The field of molecular evolution has benefited greatly from the use of ancestral sequence reconstruction as a methodology to better understand the molecular mechanisms associated with functional divergence. The method of ancestral sequence reconstruction has never been experimentally validated despite the method being exploited to generate high profile publications and gaining wider use in many laboratories. The failure to validate such a method is a consequence of 1) our inability to travel back in time to document evolutionary transitions and 2) the slow pace of natural evolutionary processes that prevent biologists from ‘witnessing’ evolution in action (pace viruses). In this thesis research, we have generated an experimentally known phylogeny of fluorescent proteins in order to benchmark ancestral sequence reconstruction methods. The tips/leaves of the fluorescent protein experimental phylogeny are used to determine the performances of various ASR methods. This is the first example of combining experimental phylogenetics and ancestral sequence reconstruction.

Ancestral Sequence Reconstruction is a technique used to statistically infer the most likely ancestor of a set of evolutionarily related sequences, but research which relies solely on protein data has the disadvantage of sequence information being lost upon translation of a protein from its gene transcript, due to the redundancy inherent in the genetic code. In this project, the amino acid sequences, and separately the corresponding codon sequences, of 184 homologous Acetylcholine receptor protein sequences were aligned, and phylogenetic analysis and ancestral sequence reconstruction was performed based on both alignments to infer several ancestral sequences representing important milestones in the evolutionary history of the homologous protein family. To further extract meaningful information from the nucleotide sequences, positive selection analysis was performed on the codon alignment using the Mixed Effects Model of Evolution method, which estimates and compares between the rates of synonymous and non- synonymous mutations across the alignment to detect the occurrence of positive selection events throughout their evolution. The Mixed Effects Model of Evolution can infer positive selection across both sites and evolutionary branches in a sequence alignment, thus highlighting residues along the evolutionary trajectory of the proteins which may have been functionally important in their evolution. Positive selection analysis detected positive selection at a multitude of sites and branches, and by mapping signatures at which selection is strongest with changes in the trajectory of ancestral states, several important sites were chosen as likely to be most valuable for future experimental testing. The implications of this study on the benefits of conducting ancestral sequence reconstruction with protein and codon sequences are discussed.

There is an unmet medical need in the treatment of gout. This type of inflammatory arthritis can be efficiently alleviated by the enzyme uricase. This enzyme breaks down uric acid, the causative agent of gout, so it can be flushed from the body. In humans and the other great apes, uricase is a pseudogene and as such is inactive. Research on therapeutic uricases has focused on using enzymes from naturally occurring sources; however, these foreign proteins can be very antigenic and present a potentially life-threatening safety risk to patients. We address the challenges of developing a safer uricase therapeutic by exploiting evidence that, while inactive, the human pseudogene is expressed in the human body and may be recognized as self by the immune system. To develop a ﾓhuman-likeﾔ? uricase we apply the hybrid computational and experimental approach of Ancestral Sequence Reconstruction to search functional sequence space of uricase proteins to engineer an enzyme with high sequence identity to the human pseudogene, and possessing therapeutic levels of activity for the breakdown of uric acid. This dissertation describes the development and characterization of several uricase leads. The most active ancestral uricase possesses both enhanced in vitro and in vivo stability (in healthy rats) when assayed head-to-head Pegloticase, the only FDA approved uricase for the treatment of gout.

Tracking the evolution of function in enzyme superfamilies is key in understanding how important biological functions and mechanisms have evolved. New genes are being sequenced at a rate that far surpasses the ability of characterization by wet-lab techniques. Moreover, bioinformatics allows for the use of methods not amenable to wet lab experimentation. We now face a situation in which we are aware of the existence of many gene families but are ignorant of what they do and how they function. Even for families with many structurally and functionally characterized members, the prediction of function of ancestral sequences can be used to elucidate past patterns of evolution and highlight likely future trajectories. In this thesis, we apply in silico structure and function methods to predict the functions of protein sequences from two diverse superfamily case studies. In the first, the metallo-β-lactamase superfamily, many members have been structurally and functionally characterised. In this work, we asked how many times the same function has independently evolved in the same superfamily using ancestral sequence reconstruction, homology modelling and alignment to catalytic templates. We found that in only 5% of evolutionary scenarios assessed, was there evidence of a lactam hydrolysing ancestor. This could be taken as strong evidence that metallo-β-lactamase function has evolved independently on multiple occasions. This finding has important implications for predicting the evolution of antibiotic resistance in this protein fold. However, as discussed, the interpretation of this statistic is not clear-cut. In the second case study, we analysed protein sequences of the DUF-62 superfamily. In contrast to the metallo-β-lactmase superfamily, very few members of this superfamily have been structurally and functionally characterised. We used the analysis of alignment, gene context, species tree reconciliation and comparison of the rates of evolution to ask if other functions or cellular roles might exist in this family other than the ones already established. We find that multiple lines of evidence present a compelling case for the evolution of different functions within the Archaea, and propose possible cellular interactions and roles for members of this enzyme family.

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.

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Användningen av biokatalysatorer, särskilt enzymer, inom kemikalie- och läkemedelsindustrin erbjuder betydande fördelar jämfört med de traditionella kemo-katalytiska metoderna som historiskt har dominerat industrin. En viktig klass av enzymer, transaminaser, spelar en central roll i tillverkningen av kirala aminer, som utgör grundläggande byggstenar i dessa industriella sektorer. Denna studie är inriktad på ett specifikt amin transaminas från Silicibacter pomeroyi. Tidigare har detta enzym visat förmåga att katalysera en mängd olika reaktioner för produktion av kirala aminer, men för att realisera dess fulla potential inom industriella tillämpningar krävs förbättrad stabilitet vid högre temperaturer. I motsats till de vanligt förekommande metoderna för proteinteknik, såsom rationell design och riktad evolution, används i denna studie släktsekvensrekonstruktion för att skapa mer temperaturtåliga varianter av detta enzym. Tidigare användning av denna metod har visat sig kunna generera proteiner med högre temperaturtålighet. Genom denna metod, där förfäder till detta enzym återskapas utifrån bevarade sekvenser, förväntas generering av varianter som kan bibehålla sin funktion vid högre temperaturer under en längre tid. Genom att utforska denna alternativa strategi för proteinteknik strävar studien efter att ge mer robusta biokatalysatorer för industriella tillämpningar. Utfallet från denna studie visade att två förfäder hade ökad termostabilitet. Detta visade sig dels genom analys av T5015 som påvisade en 3.9 och 6 C° förbättring för respektive förfader. Vidare påvisade t1/2 mätningar att dessa enzymer kunde utstå 2.06 till 3.72 gånger så lång tid vid 55 C° innan de inaktiverades. De påvisade dock lägre specifik aktivitet vid rumstemperatur, där en bidragande faktor till detta var att enbart en liten fraktion av förfäderna är korrekt veckade. Detta visar att det är nödvändigt med fortsatta förbättringar och fortsatta studier kring substratacceptans och stabilitet i olika lösningsmedel. Sammanfattningsvis påvisar resultaten att släktsekvensrekonstruktion är en proteinteknik som fungerar för att skapa proteiner med ökad termostabilitet och bör ses som ett mer självklart alternativ till riktad evolution och rationell design. / The utilisation of biocatalysts, particularly enzymes, in chemical and pharmaceutical industries presents significant advantages over the traditional chemocatalytic methods that historically dominated the industry. A crucial class of enzymes, transaminases, play a central role in the production of chiral amines, fundamental building blocks in these industrial sectors. This study focuses on a specific amine transaminase from Silicibacter pomeroyi. While this enzyme has previously demonstrated the ability to catalyse a variety of reactions for chiral amine production, realising its full potential in industrial applications requires enhanced stability at higher temperatures. In contrast to commonly employed protein engineering methods such as rational design and directed evolution, this study utilises ancestral sequence reconstruction to generate more temperature-resistant variants of this enzyme. Previous applications of this method have shown promising results in generating proteins with increased thermal stability. Through this approach, wherein ancestors of this enzyme are recreated from extant sequences, it is expected that variants capable of maintaining function at higher temperatures will be produced. By exploring this alternative strategy for protein engineering, the study aims to provide more robust biocatalysts for industrial applications. The outcome of this study is that two ancestors exhibited increased thermostability. This was evidenced by the analysis of T5015, which showed an improvement of 3.9 and 6 °C for each respective ancestor. Furthermore, t1/2 measurements indicated that they remained active for 2.06 to 3.72 fold longer at 55 °C before becoming inactive. However, they exhibited lower specific activity at room temperature, partially due to only a small fraction of the ancestral protein samples being properly folded. This suggests further improvements and continued investigations into substrate acceptance and stability in different solvents are required. In conclusion, this study demonstrates that ancestral sequence reconstruction is a protein engineering technique effective in enhancing protein thermostability and should be considered a more viable alternative to directed evolution and rational design.

La reconstruction et la résurrection moléculaire de protéines ancestrales est au coeur de cette thèse. Alors que les données moléculaires fossiles sont quasi inexistantes, il est possible d'estimer quelles étaient les séquences ancestrales les plus probables le long d'un arbre phylogénétique décrivant les relations de parentés entre séquences actuelles. Avoir accès à ces séquences ancestrales permet alors de tester de nombreuses hypothèses biologiques, de la fonction des protéines ancestrales à l'adaptation des organismes à leur environnement. Cependant, ces inférences probabilistes de séquences ancestrales sont dépendantes de modèles de substitution fournissant les probabilités de changements entre acides aminés. Ces dernières années ont vu le développement de nouveaux modèles de substitutions d'acides aminés, permettant de mieux prendre en compte les phénomènes biologiques agissant sur l'évolution des séquences protéiques. Classiquement, les modèles supposent que le processus évolutif est à la fois le même pour tous les sites d'un alignement protéique et qu'il est resté constant au cours du temps lors de l'évolution des lignées. On parle alors de modèle homogène en temps et en sites. Les modèles récents, dits hétérogènes, ont alors permis de lever ces contraintes en permettant aux sites et/ou aux lignées d'évoluer selon différents processus. Durant cette thèse, de nouveaux modèles hétérogènes en temps et sites ont été développés en Maximum de Vraisemblance. Il a notamment été montré qu'ils permettent d'améliorer considérablement l'ajustement aux données et donc de mieux prendre en compte les phénomènes régissant l'évolution des séquences protéiques afin d'estimer de meilleurs séquences ancestrales. A l'aide de ces modèles et de reconstruction ou résurrection de protéines ancestrales en laboratoire, il a été montré que l'adaptation à la température est un déterminant majeur de la variation des taux évolutifs entre lignées d'Archées. De même, en appliquant ces modèles hétérogènes le long de l'arbre universel du vivant, il a été possible de mieux comprendre la nature du signal évolutif informant de manière non-parcimonieuse un ancêtre universel vivant à plus basse température que ses deux descendants, à savoir les ancêtres bactériens et archéens. Enfin, il a été montré que l'utilisation de tels modèles pouvait permettre d'améliorer la fonctionnalité des protéines ancestrales ressuscitées en laboratoire, ouvrant la voie à une meilleure compréhension des mécanismes évolutifs agissant sur les séquences biologiques / The molecular reconstruction and resurrection of ancestral proteins is the major issue tackled in this thesis manuscript. While fossil molecular data are almost nonexistent, phylogenetic methods allow to estimate what were the most likely ancestral protein sequences along a phylogenetic tree describing the relationships between extant sequences. With these ancestral sequences, several biological hypotheses can be tested, from the evolution of protein function to the inference of ancient environments in which the ancestors were adatapted. These probabilistic estimations of ancestral sequences depend on substitution models giving the different probabilities of substitution between all pairs of amino acids. Classicaly, substitution models assume in a simplistic way that the evolutionary process remains homogeneous (constant) among sites of the multiple sequence alignment or between lineages. During the last decade, several methodological improvements were realised, with the description of substitution models allowing to account for the heterogeneity of the process among sites and in time. During my thesis, I developed new heterogeneous substitution models in Maximum Likelihood that were proved to better fit the data than any other homogeneous or heterogeneous models. I also demonstrated their better performance regarding the accuracy of ancestral sequence reconstruction. With the use of these models to reconstruct or resurrect ancestral proteins, my coworkers and I showed the adapation to temperature is a major determinant of evolutionary rates in Archaea. Furthermore, we also deciphed the nature of the phylogenetic signal informing substitution models to infer a non-parsimonious scenario for the adaptation to temperature during early Life on Earth, with a non-hyperthermophilic last universal common ancestor living at lower temperatures than its two descendants. Finally, we showed that the use of heterogeneous models allow to improve the functionality of resurrected proteins, opening the way to a better understanding of evolutionary mechanisms acting on biological sequences

Visual pigments are light sensitive receptors in the eye that form the basis of sensory visual transduction. This thesis presents three studies that explore visual pigment proteins in vertebrates using a number of computational and experimental methods in an evolutionary framework. The objective is not only to identify, but also to experimentally investigate the functional consequences of genetic variation in vertebrate visual pigments. The focus is on great bowerbirds (Chlamydera nuchalis), which are a model system in visual ecology due to their spectacular behaviour of building and decorating courtship bowers. There are 4 chapters: Chapter 1 introduces background information on visual pigments and vision in birds. Among visual pigment types, the short-wavelength-sensitive (SWS1) pigments have garnered particular interest due to the broad spectral range among vertebrates and the importance of UV signals in communication. Chapter 2 investigates the evolutionary history of SWS1 in vertebrates with a view toward its utility as a phylogenetic marker. Chapter 3 investigates SWS1 evolution and short-wavelength vision in birds, with particular focus on C. nuchalis and its SWS1. The evolution of spectral tuning mechanisms mediating UV/violet vision in passerines and parrots is elucidated in this chapter using site-directed mutagenesis, protein expression, and phylogenetic recreation of ancestral opsins. While cone opsins mediate colour vision in bright light, the rhodopsin visual pigment contained in rod photoreceptors is critical for dim light vision. Detailed characterization of rhodopsin function has only been conducted on a few model systems. Chapter 4 examines C. nuchalis RH1 using a number of functional assays in addition to absorbance spectra, including hydroxylamine sensitivity and the rate of retinal release. This chapter includes an investigation into the role of amino acid mutations typical of dim-light adapted vertebrates, D83N and A292S, in regulating functional properties of bovine and avian RH1s using site-directed mutagenesis. Together these chapters describe naturally occurring mutations in visual pigments and explore the way they can influence visual perception. These represent one of the few investigations of visual pigments from a species that is not a model lab organism and form a significant contribution to the field of visual pigment biochemistry and evolution.

Visual pigments are light sensitive receptors in the eye that form the basis of sensory visual transduction. This thesis presents three studies that explore visual pigment proteins in vertebrates using a number of computational and experimental methods in an evolutionary framework. The objective is not only to identify, but also to experimentally investigate the functional consequences of genetic variation in vertebrate visual pigments. The focus is on great bowerbirds (Chlamydera nuchalis), which are a model system in visual ecology due to their spectacular behaviour of building and decorating courtship bowers. There are 4 chapters: Chapter 1 introduces background information on visual pigments and vision in birds. Among visual pigment types, the short-wavelength-sensitive (SWS1) pigments have garnered particular interest due to the broad spectral range among vertebrates and the importance of UV signals in communication. Chapter 2 investigates the evolutionary history of SWS1 in vertebrates with a view toward its utility as a phylogenetic marker. Chapter 3 investigates SWS1 evolution and short-wavelength vision in birds, with particular focus on C. nuchalis and its SWS1. The evolution of spectral tuning mechanisms mediating UV/violet vision in passerines and parrots is elucidated in this chapter using site-directed mutagenesis, protein expression, and phylogenetic recreation of ancestral opsins. While cone opsins mediate colour vision in bright light, the rhodopsin visual pigment contained in rod photoreceptors is critical for dim light vision. Detailed characterization of rhodopsin function has only been conducted on a few model systems. Chapter 4 examines C. nuchalis RH1 using a number of functional assays in addition to absorbance spectra, including hydroxylamine sensitivity and the rate of retinal release. This chapter includes an investigation into the role of amino acid mutations typical of dim-light adapted vertebrates, D83N and A292S, in regulating functional properties of bovine and avian RH1s using site-directed mutagenesis. Together these chapters describe naturally occurring mutations in visual pigments and explore the way they can influence visual perception. These represent one of the few investigations of visual pigments from a species that is not a model lab organism and form a significant contribution to the field of visual pigment biochemistry and evolution.