Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions. The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso. The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding. We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur. In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence.

Understanding the mechanisms of virus capsid assembly has been an important research objective over the past few decades. Determining critical points along the pathways by which virus capsids form could prove extremely beneficial in producing more stable DNA vectors or pinpointing targets for antiviral therapy. The inability of current experimental technology to address this objective has resulted in a need for alternative approaches. Theoretical and computational studies offer an unprecedented opportunity for detailed examination of capsid assembly. The Schwartz Lab has previously developed a discrete event stochastic simulator to model virus assembly based upon local rules detailing the geometry and interaction kinetics of individual capsid subunits. Applying numerical optimization methods to learn kinetic rate parameters that fit simulation output to in vitro static light scattering data has been a successful avenue to understand the details of virus assembly systems; however, information describing in vitro assembly processes does not necessarily translate to real virus assembly pathways in vivo. There are a number of important distinctions between experimental and realistic assembly environments that must be addressed to produce an accurate model. This thesis will describe work expanding upon previous parameter estimation algorithms for more complex data over three model icosahedral virus systems: human papillomavirus (HPV), hepatitis B virus (HBV) and cowpea chlorotic mottle virus (CCMV). Then it will consider two important modifications to assembly environment to more accurately reflect in vivo conditions: macromolecular crowding and the presence of nucleic acid about which viruses may assemble. The results of this work led to a number of surprising revelations about the variability in potential assembly rates and mechanisms discovered and insight into how assembly mechanisms are affected by changes in concentration, fluctuations in kinetic rates and adjustments to the assembly environment.

Proteins function as worker molecules in the cell and their natural environment is crowded. How they fold in a cell-like environment and how they recognize their interacting partners in such conditions, are questions that underlie the work of this thesis. Two distinct subjects were investigated using a combination of biochemical- and biophysical methods. First, the unfolding/dissociation of a heptameric protein (cpn10) in the presence of the crowding agent Ficoll 70. Ficoll 70 was used to mimic the crowded environment in the cell and it has been used previously to study macromolecular crowding effects, or excluded volume effects, in protein folding studies. Second, the conformational changes upon interaction between the Mediator subunit Med25 and the transcription factor Dreb2a from Arabidopsis thaliana. Mediator is a transcriptional co-regulator complex which is conserved from yeast to humans. The molecular mechanisms of its action are however not entirely understood. It has been proposed that the Mediator complex conveys regulatory signals from promoter-bound transcription factors (activators/repressors) to the RNA polymerase II machinery through conformational rearrangements. The results from the folding study showed that cpn10 was stabilized in the presence of Ficoll 70 during thermal- and chemical induced unfolding (GuHCl). The thermal transition midpoint increased by 4°C, and the chemical midpoint by 0.5 M GuHCl as compared to buffer conditions. Also the heptamer-monomer dissociation was affected in the presence of Ficoll 70, the transition midpoint was lower in Ficoll 70 (3.1 μM) compared to in buffer (8.1 μM) thus indicating tighter binding in crowded conditions. The coupled unfolding/dissociation free energy for the heptamer increased by about 36 kJ/mol in Ficoll. Altogether, the results revealed that the stability effect on cpn10 due to macromolecular crowding was larger in the individual monomers (33%) than at the monomer-monomer interfaces (8%). The results from the interaction study indicated conformational changes upon interaction between the A. thaliana Med25 ACtivator Interaction Domain (ACID) and Dreb2a. Structural changes were probed to originate from unstructured Dreb2a and not from the Med25-ACID. Human Med25-ACID was also found to interact with the plant-specific Dreb2a, even though the ACIDs from human and A. thaliana share low sequence homology. Moreover, the human Med25-interacting transcription factor VP16 was found to interact with A. thaliana Med25. Finally, NMR, ITC and pull-down experiments showed that the unrelated transcription factors Dreb2a and VP16 interact with overlapping regions in the ACIDs of A. thaliana and human Med25. The results presented in this thesis contribute to previous reports in two different aspects. Firstly, they lend support to the findings that the intracellular environment affects the biophysical properties of proteins. It will therefore be important to continue comparing results between in vitro and cell-like conditions to measure the magnitude of such effects and to improve the understanding of protein folding and thereby misfolding of proteins in cells. Better knowledge of protein misfolding mechanisms is critical since they are associated to several neurodegenerative diseases such as Alzheimer’s and Parkinson's. Secondly, our results substantiate the notion that transcription factors are able to bind multiple targets and that they gain structure upon binding. They also show that subunits of the conserved Mediator complex, despite low sequence homologies, retain a conserved structure and function when comparing evolutionary diverged species.

The regulation of protein function through oligomerization is a common theme in biological systems. In this work, I have focused on the effects of the oligomeric states of the human Rad52 protein on activities related to DNA binding. HsRad52, a member of the RAD52 epistasis group, is thought to play an important and as yet undefined role in homologous recombination. HsRad52 preferentially binds to ssDNA over dsDNA and stimulates HsRad51-mediated strand exchange (Benson et al., 1998). In either the presence or absence of DNA, HsRad52 has been observed to form both 10 nm ring-like structures as well as higher order oligomers consisting of multiple 10 nm rings (Van Dyck et al., 1998; Van Dyck et al., 1999). Earlier protein-protein interaction studies mapped the domain responsible for HsRad52 self-association in the N-terminus (residues 85-159) (Shen et al., 1996). Data presented here identifies a novel self-association domain in the C-terminus of HsRad52 that is responsible for the formation of higher order oligomers. VanDyck et al. observed DNA ending binding complexes consisting of multiple rings (Van Dyck et al., 1999). They proposed that these higher order oligomers may be functionally relevant. In this work, we demonstrate that DNA binding depends on neither ring shaped oligomers nor higher order oligomers but that activities of HsRad52 that require simultaneous interaction with more than one DNA molecule depend on the formation of higher order oligomers consisting of multiple HsRad52 rings. Early studies of HsRad52 proposed that the DNA binding domain resides in the highly conserved N-terminus of the protein (Park et al., 1996). A series of studies using truncation mutants of HsRad52 have provided evidence that supports this hypothesis. For example, we demonstrated that a truncation mutant containing only the first 85 residues of the protein is still able to bind DNA (Lloyd, submitted 2002). In this study, we demonstrate that aromatic (Y65, F79 and Y81) and hydrophobic (L43, I52 and I66) residues within the N-terminus contribute to DNA binding by either directly contacting the DNA or by stabilizing the structure of the protein. In summary, through the work presented in this dissertation, we have determined that the formation of 10 nm rings is mediated by a self-association domain in the N-terminus and that the formation of higher order oligomers consisting of multiple HsRad52 rings is mediated by an additional self-association domain in the C-terminus. We have correlated the oligomeric properties of HsRad52 with its biochemical functions related to DNA binding. Additionally, we have demonstrated that aromatic and hydrophobic residues contribute to DNA binding. Further studies will differentiate between the contribution of these residues to the DNA binding by stabilizing the overall structure of the protein versus making specific DNA contacts. Additional studies will also address how the oligomeric state of HsRad52 contributes to its role in HsRad51-mediated strand exchange.

A deformação plástica de polímeros semicristalinos na região situada entre suas Tg (temperatura de transição vítrea) e T m (temperatura de fusão cristalina), modifica suas propriedades termodinâmicas e morfológicas produzindo materiais com novas propriedades e aplicações. Neste trabalho polifluoreto de vinilideno (PVF2) e polietileno de alta densidade (PEAD) foram submetidos à deformação plana por compressão na região entre suas T 9 e T m, para a investigação de dois tipos de processos em nãoequilíbrio: a deformação plástica desses polímeros e o processo de relaxação do material deformado. As mudanças das propriedades termodinâmicas, coeficientes de expansão térmica linear (ai) e capacidades térmicas a pressão constante (Cp), ocorridas no primeiro processo foram registradas através de técnicas de Análise Termomecânica (TMA) e Calorimetria Diferencial de Varredura (DSC). Foi verificado aumento de ai e Cp nas amostras deformadas. As mudanças morfológicas ocorridas no primeiro processo foram registradas através das técnicas de Difração de Raios X e Densimetria. Observou-se aumento da fase amorfa bem como transição de fase na região cristalina em ambos materiais. Medidas de DSC mostraram maior grau de cristalinidade no PEAD que no PVF2. O segundo processo foi acompanhado submetendo-se o material deformado a ciclos consecutivos de aquecimento e resfriamento, com o auxílio das técnicas de TMA e DSC. As "memórias" de forma e termomecânica foram registradas através de transições relacionadas a mudanças irreversíveis no ai ao longo das coordenadas de deformação. Também foi observado que o PVF2 possui maior tendência à recuperação da forma que o PEAD. Relaxação da Cp não foi observada no intervalo de temperatura estudado. Os resultados obtidos neste trabalho levam a concluir que o processo de deformação plana por compressão permite a obtenção de materiais fisicamente modificados com novas propriedades mecânicas e termodinâmicas, os quais possuem a tendência a recuperar parcialmente seu estado original. Também foi verificada que a recuperação da forma e da Cp não estão diretamente relacionadas: enquanto a recuperação da forma do material semicristalino parece estar mais relacionada à mobilidade da fase amorfa, a recuperação da Cp está provavelmente mais relacionada à capacidade de recuperação da fase cristalina. / The plastic deformation of semi-crystalline polymers between Tg (glass transition temperature) and Tm (crystalline melting temperature), modifies their thermodynamics and morphological properties producing materiais with new properties and applications. In this work polyvinylidene fluoride (PVF2) and high density polyethylene (HOPE) were submitted to plane-strain compression in a deep channel die in a temperature range between their Tg and T m, in order to investigate two kind of nonequilibrium processes: the plastic deformation of these polymers and the relaxation process of the deformed material. The changes of thermodynamical properties, linear thermal expansion coefficients (ai) and constant pressure heat capacities (Cp), occurred in the first process were registered by Thermomechanical Analysis (TMA) and Oifferential Scanning Calorimetry COSC) techniques. An increase in ai and Cp in deformed samples has been observed. The morphological changes occurred in the first process were registered by X-Ray Diffraction and Densimetry techniques. It was observed an amorphous phase increase as well as a phase transition in the crystalline region in both materiais. The DSC measurements have shown a better cristalinity degree in PEAO than in PVF2. The second process was followed by submitting the samples to consecutive cycles of heating and cooling using TMA and DSC techniques. The shape and thermomechanical "memory" were registered by means of transitions related to irreversible changes in the linear thermal expansion coefficient, along the deformation coordinates. It was also observed that PVF2 has a better tendency to shape recovery than PEAD. Relaxation in the heat capacity has not been observed in temperature range studied. The results obtained in this work lead to the conclusion that the plane-strain compression allows physically modified materiais with new thermodynamical and mechanical properties, which have the tendency to recover its original state, to be obtained. It was also verified that shape and Cp recovery are not directly related: while the shape recovery of semi-crystalline materiais seems to be more related to the amorphous phase mobility, the Cp recovery is most probably related to the ability of crystalline phase recovery.

SMFD techniques offer genuine detection possibilities which are often inaccessible using ensemble methods. This was demonstrated in three projects investigating translocation activity of CHD4 protein, analysis of MS2 phage capsid assembly and in-cell characterization of DNA structure. In other projects, binding interactions between two fluorescent probes and a short oligonucleotide were characterized and all optical depth of focus extended microscope configuration for imaging of individual molecules inside bacterial cells was developed and tested.

A deformação plástica de polímeros semicristalinos na região situada entre suas Tg (temperatura de transição vítrea) e T m (temperatura de fusão cristalina), modifica suas propriedades termodinâmicas e morfológicas produzindo materiais com novas propriedades e aplicações. Neste trabalho polifluoreto de vinilideno (PVF2) e polietileno de alta densidade (PEAD) foram submetidos à deformação plana por compressão na região entre suas T 9 e T m, para a investigação de dois tipos de processos em nãoequilíbrio: a deformação plástica desses polímeros e o processo de relaxação do material deformado. As mudanças das propriedades termodinâmicas, coeficientes de expansão térmica linear (ai) e capacidades térmicas a pressão constante (Cp), ocorridas no primeiro processo foram registradas através de técnicas de Análise Termomecânica (TMA) e Calorimetria Diferencial de Varredura (DSC). Foi verificado aumento de ai e Cp nas amostras deformadas. As mudanças morfológicas ocorridas no primeiro processo foram registradas através das técnicas de Difração de Raios X e Densimetria. Observou-se aumento da fase amorfa bem como transição de fase na região cristalina em ambos materiais. Medidas de DSC mostraram maior grau de cristalinidade no PEAD que no PVF2. O segundo processo foi acompanhado submetendo-se o material deformado a ciclos consecutivos de aquecimento e resfriamento, com o auxílio das técnicas de TMA e DSC. As "memórias" de forma e termomecânica foram registradas através de transições relacionadas a mudanças irreversíveis no ai ao longo das coordenadas de deformação. Também foi observado que o PVF2 possui maior tendência à recuperação da forma que o PEAD. Relaxação da Cp não foi observada no intervalo de temperatura estudado. Os resultados obtidos neste trabalho levam a concluir que o processo de deformação plana por compressão permite a obtenção de materiais fisicamente modificados com novas propriedades mecânicas e termodinâmicas, os quais possuem a tendência a recuperar parcialmente seu estado original. Também foi verificada que a recuperação da forma e da Cp não estão diretamente relacionadas: enquanto a recuperação da forma do material semicristalino parece estar mais relacionada à mobilidade da fase amorfa, a recuperação da Cp está provavelmente mais relacionada à capacidade de recuperação da fase cristalina. / The plastic deformation of semi-crystalline polymers between Tg (glass transition temperature) and Tm (crystalline melting temperature), modifies their thermodynamics and morphological properties producing materiais with new properties and applications. In this work polyvinylidene fluoride (PVF2) and high density polyethylene (HOPE) were submitted to plane-strain compression in a deep channel die in a temperature range between their Tg and T m, in order to investigate two kind of nonequilibrium processes: the plastic deformation of these polymers and the relaxation process of the deformed material. The changes of thermodynamical properties, linear thermal expansion coefficients (ai) and constant pressure heat capacities (Cp), occurred in the first process were registered by Thermomechanical Analysis (TMA) and Oifferential Scanning Calorimetry COSC) techniques. An increase in ai and Cp in deformed samples has been observed. The morphological changes occurred in the first process were registered by X-Ray Diffraction and Densimetry techniques. It was observed an amorphous phase increase as well as a phase transition in the crystalline region in both materiais. The DSC measurements have shown a better cristalinity degree in PEAO than in PVF2. The second process was followed by submitting the samples to consecutive cycles of heating and cooling using TMA and DSC techniques. The shape and thermomechanical "memory" were registered by means of transitions related to irreversible changes in the linear thermal expansion coefficient, along the deformation coordinates. It was also observed that PVF2 has a better tendency to shape recovery than PEAD. Relaxation in the heat capacity has not been observed in temperature range studied. The results obtained in this work lead to the conclusion that the plane-strain compression allows physically modified materiais with new thermodynamical and mechanical properties, which have the tendency to recover its original state, to be obtained. It was also verified that shape and Cp recovery are not directly related: while the shape recovery of semi-crystalline materiais seems to be more related to the amorphous phase mobility, the Cp recovery is most probably related to the ability of crystalline phase recovery.

L'objectif de la thèse était d'étudier les propriétés structurales et dynamiques de deux systèmes biologiques, avec une approche centrée sur les techniques de diffusion de neutrons. Les premier système biologique porte sur le système de transport FHA/FhaC de la bactérie à Gramm négatif Bordetella pertussis. C'est un modèle pour le système de transport bactérien à deux partenaires (TPS), qui est largement utilisé par de nombreuses bactéries à Gramm negatif, essentiellement pour excréter des facteurs de virulence. Cette thèse comporte une analyse structurale sur le complexe chaperonneprotéine client Fha30/Par27 mais aussi une étude sur les propriétés mécaniques de Fha30, qui possède une structure particulière en hélice beta. Le deuxième système porte sur le complexe de réplication des virus de la famille des Rhabdoviridae. Outre son importance en termes de santé humaine, le complexe de réplication comporte des protéines aux propriétés qui, sur le plan structural, sont très intéréssantes, comme par exemple la phosphoprotéine qui comporte des parties structurées reliées par des parties dépourvues de structure. On trouvera dans cette thèse une étude des propriétés dynamiques des différents domaines de la phosphoprotéine, mais aussi une étude structurale sur le complexe que celle-ci forme avec la nucleoprotéine. / The main goal of this thesis is to study the dynamical and structural properties of two biological systems, with an aproach centered on neutron scaterring techniques. The first system consists of the transport system FhaB/FhaC from Gramm negative bacteria Bordetella pertussis. It is a model system for the so called Two Partner Secretion (TPS) system, wich is widespread among Gramm negative bacteria to export virulence factors to the medium. This thesis emphasizes on the complexe Fha30, a N-terminal fragment of FHA, forms with the periplasmic chaperonne Par27, and on the mechanical properties of FHA, which has an uncanny beta helix structure. The other system is the replication complex of the Rhabdoviridae virus family. Apart from it's importance in humant health issues, the replication complex is composed of proteins with interesting structural and dynamical properties, specially the phosphoprotein which is composed of structured parts linked by flexible linkers. This thesis emphasizes on the dynamical properties of different fragments of the phosphoprotein, but also on the structural aspects of the complex the latter forms with the nucleoprotein. . Le deuxième système porte sur le complexe de réplication des virus de la famille des Rhabdoviridae. Outre son importance en termes de santé humaine, le complexe de réplication comporte des protéines aux propriétés qui, sur le plan structural, sont très intéréssantes, comme par exemple la phosphoprotéine qui comporte des parties structurées reliées par des parties dépourvues de structure. On trouvera dans cette thèse une étude des propriétés dynamiques des différents domaines de la phosphoprotéine, mais aussi une étude structurale sur le complexe que celle-ci forme avec la nucleoprotéine.

Il est généralement admis que la qualité des données de diffraction de rayons X peut être améliorée par la fusion des ensembles de données à partir de plusieurs cristaux. Toutefois, cet effet n'est valable que si les ensembles de données utilisés sont des cristaux qui sont structurellement identiques. Donc les cristaux macromoléculaire congeles on tres raremente une identité strcuturele comune. Donc, pour obtenir un gain réel a partir da une paquete de multi-cristoux. Il est necessaire de faire une selection approprieé des cristaux. Les études statistiques ont été effectuées pour trouver une méthode appropriée pour la description sur le non-isomorphisme. L'application d'analyse par grappes hiérarchiques, sur la base de la matrice du coefficient de corrélation entre les intensités d'échelle est proposée pour l'identification d'ensembles de données isomorphes. Multi-monocristallines de données de longueur d'onde de dispersion anomale ensembles de quatre molécules de protéines différentes ont été sondés pour tester l'applicabilité de cette méthode. L'utilisation de l'"hierarchical cluster analysis" a permi d'ameliorer de facon significative les indicatours crystallohraphique des donnée et de resoudre la structure. / It is generally assumed that the quality of X-ray diffraction data can be improved by merging data sets from several crystals. However this effect is only valid if the data sets used are from crystals that are structurally identical. It is found that frozen macromolecular crystals very often have relatively low structure identity (and are therefore not isomorphous) thus to obtain a real gain from multi-crystal data sets one needs to make a appropriate selection of structurally similar crystals. Statistical studies has been performed to find a suitable method for the description on non-isomorphism. The application of hierarchical cluster analysis, based on the matrix of the correlation coefficient between scaled intensities is proposed for the identification of isomorphic data sets. Multi-crystal single wavelength anomalous dispersion data sets from four different protein molecules have been probed to test for the applicability of this method. The use of hierarchical cluster analysis has permitted the selection of batches of data sets, which when merged together significantly improved the crystallographic indicators of the merged data and allowed the solution of the structure.

A importância do estudo das celulases não se limita à aquisição de conhecimento científico, mas também ao grande potencial biotecnológico que elas representam. Isso se deve ao fato da celulose ser a molécula mais abundante presente na natureza e prover uma vasta gama de produtos e processos sustentáveis. Muitas famílias de celulases já foram bem caracterizadas, enquanto outras permanecem ainda desconhecidas. Dentre estas últimas, a família 45 das hidrolases de glicosídeos é a família de celulases fúngicas menos caracterizada tanto estruturalmente quanto funcionalmente. Recentemente foi proposta a divisão dessa família em três subfamílias e, até agora, apenas membros da subfamília A tiveram enzimas estruturalmente elucidadas. Nesse trabalho reportamos a estrutura cristalográfica da proteína recombinante endoglucanase de Phanerochaete chrysosporium (PcCel45A), a primeira das hidrolases de glicosídeos da subfamília C, e seu complexo com celobiose a 1,4 Å e 1,7 Å de resolução, respectivamente. A PcCel45A é uma enzima de domínio único, com uma estrutura em β-barril e seu empacotamento geral remete ao formato de âncora. O sítio ativo da enzima forma um longo sulco na superfície da estrutura, sendo que o seu centro catalítico é diferente das outras enzimas publicadas dessa família e o aspartato catalítico, que atua como aceptor de próton na reação de inversão, (Asp10) não é conservado. Adicionalmente, a estrutura cristalográfica dessa enzima apresenta mais similaridades com as β-expansinas (proteínas de plantas) e transglicosilases líticas (proteínas que clivam o peptidoglicano de bactérias) do que com as outras representantes da família 45, o que a torna ainda mais singular. Para entendermos melhor seu funcionamento foram realizadas mutações sítio-dirigidas nos principais resíduos do sítio ativo. O Asp121, conhecido por participar da reação de inversão das outras enzimas da família como doador de próton, mostrou-se essencial para a atividade da enzima, enquanto que outros resíduos conservados como a Tyr25, o Trp161 e o Asp92 afetaram, mas não aniquilaram a atividade da enzima, apresentando aproximadamente 20%, 50% e 10% da atividade da enzima nativa, respectivamente. / The importance of the study of the cellulases is not limited to generating significant scientific knowledge, since these enzymes represents an enormous potential in biotechnology. This is partly because cellulose is the most abundant molecule in nature and provides a wide range of products and sustainable process. Many cellulases families have been well characterized, while others still remain unknown. Among them, the glycoside hydrolase family 45 is the least well characterized both structurally and functionally, between fungal cellulases. It was recently proposed the subdivision of this family into three subfamilies, with structural information available only for subfamily A. In this work, we report the chrystallographic structure of the recombinant endoglucanase from Phanerochaete chrysosporium (PcCel45A), the first GH45 subfamily C and its complex with cellobiose at 1.4 Å and 1.7 Å respectively. The PcCel45A is a single domain enzyme, which has a β-barrel structure with the overall shape resembling an anchor. The active site of the enzyme has a long cleft on the surface, being remarkably different from those members of subfamily A, and the catalytic aspartate responsible for acting as proton acceptor (Asp10) is not present. Additionally, the chrystallographic structure of this enzyme has shown more similarity with β -expansins (plant proteins) and lytic transglycosylase (proteins that cleave the peptidoglycan of bacteria) than others representants of family 45, which makes it more singular. For a better understanding of its function, we perform pontual mutations in the main residues from active site. The Asp121, known for acting as proton acceptor in the inversion reaction of others enzymes, proved to be essential for the enzyme activity, while others conserved residues as Tyr25, Trp161 and Asp92 affected but not annihilated the enzyme activity, leaving approximately 20%, 50% and 10% of the native enzyme activity.