1 mutant with improved binding affinity. / QC 20100924

Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the ZSPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: ZTaq, originally selected to bind Taq DNA polymerase, and anti-ZTaq, an anti-idiotypic binder to ZTaq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free ZSPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Å2 total surface area and 10 out of 13 varied residues in ZSPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of ZSPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free ZSPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of ZSPA-1 upon binding seriously reduces the binding affinity. The ZTaq:anti-ZTaq complex buries in total 1672 Å2 surface area and all varied positions in anti-ZTaq are directly involved in binding. The main differences between the Z:ZSPA-1 and the ZTaq:anti-ZTaq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither ZTaq nor anti-ZTaq shows the molten globule behaviour seen for ZSPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general. / QC 20101025

Le but du projet est de mettre au point de nouveaux outils enzymatiques permettant la production de chitinoligosaccharides de taille et de degré d'acétylation parfaitement contrôlés pour l'étude d'enzymes impliquées dans la biosynthèse, la biodégradation et la modification de la chitine, et pour l'étude de récepteurs protéiques d'origine animale ou végétale. Des études récentes ont montré que les oligomères de la chitine et leurs dérivés sont des molécules qui interviennent dans les phénomènes symbiotiques et de reconnaissance hôte-pathogène dans le règne végétal. Ces molécules sont utilisées en agrochimie comme biofertilisants, et potentiellement en phytosanitaire. Ils sont connus également pour posséder de nombreuses activités biologiques dans le domaine de la santé (effets antimicrobiens, anticancéreux, anti-inflammatoires, immunostimulants...). Si les activités de cette classe d'oligosaccharides sont parfaitement reconnues, leurs modes d'actions restent encore à éclaircir, ce qui nécessite de disposer de molécules pures aux structures chimiques parfaitement contrôlées. La production d'oligomères de la chitine nécessite traditionnellement la mise en œuvre d'une chimie fastidieuse, qui peut être facilitée par une approche chimio-enzymatique.Dans le cadre de cette thèse, nous souhaitons donc développer des outils enzymatiques permettant la synthèse d'une bibliothèque de molécules de taille et de degré d'acétylation contrôlés en vue d'études structure-activité biologique. Pour cela, nous chercherons à produire des N-désacétylases et des chitinases dans différents systèmes d'expression et à caractériser leur activité afin de générer une panoplie de molécules de structure moléculaire parfaitement définie à partir de fragments saccharidiques issus de la biomasse. Les molécules ainsi préparées pourront ensuite être modifiées de façon chimio-sélective afin d'obtenir des sondes photoactivables et/ou biotinylées pour la caractérisation de récepteurs, des substrats fluoro- ou chromogéniques pour le dosage spécifique d'activités enzymatiques ou encore des lipochitinoligosaccharides capables de favoriser la croissance des plantes. Les approches utilisées pour mener à bien ce projet pluridisciplinaire seront : l'ingénierie et la production de protéines recombinantes, la caractérisation biochimique d'activités enzymatiques ainsi que la synthèse chimio-enzymatique et la modification chimique d'oligosaccharides qui devront être caractérisés d'un point de vue physicochimique. Il s'agit d'un projet intégré dans une collaboration nationale financée par l'ANR réunissant des équipes de l'Université de Grenoble, de Lyon, d'Orsay, et l'entreprise Bayer CropScience. / The aim of the project is to develop new tools for enzymatic production of chitooligosaccharides size and degree of acetylation perfectly controlled for the study of enzymes involved in biosynthesis, biodegradation and modification of chitin and for the study of protein receptors of animal or vegetable origin. Recent studies have shown that oligomers of chitin and its derivatives are molecules involved in the phenomena of symbiotic and host-pathogen recognition in plants. These molecules are used as agrochemicals biofertilizers. They are also known to possess numerous biological activities in the field of health (anti-microbial, anti-cancer, anti-inflammatory, immunostimulant ...). If the activities of this class of oligosaccharides are well recognized, their modes of action remain to be clarified, which requires having pure molecules with chemical structures perfectly controlled. The production of chitin oligomers traditionally requires the implementation of a tedious chemistry, which can be facilitated by a chemoenzymatic approach.As part of this thesis, we want to develop enzymatic tools for the synthesis of a library of molecules of size and degree of acetylation controlled studies for structure-biological activity. For this, we will seek to produce N-deacetylase and chitinases in different expression systems and characterize their activity to generate a variety of molecules well-defined molecular structure from saccharide fragments derived from biomass. The molecules thus prepared can then be modified so as to achieve chemoselective photoactivatable probes and / or biotinylated for the characterization of receptors, substrates or fluoro-chromogenic assay for specific enzyme activities or lipo-chitooligosaccharides can promote plant growth. The approaches used to complete this multidisciplinary project are: engineering and production of recombinant proteins, the biochemical characterization of enzymatic activities and chemoenzymatic synthesis and chemical modification of oligosaccharides to be characterized from the point of physicochemical view. This is an integrated project in a national collaboration funded by the ANR with teams from the University of Grenoble, Lyon, Orsay, and Bayer CropScience.

Submitted by Marco Antonio Oliveira da Silva (maosilva@ipen.br) on 2016-12-22T11:54:03Z No. of bitstreams: 0 / Made available in DSpace on 2016-12-22T11:54:03Z (GMT). No. of bitstreams: 0 / A endostatina (ES) é uma proteína inibidora da angiogênese, com ação específica sobre células endoteliais em proliferação, utilizada para tratamento de tumores sólidos. No entanto, o elevado efeito antitumoral da ES observado em animais não é reproduzido em humanos. Com o intuito de potencializar a eficácia terapêutica da ES, produzimos duas proteínas híbridas com dois domínios funcionais. O primeiro domínio é a ES, que apresenta especificidade por células endoteliais ativadas, dirigindo estas proteínas de fusão às células endoteliais em proliferação, promovendo sua internalização e seu efeito inibitório. Como segundo domínio funcional utilizamos os domínios BH3 próapoptóticos de duas proteínas BH3-only com o objetivo de promover a liberação de citocromo C e desencadear o processo de apoptose, aumentando a ação antiangiogênica da ES. Neste trabalho, foram desenhadas duas proteínas de fusão que contêm o domínio BH3 das potentes proteínas pró apoptóticas PUMA e BIM (ES-PUMA e ES-BIM), que deveriam apresentar efeito antiangiogênico potencializado em relação à ES selvagem. A inserção dos fragmentos de DNA codificantes para os domínios BH3 de PUMA e BIM no vetor contendo o gene da ES (pET-ES) foram realizadas por mutagênese sítiodirigida. Estas proteínas de fusão recombinantes foram expressas como corpos de inclusão em E.coli, renaturadas utilizando processo que utiliza alta pressão e purificadas em resina de afinidade por heparina. O tratamento de células endoteliais com as proteínas ES-PUMA e ES-BIM não levou à queda de viabilidade em ensaio de MTS ou de apoptose avaliado por citometria de fluxo, em comparação com os resultados obtidos pelo tratamento com ES. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

This thesis discusses the issue of predicting of the effect of amino acid substitutions on protein funkcion, based on phylogenetic analysis method, inspired by tool MAPP. Significant number of genetic diseases is caused by nonsynonymous SNPs manifested as single point mutations on the protein level. The ability to identify deleterious substitutions could be useful for protein engineering to test whether the proposed mutations do not damage protein function same as for targeting disease causing harmful mutations. However the experimental validation is costly and the need of predictive computation methods has risen. This thesis describes desing and implementation of a new in silico predictor based on the principles of evolutionary analysis and dissimilarity between original and substituting amino acid physico-chemical properties. Developed algorithm was tested on four datasets with 74,192 mutations from 16,256 sequences in total. The predictor yields up to 72 % accuracy and in the comparison with the most existing tools, it is substantially less time consuming. In order to achieve the highest possible efficiency, the optimization process was focused on selection of the most suitable (a) third-party software for calculation of a multiple sequence alignment, (b) overall decision threshold and (c) a set of physico-chemical properties.

HIV-1 is a dangerous retrovirus which represents one of the world's leading health problems. HIV-1 infection is incurable and without proper treatment by antiretroviral therapy it leads to death within several years. Despite intensive research, no HIV vaccine is currently available. This thesis presents a new and unique approach which has not been used for vaccine development yet. The promising strategy is based on small binding proteins that can elicit broadly neutralizing HIV-1 antibodies by mimicking their epitopes. The aim of this project was to select and characterize small binding proteins that can successfully mimic the surface of viral envelope glycoproteins that is recognized by the broadly neutralizing HIV-1 antibodies PGT121 and PGT126. Proteins were selected from a highly complex combinatorial protein library derived from a new type of scaffold called Myomedin. Firstly, the extent of the protein library was narrowed down using the ribosome display. Then the direct sandwich ELISA screening was applied to select scaffold variants that interact with the target antibodies. In total over 200 variants were tested and several promising candidates were found. These Myomedin variants were purified, biochemically and biophysically characterised and the best ones were used to immunize mice....

Stabilní proteiny nacházejí široké uplatnění v řadě medicínských a biotechnologických aplikacích. Přírodní proteiny se vyvinuly tak, aby fungovaly převážně v mírných podmínkách uvnitř buněk. V důsledku toho vzniká zájem o stabilizaci proteinů za účelem jejich širšího uplatnění také v průmyslovém prostředí. Obor proteinového inženýrství se v posledních letech rozvinul do úrovně umožňující modifikovat proteiny pro různá využití, ačkoliv identifikace stabilních mutací je stále zatížená drahou a časově náročnou experimentální prací. Výpočetní metody se proto uplatňují jako atraktivní alternativa, která dovoluje prioritizovat potenciálně stabilizující mutace pro laboratorní práci. Během posledních let bylo vyvinuto velké množství výpočetních strategií: i) výpočty energie pomocí silových polí, ii) evoluční metody, iii) strojové učení a iv) kombinace více přístupů. Spolehlivost a využití nástrojů jsou často limitovány predikcí pouze jednobodových mutací, které mají malý dopad na stabilitu proteinů, zatímco sofistikovanější metody pro predikci multibodových mutací vyžadují větší množství práce na straně uživatele. Hlavním záměrem této práce je poskytnout uživatelům plně automatizované metody, umožňující návrh vysoce stabilních vícebodových mutantů bez potřeby pokročilých znalostí bioinformatických nástrojů a zkoumaného proteinu. V této práci jsou prezentovány následující nástroje a databáze:  FireProt je plně automatizovaná metoda pro návrh stabilních vícebodových mutantů z kategorie tzv. hybridních přístupů. Ve svém výpočetním jádře spojuje jak energetické tak i evoluční metody, přičemž evoluční informace jsou užívány především jako filtry pro časově náročné výpočty energií. Kromě detekce potenciálně stabilizujících mutací se FireProt rovněž snaží spojit tyto mutace do jednoho vícebodového mutanta s minimalizací rizika vzniku antagonistických efektů. FireProt-ASR je plně automatizovaná platforma pro rekonstrukci ancestrálních sekvencí, která dovoluje uživatelům využít tuto strategii bez nutnosti velkého objemu manuální práce a hluboké znalosti zkoumaného proteinu. FireProt-ASR řeší všechny kroky ancestrální rekonstrukce, včetně sběru biologicky relevantních sekvencí, konstrukce zakořeněného fylogenetického stromu a rekonstrukce ancestrálních sekvencí.HotSpotWizard je nástroj pro návrh mutací a mutačních knihoven za účelem zlepšení stability a aktivity zkoumaných proteinů. Nástroj dovoluje provést i širší analýzu za využití čtyř různých strategií běžně používaných v oboru proteinového inženýrství: i) identifikace evolučně variabilních pozic v blízkosti katalytických kapes a tunelů, ii) identifikace pohyblivých regionů, iii) výpočet sekvenčního konsensu a iv) identifikace korelovaných pozic.FireProt-DB je databáze dostupných experimentálních dat popisujících stabilitu proteinů. Hlavním účelem této databáze je standardizovat data v oblasti proteinové stability, poskytnout uživatelům platformu k jejich snadnému ukládání a umožnit intuitivní vyhledávání, které by mohly být využité k trénování nových nástrojů s využitím technik strojového učení.

Peptide binders are short proteins that bind to larger proteins. Due to peptide binders having high specificity and being cheap to synthesize, they are a prime candidate for drug design. Creating new proteins in silico can be divided into three steps: protein backbone generation, sequence design, and computational filtering. With the release of AlphaFold2 (AF2), protein structure prediction is possible with atomic accuracy, even for peptide-protein complexes. Structure predictions enables some important computational filtering, which saves time and resources before doing experimental validation. After the release of AF2 together with the advancements in generative AI, new computational methods for the first two design steps have been developed. In this report, three different methods for backbone generation and sequence design were evaluated and compared: EvoBind, RFdiffusion, and ProteinMPNN. The latter two were developed solely for protein design. However, their broad application capabilities allowed for peptide development, which was implemented in this report. In total, 5500 peptides for 55 different protein targets were designed by each method, with the purpose to evaluate the performance and identify advantages of the methods. Combining the three methods in unexplored ways allowed for additional evaluation as well as gaining deeper understanding of how the methods worked. Whilst not being one-shot design approaches, all methods used in the report showed potential of being able to design de novo peptide binders with varying degree of in silico success. The methods’ peptide design success rate ranged from 16% to 2.6%. The direct evolution approach applied with EvoBind generated most peptide binder backbones with close binding to the specified interfaces. Using the message passing neural network (MPNN) in ProteinMPNN, the sequences designed were optimized for binding affinity and resulted in sequences that were easier for AF2 to predict. The methods allow for the potential development of peptide binder therapeutics to become more cost- and time efficient, on the basis that AF2’s predictions are aligned with the expressed peptides’ bindings and structures.

Tyrosine site-specific recombinases (Y-SSRs) are DNA editing enzymes that play a valuable role for the manipulation of genomes, due to their precision and versatility. They have been widely used in biotechnology and molecular biology for various applications, and are slowly finding their spot in gene therapy in recent years. However, the limited number of available Y-SSR systems and their often narrow target specificity have hindered the full potential of these enzymes for advanced genome engineering. In this PhD thesis, I conducted a comprehensive investigation of novel Y-SSRs and their potential for advancing genome engineering. This PhD thesis aims to address the current limitations in the genetic toolbox by identifying and characterizing novel Cre-type recombinases and demonstrating their impact on the directed evolution of designer recombinases for precise genome surgery. To achieve these aims, I developed in a collaboration a comprehensive prediction pipeline, combining a rational bioinformatical approach with knowledge of the biological functions of recombinases, to enable high success rate and high-throughput identification of novel tyrosine site-specific recombinase (Y-SSR) systems. Eight putative candidates were molecularly characterized in-depth to ensure their successful integration into future genome engineering applications. I assessed their activity in prokaryotes (E. coli) and eukaryotes (human cell lines), and determined their specificity in the sequence space of all known Cre- type target sites. The potential cytotoxicity associated with cryptic genomic recombination sites was also explored in the context of recombinase applicability. This approach allowed the identification of novel Y-SSRs with distinct target sites, enabling simultaneous use of multiple Y-SSR systems, and provided knowledge that will facilitate the assignment of novel and known recombinases to specific uses or organisms, ensuring their safe and effective implementation. The introduction of these novel Y-SSRs into the genome engineering toolbox opens up new possibilities for precise genome manipulation in various applications. The broader targetability offered by these enzymes could accelerate the development of novel gene therapies, as well as advance the understanding of gene function and regulation. Moreover, these recombinases could be used to design custom genetic circuits for synthetic biology, allowing researchers to create more complex and sophisticated cellular systems. Finally, I introduced the novel Y-SSRs into efforts aimed at developing designer recombinases for precise genome surgery, demonstrating their impact on accelerating the directed evolution process. Therapeutically relevant recombinases with altered DNA specificity have been developed for excision or inversion of specific DNA sequences. However, the potential for evolving recombinases capable of integrating large DNA cargos into naturally occurring lox-like sites in the human genome remained untapped so far. Thus, I embarked on evolving the Vika recombinase to mediate the integration of DNA cargo into a native human sequence. I discovered that Vika could integrate DNA into the voxH9 site in the human genome, and then, I enhanced the process through directed evolution. The evolved variants of Vika displayed a marked improvement in integration efficiency in bacterial systems. However, the translation of these results into mammalian systems has not yet been entirely successful. Despite this, the study laid the groundwork for future research to optimize the efficiency and applicability of Y-SSRs for genomic integration. In summary, this thesis made significant strides in the identification, characterization, and development of novel Y-SSRs for advanced genome engineering. The comprehensive prediction pipeline, combined with in-depth molecular characterization, has expanded the genetic toolbox to meet the growing demand for better genome editing tools. By exploring efficiency, cross-specificity, and potential cytotoxicity, this research lays the foundation for the safe and effective application of novel Y-SSRs in various therapeutic settings. Furthermore, by demonstrating the potential of these recombinases to improve efforts in creating designer recombinases through directed evolution, this research has opened new avenues for precise genome surgery. The successful development and implementation of these novel recombinases have the potential to revolutionize gene therapy, synthetic biology, and our understanding of gene function and regulation.