The relationship between enzyme dynamics and enzymatic catalysis has become a central topic in modern enzymology, and studies in this area promise to enrich our current understanding of catalysis in biological systems. Escherichia coli dihydrofolate reductase (EcDHFR) has been a frequent subject of study in the context of protein dynamics, due to its small size, biological ubiquity, and the fact that its structural, kinetic and mechanistic characteristics are well established. Intrinsic kinetic isotope effects (KIEs) have proven to be highly sensitive probes of the role of dynamics in EcDHFR catalyzed reaction, as they circumvent the kinetic complexity of the enzyme-catalyzed reactions, and extract information directly pertaining to the chemical step. Previously, studies of their temperature-dependence were used to probe the effect of mutations at residues distant from the active site upon the hydride-transfer reaction catalyzed by EcDHFR. The results of these experiments supported the presence of a network of residues that were dynamically linked to the hydride-transfer step, and were in excellent agreement with computational studies predicting the presence of such a network. This thesis aims to extend upon these results to study the nature and extent of the dynamic network in EcDHFR, both by using an established experimental methods and by developing new biophysical probes of protein dynamics in this system. The major experimental methodology utilized in the following chapters is the determination and analysis of KIEs in a variety of EcDHFR mutants. To facilitate these measurements, new synthetic routes to a range of isotopically labeled nicotinamide cofactors have been developed. Some of the labeled materials have been used to establish a sensitive, triple-isotope technique to competitively measure deuterium isotope effects in enzyme-catalyzed reactions in EcDHFR. Synthesized materials were usd to measure the temperature dependence of intrinsic KIEs in selected dynamically altered mutants of EcDHFR, viz. W133F and F125M DHFR. Crystal structures have been obtained for both these mutants as well as for the previously studied G121V isozyme, and the combination of kinetic and structural information discussed in the context of catalytically important dynamic fluctuations in EcDHFR. Pressure-dependence of deuterium KIEs is also developed as a tool to probe the role of dynamics and tunneling in the EcDHFR reaction, with the ultimate aim of establishing high-pressure KIE measurements as a complementary method to variable temperature measurements. Finally, molecular recognition force spectroscopy (MRFS) measurements of an EcDHFR self-assembled monolayer (SAM) on gold are described. The surprisingly active enzymatic SAM has been shown to be a promising platform for future MRFS experiments to measure the forces involved in EcDHFR dynamics. All together, these studies advanced our ability to study the role of enzyme dynamics and quantum tunneling in enhancing their chemistry.

The studies of enzymes from extreme sources have gained significance due to their increasing potential applications. The proteins from halophiles (salt loving) have adapted to challenging environmental conditions and require salt for their structure and function. How halophilic proteins adapt to a hypersaline environment is still an intriguing question. It is important to mimic the environmental conditions of the sample under investigation with experimental techniques. In this study, structure and dynamic features of a halophilic enzyme have been investigated under high salt conditions. The acquisition of NMR data on high salt samples has always been problematic. We have devised a simple and elegant approach for obtaining NMR data for a protein in a high salt buffer that allows for virtually complete 1H, 13C, and 15N assignments. These data were then used to calculate the NMR derived structure of Haloferax volcanii dihydrofolate reductase in 3.5 M NaCl. Structure calculations showed that this protein folds in a similar manner as investigated in the crystal structures of Haloferax volcanii dihydrofolate reductase and Escherichia coli dihydrofolate reductase. To understand the effect of salt on flexibility as well as activity, NMR relaxation studies at 3.5 M and 1.0 M salt concentration were carried out. NMR dynamics of this enzyme revealed that the loss of activity as the salt concentration is lowered is due to lose in the inherent flexibility across the backbone, particularly in the catalytic loops.

No description available.

Dihydrofolate reductase is an enzyme that catalyzes the reaction of dihydrofolate to tetrahydrofolate with nicotinamide adenine dinucleotide phosphate. Haloferax volcanii is a “salt-loving” microorganism found in the Dead Sea. Such microorganisms have adapted to their extreme environments and now require extremely high salt concentrations to survive. The study of dihydrofolate reductase from Haloferax volcanii with the substrate folate bound, which is the focus of the work presented in this dissertation, offers further understanding of this adaptation. The effect that high salt concentration has on this enzyme is not fully understood; however, this dissertation includes the investigation of the structure of a halophilic enzyme with substrate bound, providing new information in this uncertain field of research. In particular, the results shown here (along with future studies) can be applied towards the question of how an extremely salty environment affects enzyme function, stability, solubility, and flexibility.Triple resonance nuclear magnetic resonance spectra of the 17.9 kDa enzyme with bound substrate have been interpreted. 1H, 13C, and 15N backbone and side chain chemical shifts were specifically assigned to the amino acids that make up the enzyme. These assignments revealed overall similarities to the chemical shifts of the apo enzyme, with some exceptions. These exceptions are of particular interest as they are due to the binding of folate. Secondary structural analysis of the nuclear magnetic resonance data based on the chemical shift index showed that the binary complex has similar secondary structural features to the folate-bound dihydrofolate reductase from mesophilic Escherichia coli.The structure of dihydrofolate reductase from Haloferax volcanii with folate bound has been investigated using Crystallography and nuclear magnetic resonance system structure calculations, suggesting an overall similarity to the X-ray crystallography and nuclear magnetic resonance structures of apo dihydrofolate reductase from Haloferax volcanii. The results of the structure calculation and secondary structural analysis of the chemical shift assignments suggest secondary structural features including a beta-sheet in the core of the enzyme composed of 8 beta-strands, surrounded by 4 alpha-helices, and 4 major loops. The structure of the binary complex was compared to its mesophilic counterpart, the folate-bound dihydrofolate reductase from Escherichia coli, which was investigated by X-ray crystallography. The results of the work described in this dissertation do not agree with loop structure of the mesophilic complex.

Protein motions are complex, including occurring at different time scales, and their roles in enzyme-catalyzed reactions have always been of great interest among enzymologists. In order to characterize the potential factors that play a role on the chemical step of enzymatic reactions, variants of dihydrofolate reductase have been used as a benchmark system to study the motions of proteins correlated with the chemical step. A “global dynamic network” of coupled residues in Escherichia coli dihydrofolate reductase (ecDHFR), which assists in catalyzing the chemical step, has been demonstrated through quantum mechanical/molecular mechanical and molecular dynamic (QM/MM/MD) simulations, as well as bioinformatic analyses. A few specific residues — M42, G121, and I14 — were shown to function synergistically with measurements of single turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. Although similar networks have been found in other enzymes, the general features of these networks are still unclear. This project focuses on exploring homologous residues of the proposed global network in human DHFR through computer simulations and measurements of the temperature dependence of KIEsint. The mutants M53W and S145V, both remote residues, showed significant decreases in catalytic efficiency. Non-additive isotope effects on activation energy were observed between M53 and S145, indicating their synergistic effect on hydride transfer in human DHFR. Apart from the effects of the conserved residues, we also extend our studies to exploring three potential phylogenetic events that account for the discrepancies between E. coli and human DHFR. They are L28, PP insertion and PEKN insertions by phylogenetic sequence analysis. Two of them (N23PP and G51PEKN E. coli DHFR) have been proved to be important both by MD simulation and experimental probe of KIEs measurement. The experiments have found that PP insertion itself rigidified the M20 loop and motions coupled to hydride transfer were impaired, however, loop rigidification was improved after incorporating PEKN. Furthermore, deletion of PP and PEKN of the engineered human enzyme also show a similar outcome. However, the effect of the key residue of L28 is not clear. In this project, we have step-wise engineered the human DHFR to be like hagfish (F31M) and E. coli (F32L). And it is found out that there is an increase in the temperature dependence of KIEs when the enzyme was bacterilized into a more primitive variant. This indicates that not only is residue F32 important and correlated with the chemical step as indicated by bioinformatic studies, but it is possible to trace the evolutionary trajectory. A triple mutation F32L-PP26N-PEKN62G on the human DHFR was also conducted, and it is not surprising to find out that the temperature dependence of KIEs has retained its behavior like wild-type human DHFR. These results suggest that the three predicted phylogenetically coherent events coevolved together to maintain the evolutionary preservation of the protein dynamics to enable H-tunneling from well-reorganized active sites. As has been indicated by the previous project, as the enzyme evolves, the active site of the enzyme will “reorganize” to form the optimal transition state for chemical step (from F32L-F32M-wild type DHFR). Here in this project, we aimed to systematically address this point of view through a series of cyclic permutation DHFR from directed evolutions. As this primitive enzyme is 7 orders of magnitude less efficient than the well-evolved human DHFR, together with four generations of evolved variants (cp, cp’ and cp”), this provides a good model system for explorations of the molecular basis of enzyme evolution. It is found that the organizations of transition state are improved before the catalytic efficiency is enhanced as the enzyme evolves.

Utilitzant un mètode de síntesi prèviament desenvolupat s'han obtingut una sèrie d'oligodesoxiribonucleòtids cíclics de seqüència complementària al mRNA de la dihidrofolat reductasa (DHFR). Els oligonucleòtids cíclics sintetitzats han mostrat activitat citotòxica sobre cèl·lules de hàmster xinès, presumiblement per un mecanisme antisentit que implica la inhibició de l'expressió de la DHFR. La mida de l'oligonucleòtid cíclic influeix en la seva activitat citotòxica i per a tots ells existeix una concentració a la que l'activitat és més gran. L'activitat dels oligonucleòtids cíclics, que és menor que la dels oligonucleòtids lineals amb unions internucleosídiques de tipus fosforotioat, i és molt major que la dels lineals que contenen unions naturals fosfodiester, és atribuïble al seu caràcter cíclic i a la conseqüent resistència front a les exonucleases.Per a possibilitar l'obtenció d'oligonucleòtids cíclics que també siguin resistents a l'acció de les endonucleases, s'ha comprovat que la metodologia general de síntesi és aplicable a l'obtenció d'oligonucleòtids cíclics amb unions internucleosídiques fosforotioat en totes les posicions, excepte en la que uneix l'oligòmer al suport sòlid en el precursor lineal.D'altra banda, s'ha desenvolupat un esquema de síntesi per a, a partir de 5-bromouracil i en 8 etapes, obtenir dos nous monòmers d'àcid nucleic peptídic que incorporen com a nucleobase modificada a anàlegs de citosina, anomenats abraçadores de guanina. Aquests són potencialment capaços de formar fins a quatre i cinc enllaços d'hidrogen amb una guanina d'una seqüència complementària.L'elaboració de l'anell de fenoxazina de les abraçadores de guanina té com a etapa clau la incorporació del 2-aminoresorcinol en la posició 4 de l'uracil. Per a això, es parteix del N1-tert-butoxicarbonilmetil derivat del 5-bromouracil i s'activa la posició 4 del 5-bromouracil en forma de triazole derivat per a la substitució nucleòfila amb el 2-aminoresorcinol.En l'esquema desenvolupat, la ciclació de l'aminoresorcinol derivat del 5-bromouracil per a formar la fenoxazina i la incorporació dels apèndixs de 2-etanolamina i 2-hidroxietilguanidina, condueix a millors resultats si es duu a terme en aquest ordre degut a que els esquemes de síntesi de les dues abraçadores tenen un intermedi més comú i els rendiments globals són superiors. S'ha dut a terme la síntesi en fase sòlida mitjançant l'estratègia Fmoc/Bhoc d'una sèrie de decàmers de PNA, uns que contenen únicament nucleobases naturals i uns altres en els que alguna citosina de les anteriors seqüències ha estat substituïda per abraçadores de guanina. En els acoblaments dels monòmers s'ha emprant HATU com a agent activant i han tingut lloc amb rendiments entre el 95 i el 98% per als que contenen nucleobases naturals i lleugerament inferiors (aproximadament del 90%) per als monòmers modificats amb les abraçadores. Per primer cop s'ha posat a punt la utilització de l'energia de microones per a activar les reaccions d'acoblament en la síntesi de PNA. La qualitat dels productes crus de síntesis de PNA és millor que quan les reaccions es realitzen a temperatura ambient. Finalment, per tal d'avaluar l'afinitat dels decàmers de PNA sintetitzats per les seves seqüències de DNA complementàries s'han enregistrat les corbes de fusió per espectroscòpia d'UV dels corresponents dúplexs PNA-DNA a partir de les quals s'han determinat les temperatures de fusió, TM.Per als PNAs que contenen únicament nucleobases naturals s'ha observat una bona correlació lineal entre les temperatures de fusió i el nombre de parells de bases G-C del dúplex.La substitució en els PNAs d'alguna citosina per abraçadores de guanina, tant si contenen un grup amino com un grup guanidino, produeix una pronunciada desestabilització dels corresponents dúplexs. S'interpreta que la capacitat de les abraçadores de guanina per a estabilitzar dúplexs PNA-DNA és enormement dependent del seu entorn (seqüència i tipus d'àcid nucleic).

In prokaryotes, protein synthesis and folding are often coupled, and the protein begins to fold from the N-terminus as it is being synthesized. It has been hypothesised that there could be kinetic coupling of the speed of translation and the folding, which means that an altered rate of synthesis can cause a possible misfolding of the protein. Testing this hypothesis will be impactful for protein misfolding diseases such as Alzheimer’s, Parkinson’s, Huntington’s etc., and also help in the study of the effect of synonymous, non-synonymous and rare codon changes on a protein. However, research works in this regard are far and few and none of them have been carried out in a homologous in vitro system. This project is an attempt to study the co-translational folding of Escherichia coli protein dihydro folate reductase (DHFR) using an E. coli reconstituted transcription/ translation system (RTTF) in vitro. The preparatory phase involves: preparation of UAG mutants of the DHFR DNA (for site-specific incorporation of fluorescent dyes), preparation of amber tRNAs which recognise the UAG codons, aminoacylation of the tRNAs and labelling the amino acids with fluorescent dyes. The experimental phase involves: incorporation of each of the fluorescent amino acids in the protein during in vitro synthesis in steady-state, observing incorporation of the same in stopped-flow spectrofluorimeter, attempting to observe fluorescent resonance energy transfer (FRET) between the two dyes due to co-translational folding. The preparatory and experimental phases were completed successfully, and it has been established that the amino acids with the fluorescent moieties can be incorporated site specifically in the mutant protein. The synthesis of the protein was observed using stopped-flow spectrometer for each of the fluorescent amino acids individually.  The synthesis of the mutants using two sets of dye pairs was also observed using a steady-state fluorimeter as well as stopped-flow spectrofluorimeter and the FRET between the two fluorophores was obtained. Although further experiments are required to fully validate and standardize this technique,  it will, even now,  aid in the study of the folding of proteins in a cell-free system.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Le dihydrofolate réductase (DHFR) est la principale cible du méthotrexate, un important composant du traitement de la leucémie lymphoblastique aiguë (LLA). Une association des polymorphismes du promoteur de DHFR avec l’issue de la LLA a été mise en évidence au laboratoire. Une survie sans événement (EFS) réduite corrélait avec les allèles A -317 et C -1610, et l’haplotype *1, défini par ces allèles. L’haplotype *1 était aussi associé à une expression élevée du DHFR. Dans cette étude, nous étendons l’analyse à la région régulatrice adjacente, d’environ 400 pb, correspondant au transcrit mineur non-codant du DHFR, qui joue un rôle essentiel dans la régulation de la transcription au niveau du promoteur majeur. Six polymorphismes ont été identifiés, parmi lesquels 5 étaient des SNPs et un polymorphisme de longueur composé d’un nombre variable d’éléments de 9 pb et d’une insertion/délétion de 9 pb. L’analyse d’haplotype, incluant tous les polymorphismes promoteurs, a révélé une diversification de l’haploytpe *1 en 5 sous-types (*1a à *1e). Les variations du promoteur majeur et les sous-types de l’haplotype *1 ont été par la suite analysés pour l’association avec l’issue de LLA. Un EFS réduit corrélait avec l’allèle A du polymorphisme G308A (p=0,02) et avec l’haplotype *1 (p=0,01). Des niveaux élevées d’ARNm étaient trouvés chez les porteurs de l’haplotype *1b (p=0,005) et pas pour les autres sous-types de l’haplotype *1. Alors, la mauvaise issue de LLA associée avec l'haplotype *1 est en effet déterminée par le sous-type *1b. Cette étude donne un nouvel aperçu des polymorphismes régulateurs du DHFR définissant plus précisément les variations du DHFR prédisposant un événement. / Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. We recently reported an association of DHFR promoter polymorphisms with ALL outcome. Lower event free survival (EFS) correlated with the alleles A -317 and C -1610, and with haplotype *1, defined by these alleles. Haplotype*1 was also associated higher DHFR expression. Here we extended the analysis to adjacent 400bp regulatory region corresponding to non-coding minor DHFR transcript which plays an essential role in the regulation of transcription from the major promoter. Six polymorphisms were identified, of which 5 were SNPs and one length polymorphism composed of variable number of 9bp elements and 9bp insertion/deletion. Haplotype analysis including all promoter polymorphisms revealed diversification of haplotype *1 into 5 subtypes (*1a to *1e). Major promoter variations and haplotype *1 subtypes were subsequently analyzed for the association with ALL outcome. Lower EFS correlated with an A allele of G308A polymorphism (p=0.02) and with *1b haplotype (p=0.01). Higher mRNA levels were found in the carriers of *1b haplotype (p=0.005) and not for remaining haplotype *1 subtypes. So, the worse ALL outcome associated with haplotype *1 is actually determined by the subtype *1b. The study provides a new insight into DHFR regulatory polymorphisms defining more precisely event–predisposing DHFR variations.

Deficiência na enzima lisossomal glucocerebrosidase (GCR) resulta na doença de Gaucher. O tratamento atual consiste na administração da enzima exógena, produzida em células CHO. Porém, o medicamento disponível no mercado é extremamente custoso. Neste trabalho, propusemos a clonagem e a expressão da GCR humana em células CHO, visando a obtenção de um clone celular produtor para viabilizar a produção futura da enzima, a um custo menor, no Instituto Butantan. A expressão estável da GCR recombinante foi obtida a partir da transfecção de células CHO-dhfr- com o plasmídeo pED de expressão em células de mamíferos contendo o cDNA da GCR, seguido de amplificação gênica por MTX. A GCR foi detectada no extrato celular (~ 64 kDa) e secretada para o sobrenadante (63-69 kDa) em ensaios de western blotting, usando o anticorpo policlonal anti-GCR gerado neste trabalho. A enzima secretada hidrolisou o substrato 4-MUG e a sua produtividade foi estimada em 5,14 pg/célula/dia para o melhor subclone produtor, selecionado para a produção futura da GCR em larga escala. / Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher\'s disease. Current treatment consists on enzyme replacement therapy by the administration of recombinant GCR produced in CHO cells. However, the medicine available in the market is extremely expensive. In this work, we proposed the cloning and expression of human GCR in CHO cells, in order to obtain a productive cellular clone for future production of GCR enzyme at a lower cost at the Butantan Institute. The stable expression of recombinant GCR was obtained after transfection of CHO-dhfr- cells with pED mammalian expression vector containing the GCR cDNA, followed by gene amplification with MTX. The GCR was detected by western blotting analysis, either as cell-associated (~ 64 kDa) or as secreted forms (63-69 kDa), using the anti-GCR polyclonal antibody produced in this work. The secreted enzyme was active on 4-MUG and was produced at a level of about 5,14 pg/cell/day for the best producer subclone, selected for subsequent steps of GCR production on large scale in next future.