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The ability of mutations to change the fold of a protein provides evolutionary pathways to new structures. To study hypothetical pathways for protein fold evolution, we designed intermediate sequences between Xfaso1 and Pfl6, two homologous Cro proteins that have 40% sequence identity but adopt all–α and α+β folds, respectively. The designed hybrid sequences XPH1 and XPH2 have 70% sequence identity to each other. XPH1 is more similar in sequence to Xfaso1 (86% sequence identity) while XPH2 is more similar to Pfl6 (80% sequence identity). NMR solution ensembles show that XPH1 and XPH2 have structures intermediate between Xfaso1 and Pfl6. Specifically, XPH1 loses α-helices 5 and 6 of Xfaso1 and incorporates a small amount of β-sheet structure; XPH2 preserves most of the β-sheet of Pfl6 but gains a structure comparable to helix 6 of Xfaso1. These findings illustrate that the sequence space between two natural protein folds may encode a range of topologies, which may allow a protein to change its fold extensively through gradual, multistep mechanisms. Evolving a new fold may have consequences, such as a strained conformation. Here we show that Pfl6 represents an early, strained form of the α+β Cro fold resulting from an ancestral remnant of the all-α Cro proteins retained after the fold switch. This nascent fold can be stabilized through deletion mutations in evolution, which can relieve the strain but may also negatively affect DNA-binding function. Compensatory mutations that increase dimerization appear to offset these effects to maintain function. These findings suggest that new folds can undergo mutational editing through evolution, which may occur in parallel pathways with slightly different outcomes.

Human acidic fibroblast growth factor (FGF-1) is a member of the £]-trefoil superfamily and exhibits a characteristic three-fold tertiary structure symmetry. However, evidence of this symmetry is not readily apparent at the level of the primary structure. This suggests that while selective pressures may exist to retain (or converge upon) a symmetric tertiary structure, other selective pressures have resulted in divergence of the primary structure during evolution. Using intra-chain and homologue sequence comparisons for 19 members of this family of proteins, we have designed mutants of FGF-1 that constrain a subset of core-packing residues to three-fold symmetry at the level of the primary structure. The consequences of these mutations upon structure, stability, folding and unfolding kinetics have been evaluated using a combination of x-ray crystallography, differential scanning calorimetry, isothermal equilibrium denaturation and stopped flow protein refolding/unfolding kinetics. An alternative core packing group has been introduced into FGF-1. The alternative core is very similar from the wild type (WT) core with regard to structure, stability, folding and unfolding kinetics. The remaining asymmetry within the protein core is related to asymmetry in the tertiary structure. The removal of tertiary structure asymmetry greatly increases protein stability and results in a conversion from three-state to a two-state folding pathway. The tertiary structure asymmetry is intimately linked to functional regions of the protein. Surprisingly, upon deletion of the functional insertions, the mutant protein is approximately 80 times more potent than the wild type form as determined by functional bioassays. The results show that the ƒÒ-trefoil superfold is compatible with a three-fold symmetric constraint upon the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new protein arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of ƒÒ-trefoil proteins by utilizing symmetric structural complementarity. This study implies that a symmetric architecture of the £]-trefoil fold is kinetically and thermodynamically ¡§fit¡¨. / Dissertation / PhD

A L-Asparaginase (L-ASNase) é uma enzima tetramérica bacteriana, utilizada em sessões de quimioterapia. Essa enzima depleta os aminoácidos asparagina (Asn) e glutamina (Gln), transformando-os em aspartato (Asp) ou glutamato (Glu), respectivamente, e em amônia. Contudo, a L-ASNase pode induzir resposta imune, levando à produção de anticorpos antiasparaginase, uma causa importante de resistência ao medicamento. Uma L-ASNase ideal seria aquela com alta atividade e estabilidade e baixo potencial imunogênico, porém, as L-ASNases utilizadas na terapêutica não reúnem essas características simultaneamente. Por essa razão, o presente trabalho utilizou técnicas de mutagênese randômica, a fim de criar uma nova proteoforma de L-ASNase de E. chrysanthemi com uma melhor atividade e estabilidade. Além disso, foram estudadas condições de cultivo em agitador metabólico, visando à otimização de condições de produção. Foi criada uma biblioteca com 1.056 clones, e desses, 19 foram selecionados por apresentarem atividade superior ou igual à enzima selvagem quando dosada em extrato bruto. Dentre eles, dois mutantes se destacaram por apresentarem a atividade específica glutaminásica diferente da enzima selvagem. Análises in silico indicam que o mutante 9-6D apresentou diminuição de desordem estrutural e epítopos imunogênicos. O mutante 9-5F demonstrou uma diminuição da porcentagem da atividade glutaminásica quando comparada a enzima selvagem. O estudo de produção do mutante 9-5F indicou que a temperatura de indução, seguida da concentração do indutor, são os parâmetros mais relevantes para a otimização da produção de L-ASNase de E. chrysanthemi mutante. / L-Asparaginase (L-ASNase) is a bacterial tetrameric enzyme used in chemotherapy sessions that deplete asparagine (Asn) and glutamine (Gln), transforming them into Aspartate (Asp) or glutamate (Glu), respectively, and ammonia. However, L-ASNase can induce immune response leading to the production of anti-asparaginase antibody, an important cause of drug resistance. Ideally, L-ASNase would be one with high activity, high stability and low immunogenic potential, but the L-ASNases commercially available today do not present these characteristics simultaneously. For this reason, this study used techniques of random and site-directed mutagenesis in order to create a new proteoform of E. chrysanthemi L-ASNase with improved activity and stability. In addition, culture conditions were studied in a metabolic shaker, aiming at the optimization of production conditions. A library with 1,056 clones was created, and of these clones, 19 were selected because they had activity superior or equal to the wild-type enzyme in crude protein extract. Among them, 2 mutants stood out for having different glutaminase specific activity in relation to wild-type enzyme. The 9-6D mutant also showed decreased structural disorder and immunogenic epitopes. The 9-5F mutant demonstrated a decrease in percentage of glutaminase activity when compared to the wild-type enzyme. The production study of 9-5F mutant indicated that the induction temperature followed by the inductor concentration are the most relevant parameters for the production optimization of E. chrysanthemi mutant L-ASNase.

A L-Asparaginase (L-ASNase) é uma enzima tetramérica bacteriana, utilizada em sessões de quimioterapia. Essa enzima depleta os aminoácidos asparagina (Asn) e glutamina (Gln), transformando-os em aspartato (Asp) ou glutamato (Glu), respectivamente, e em amônia. Contudo, a L-ASNase pode induzir resposta imune, levando à produção de anticorpos antiasparaginase, uma causa importante de resistência ao medicamento. Uma L-ASNase ideal seria aquela com alta atividade e estabilidade e baixo potencial imunogênico, porém, as L-ASNases utilizadas na terapêutica não reúnem essas características simultaneamente. Por essa razão, o presente trabalho utilizou técnicas de mutagênese randômica, a fim de criar uma nova proteoforma de L-ASNase de E. chrysanthemi com uma melhor atividade e estabilidade. Além disso, foram estudadas condições de cultivo em agitador metabólico, visando à otimização de condições de produção. Foi criada uma biblioteca com 1.056 clones, e desses, 19 foram selecionados por apresentarem atividade superior ou igual à enzima selvagem quando dosada em extrato bruto. Dentre eles, dois mutantes se destacaram por apresentarem a atividade específica glutaminásica diferente da enzima selvagem. Análises in silico indicam que o mutante 9-6D apresentou diminuição de desordem estrutural e epítopos imunogênicos. O mutante 9-5F demonstrou uma diminuição da porcentagem da atividade glutaminásica quando comparada a enzima selvagem. O estudo de produção do mutante 9-5F indicou que a temperatura de indução, seguida da concentração do indutor, são os parâmetros mais relevantes para a otimização da produção de L-ASNase de E. chrysanthemi mutante. / L-Asparaginase (L-ASNase) is a bacterial tetrameric enzyme used in chemotherapy sessions that deplete asparagine (Asn) and glutamine (Gln), transforming them into Aspartate (Asp) or glutamate (Glu), respectively, and ammonia. However, L-ASNase can induce immune response leading to the production of anti-asparaginase antibody, an important cause of drug resistance. Ideally, L-ASNase would be one with high activity, high stability and low immunogenic potential, but the L-ASNases commercially available today do not present these characteristics simultaneously. For this reason, this study used techniques of random and site-directed mutagenesis in order to create a new proteoform of E. chrysanthemi L-ASNase with improved activity and stability. In addition, culture conditions were studied in a metabolic shaker, aiming at the optimization of production conditions. A library with 1,056 clones was created, and of these clones, 19 were selected because they had activity superior or equal to the wild-type enzyme in crude protein extract. Among them, 2 mutants stood out for having different glutaminase specific activity in relation to wild-type enzyme. The 9-6D mutant also showed decreased structural disorder and immunogenic epitopes. The 9-5F mutant demonstrated a decrease in percentage of glutaminase activity when compared to the wild-type enzyme. The production study of 9-5F mutant indicated that the induction temperature followed by the inductor concentration are the most relevant parameters for the production optimization of E. chrysanthemi mutant L-ASNase.

Many alpha-helical membrane proteins contain internal symmetries, indicating that they might have evolved through a gene duplication and fusion event Here, we have characterized internal duplications among membrane proteins of known structure and in three complete genomes We found that the majority of large transmembrane (TM) proteins contain an internal duplication The duplications found showed a large variability both in the number of TM-segments included and in their orientation Surprisingly, an approximately equal number of antiparallel duplications and parallel duplications were found However, of all 11 superfamilies with an internal duplication, only for one, the AcrB Multidrug Efflux Pump, the duplicated unit could be found in its nonduplicated form An evolutionary analysis of the AcrB homologs indicates that several independent fusions have occurred, including the fusion of the SecD and SecF proteins into the 12-TM-protein SecDF in Brucella and Staphylococcus aureus In one additional case, the Vitamin B-12 transporter-like ABC transporters, the protein had undergone an additional fusion to form protein with 20 TM-helices in several bacterial genomes Finally, homologs to all human membrane proteins were used to detect the presence of duplicated and nonduplicated proteins This confirmed that only in rare cases can homologs with different duplication status be found, although internal symmetry is frequent among these proteins One possible explanation is that it is frequent that duplication and fusion events happen simultaneously and that there is almost always a strong selective advantage for the fused form / <p>authorCount :4</p>

alpha-Helical membrane proteins are important for many biological functions. Due to physicochemical constraints, the structures of membrane proteins differ from the structure of soluble proteins. Historically, membrane protein structures were assumed to be more or less two-dimensional, consisting of long, straight, membrane-spanning parallel helices packed against each other. However, during the past decade, a number of the new membrane protein structures cast doubt on this notion. Today, it is evident that the structures of many membrane proteins are equally complex as for many soluble proteins. Here, we review this development and discuss the consequences for our understanding of membrane protein biogenesis, folding, evolution, and bioinformatics. / <p>authorCount :3</p>

How do microbes adapt to challenges from the environment? In this thesis, two distinct cases were examined through structural and biochemical methods. In the first, we followed a real-time protein evolution of HisA to a novel function. The second case was fusidic acid (FA) resistance mediated by the protein FusB in Staphylococcus aureus. In the first study, the aim was to understand how mutants of HisA from the histidine biosynthetic pathway could evolve a novel TrpF activity and further evolve to generalist or specialist enzymes. We solved the crystal structure of wild type Salmonella enterica HisA in its apo-state and the structures of the mutants D7N and D7N/D176A in complex with the substrate ProFAR. These two distinct complex structures showed us the coupled conformational changes of HisA and ProFAR before catalysis. We also solved crystal structures of ten mutants, some in complex with substrate or product. The structures indicate that bi-functional mutants adopt distinct loop conformations linked to the two functions and that mutations in specialist enzymes favor one of the conformations. We also observed biphasic relationships in which small changes in the activities of low-performance enzymes had large effects on fitness, until a threshold, above which large changes in enzyme performance had little effect on fitness. Fusidic acid blocks protein translation by locking elongation factor G (EF-G) to the ribosome after GTP hydrolysis in elongation and recycling of bacterial protein synthesis. To understand the rescue mechanism, we solved the crystal structure of FusB at 1.6Å resolution. The structure showed that FusB is a two-domain protein and C-terminal domain contains a treble clef zinc finger. Using hybrid constructs between S. aureus EF-G that binds to FusB, and E. coli EF-G that does not, the binding determinants were located to domain IV of EF-G. This was further supported by small-angle X-ray scattering studies of the FusB·EF-G complex. Using single-molecule methods, we observed FusB frequently binding to the ribosome and rescue of FA-inhibited elongation by effects on the non-rotated state ribosome. Ribosome binding of FusB was confirmed by isothermal titration calorimetry.

For my dissertation research I explored the diversity and functional evolution of steroid hormone receptors (SRs) in animals and the physiological implications of the evolution of antifreeze proteins in Antarctic notothenioid fishes. For the former, I discovered multiple new SRs from the vast and under-sampled swath of animal diversity known as invertebrates. I used the sequences of these and other newly discovered related receptors in combination with genomic data and molecular phylogenetic techniques to revise the understanding of the evolutionary history of this important gene family. While previous studies have suggested that vertebrate SR diversity arose from a gene duplication in an ancestor of all bilaterian animals, my work presents strong evidence that this duplication occurred much later, at the base of the chordates. Furthermore, to determine the implications of added diversity and a revised phylogeny on inferences of the functional evolution of SRs, I functionally characterized heretofore-unknown SRs from hemichordates, an acoelomate flatworm, and a chaetognath and statistically reconstructed and functionally characterized ancestral SRs. My results expand the known sequence and functional repertoire of SRs in animals while reinforcing the previous inference that all SRs evolved from an estrogen-sensitive ancestral receptor. I also explored the consequences of the evolution of antifreeze proteins in Antarctic notothenioid fishes, a crucial adaptation to their icy, polar environment. These special proteins adsorb to ice crystals that enter a fish's body and prevent further growth, thereby averting death. I discovered that, in addition to their lifesaving growth-inhibiting ability, AFPs also prevent the melting of internal ice crystals at temperatures above the expected equilibrium melting point. Together with a decade-long temperature record of one of the coldest fish habitats on earth, my experimental results show that the evolution and expression of antifreeze proteins is accompanied by a potentially detrimental consequence: the lifelong accumulation of ice inside these fishes' bodies. This dissertation includes previously published co-authored material as well as unpublished co-authored material. / 2017-01-14

Proteins perform an incredible array of functions facilitated by a diverse set of biochemical properties. Changing these properties is an essential molecular mechanism of evolutionary change, with major questions in protein evolution surrounding this topic. How do new functional biochemical features evolve? How do proteins change following gene duplication events? I used the S100 protein family as a model to probe these aspects of protein evolution. The S100s are signaling proteins that play a diverse range of biological roles binding Calcium ions, transition metal ions, and other proteins. Calcium drives a conformational change allowing S100s to bind to diverse peptide regions of target proteins. I used a phylogenetic approach to understand the evolution of these diverse biochemical features. Chapter I comprises an introduction to the disseration. Chapter II is a co-authored literature review assessing available evidence for global trends in protein evolution. Chapter III describes mapping of transition metal binding onto a maximum likelihood S100 phylogeny. Transition metal binding sites and metal-driven structural changes are a conserved, ancestral features of the S100s. However, they are highly labile at the amino acid level. Chapter IV further characterizes the biophysics of metal binding in the S100A5 lineage, revealing that the oft–cited Ca2+/Cu2+ antagonism of S100A5 is likely due to an experimental artifact of previous studies. Chapter V uses the S100 family to investigate the evolution of binding specificity. Binding specificity for a small set of peptides in the duplicate S100A5 and S100A6 clades. Ancestral sequence reconstruction reveals a pattern of clade-level conservation and apparent subfunctionalization along both lineages. In chapter VI, peptide phage display, deep-sequencing, and machine-learning are combined to quantitatively reconstruct the evolution of specificity in S100A5 and S100A6. S100A5 has subfunctionalized from the ancestor, while S100A6 specificity has shifted. The importance of unbiased approaches to measure specificity are discussed. This work highlights the lability of conserved functions at the biochemical level, and measures changes in specificity following gene duplication. Chapter VII summarizes the results of the dissertation, considers the implications of these results, and discusses limitations and future directions. This dissertation includes both previously published/unpublished and co- authored material.