O pH final (pHf) no músculo post mortem é amplamente utilizado como um indicador potencial de maciez e é um fator importante associado à qualidade de carne. O Brasil é líder mundial nas exportações de carne bovina, porém não são conhecidos os valores de pHf do músculo pós-abate e seu impacto na qualidade da carne de animais machos inteiros Bos taurus indicus. Baseado no exposto, objetivou-se com este trabalho identificar o atual \"status\" dos valores de pHf 24 h post mortem apresentados no frigorífico comercial, e a influência do pHf na proteólise muscular e parâmetros de qualidade como perda por gotejamento, cor e maciez de machos inteiros avaliados aos 0, 7, 14, 21 e 28 dias de maturação. Foi realizado um estudo preliminar, com 399 carcaças em abatedouro comercial, para conhecer a ocôrrencia pHf de animais machos inteiros abatidos no Brasil, podendo-se identificar três faixas de pHf: pHf 5,5 até 5,8 (baixo-normal); pHf 5,81 até 6,3 (intermediário); pHf > 6,3. Músculos Longissimus dorsi (n=12) foram porcionados em bifes, embalados a vácuo, classificados nos três diferentes grupos de pHf e maturados a 2°C por 0 (48h), 7, 14, 21 e 28 dias. Foram avaliadas características de perda por gotejamento, cor, força de cisalhamento, índice de fragmentação miofibrilar, colágeno total e solúvel, assim como proteólise miofibrilar. Carnes pertencentes ao grupo de pHf alto, apresentaram maior maciez (P<0,05) desde o início do experimento, quando comparado aos outros grupos do estudo. Bifes de pHf intermediário apresentaram os valores de força de cisalhamento mais elevados (P<0,05), o que indica um processo de maciez mais lento quando comparado com os outros grupos de pHf. Perdas por gotejamento, diminuem com o aumento nos valores de pHf (P< 0,05). Valores de L*, a*, b*, não apresentam diferenças entre os grupos de pH. O índice de fragmentação miofibrilar (MFI) foi maior em carne de pHf alto, seguido pelo grupo de pHf baixo-normal, e os menores valores pertenceram ao grupo de pHf intermediário (P<0,05) através do tempo de maturação. A degradação das proteínas como desmina e troponina-T foi maior e mais visível no pHf alto e baixo-normal desde as 48 h post mortem em comparação com o pHf intermediário, onde foram observadas as bandas de sua degradação quase ao final do período experimental. Proteínas chave como filamina e nebulina apresentaram maior degradação desde as 48 horas post mortem no pHf alto e mais lenta no pHf intermediário. Não foram observadas diferenças (P> 0,05) nos valores de colágeno total e solúvel nos diferentes grupos de pHf e nem através do tempo de maturação. Em geral, o grupo de pHf intermediário (5,8-6,3) apresentou maior inconsistência quanto às características de maciez e fragmentacão miofibrilar, consequência de uma proteólise tardia. / The ultimate pH (pHu) of the postmortem muscle is broadly use as a potential meat tenderness indicator and is an important factor related to meat quality. Brazil is the bovine meat exporter world leader; nevertheless the values of the postmortem pHu of the muscle and its impact on the meat quality of Bos indicus taurus bulls, are unknown. Therefore, the objective of this research was to establish the values of pHu after 48 hours postmortem and its impact over the meat quality through the characterization of the biochemical processes that occurs in the muscle Longissimus dorsi of Bos taurus indicus bulls. In order to know the actual pHu of the Brazilian bulls, a preliminary study were made, using 399 carcasses of a commercial slaughter house and resulting three levels of pHu: pHu 5,5 to 5,8 (low-normal); pHu 5,81 to 6,3 (intermediate); pHu > 6,3 (high). Twelve muscles Longissimus dorsi were cut into steaks, classified into the three groups of pHu, vacuum packed up and matured at 2°C for 0 (48h), 7, 14, 21 and 28 days. Drip loss, color, shear force, myofibril fragmentation index, total and soluble collagen and miofibrillar proteolysis were evaluated. The high pHu meat presented more tenderness from the beginning of the experiment (P<0,05) when compared with the other groups. The medium pHu steaks presented the highest shear force values (P<0,05), indicating a slower tenderization process in relation to the other pHu groups. The drip loss values diminished as the value of pHu rised (P< 0,05). The values of L*, a*, b* did not show significant differences within the groups of pHu. The highest miofibrillar fragmentation index (MFI) through the maturation time, was found in the high pHu meat, followed by the low pHu group and then by the intermediate pHu group (P<0,05). At 48 hours postmortem, degradation of proteins, like desmin and troponin-T, was higher and more evident in the high and low-normal pHu when compared to the intermediate pHu, where the bands and its degradation were only observed by the end of the experiment. Key proteins like filamin and nebulin, showed higher degradation rate from the 48 h postmortem in the high pHu group, and a slower degradation in the intermediate pHu group. No differences were observed through the pHu groups, nor through the maturation time (P<0,05), for total and soluble collagen values. Then, it is possible to say that the meat quality of Bos taurus indicus bulls, especially the tenderness, is related to the pHu, and can be affected by the proteolytic systems activity. In general, the tenderness and the MFI were more inconsistent in the medium pHu group (5,8-6,3) than in the two other pHu groups, as a consequence of a late proteolysis.

Le premier acide aminé incorporé dans une chaîne polypeptidique naissante est toujours la méthionine. On identifie donc toujours ce premier résidu à la méthionine N-terminale. Cependant, les deux tiers des protéines accumulées à l’état stationnaire ne présentent plus leur méthionine initiatrice. Cet enlèvement résulte essentiellement d’une maturation protéolytique affectant chaque protéine. Ainsi, l’Excision de la Méthionine N-terminale (NME) concerne la majorité des protéines et ce dès que les premiers résidus émergent du ribosome. Ce mécanisme est retrouvé dans tous les compartiments cellulaires où une synthèse protéique a lieu : le cytoplasme, les plastes et les mitochondries. Les enzymes responsables du clivage de la méthionine initiatrice sont les METhionine AminoPeptidases (METAPs) ; les METAPs sont conservées dans le Règne vivant. Des études fonctionnelles de délétions géniques ont montré le caractère létal du maintien de la première méthionine dans tous les organismes. Il y a plus de dix ans, les METAPs ont été identifiées comme étant la cible de composés naturels ayant des effets anticellulaires. Aujourd’hui un nombre croissant d’études rapportent que la NME est une cible prometteuse pour le traitement de nombreuses pathologies. Néanmoins, les bases moléculaires qui expliquent le caractère essentiel de la NME restent très peu comprises, en particulier dans le cytoplasme des eucaryotes supérieurs. Grâce à un système inductible permettant de moduler finement la NME cytoplasmique dans la plante modèle Arabidopsis thaliana et différentes approches incluant des analyses protéomiques et métabolomiques, j’ai pu étudier les événements moléculaires précoces associés à l’inhibition de la NME cytoplasmique. J’ai également caractérisé la contribution relative des deux types de METAP cytoplasmiques au processus. Dans ce contexte, j’ai pu démontrer chez A. thaliana que la NME cytoplasmique agit sur deux voies de signalisation fréquemment dérégulées lors de conditions pathologiques : le statut des composés thiolés et la protéolyse. La diminution de la NME cytoplasmique induit une protéolyse accrue principalement via une augmentation du nombre de protéines destinées à une dégradation rapide. Ainsi, l’activité de la NME, en modulant la sensibilité de nombreuses protéines à subir la protéolyse, est un élément fondamental de la régulation de la demi-vie protéique. Finalement, mes résultats simialires obtenus également chez les Archées, levures et les lignées de cellules humaines suggèrent l’existence d’un mécanisme ubiquitaire associé à la NME. / The first amino acid incorporated in nascent polypeptide chain is always methionine so called N-terminale methionine. However, in a given proteome, more than fifty percent of proteins have not this first methionine. Indeed, the early proteolytic event affecting a majority of proteins is N-terminal Methionine Excision (NME) as soon as few residues exit from the ribosome. Enzymes ensuring NME process are conserved along species. This mechanism takes place in all compartments where protein synthesis occurs including cytoplasm, plastids and mitochondria and the enzymes responsible of N-methionine excision are METhionine AminoPeptidases (METAP). Early functional studies of gene deletion has quickly showed that NME is an essential process. Ten years ago, METAPs have been identified as the molecular target of natural compounds with anticancer activities. Now, a growing number of studies suggest that NME is a promising target for treatment of various deseases. Nevertheless, molecular mechanisms making NME an essential process is poorly understood in particular in higher eukaryote cytoplasms.Using a dedicated inducible system in the model organism Arabidopsis thaliana and multiple approaches, including proteomics and metabolomics, I examined the earliest molecular events associated with the inhibition of this process and the contribution of both METAP to NME process. In this context, I demonstrated that cytoplasmic NME in A. thaliana orchestrates a cross-talk between two fundamental signaling pathways frequently deregulated in pathological conditions: thiol status and proteolysis. In these studies, we demonstrated that developmental defects induced by cytoplasmic NME inhibition are associated with an increase of the proteolytic activity due to an increase of the proteins available for rapid degradation. Thus, NME activity that modifies the availability of several proteins for degradation is an integral and fundamental element protein turnover regulation. Finally my preliminary results obtained in Archea, Fungi and human cells seem to suggest the existence of a ubiquitous mechanism associated with NME process.

O fator de indução de proteólise (proteolysis-inducing factor) ou PIF é uma glicoproteína de 24 kDa encontrada no plasma de pacientes com câncer e responsável pelo catabolismo de proteínas musculares associado a caquexia neoplásica. O presente trabalho investigou pelas técnicas de RT-PCR, RACE-PCR e Real-time PCR a presença de formas de expressão de mensagens do PIF em vários tipos celulares derivados de tecido normal e tumoral. A expressão de mRNA do gene foi testada em 37 amostras de tumores e 4 tecidos normais, sendo detectada em 7 de 20 linhagens tumorais de mama, uma linhagem tumoral de cólon e no tecido normal da mama, placenta e testículo. A expressão significativa do gene PIF entre as linhagens metastáticas da mama foi confirmada pela técnica de Real-time PCR. Uma nova variante da mensagem (PIF-SV), contendo um novo éxon de 64 bp, inserido entre os éxons 4 e 5, foi identificada em tecido normal de mama pela técnica de RACE-PCR. Esta forma variante de PIF é expressa concomitante com a forma principal de PIF em tumores primários da mama, linhagens tumorais de mama e nos tecidos normais da mama e placenta. Parece que o PIF exerce funções fisiológicas nos tecidos normais. / Proteolysis-inducing factor (PIF) is a 24 kDa glycoprotein identified in plasma of cancer patients and responsible for muscle catabolism associated with the process of cancer cachexia. The present study has investigated, using the RT-PCR, RACE-PCR and Real-time PCR techniques, the presence of PIF messages in different cell types derived from normal and tumor tissues. The PIF mRNA expression was examined in 37 tumor and 4 normal tissue samples and detected in 7 of 20 breast tumor cell lines, one colon tumor cell line and in normal breast, placenta and testis tissues. The differential expression of PIF message in metastatic breast cell lines was confirmed by the Real-time PCR technique. A new splicing form of the message, containing one new exon of 64bp, inserted between the exons 4 and 5, was identified in normal breast tissue. This splicing form of PIF is expressed concomitantly with a main form of PIF in breast tumor cell lines and also in normal breast and placenta tissue. These data suggest that PIF exhibits physiological functions in normal tissues. An over-expression and a production of a new splicing form (PIF-SV), seem to contribute in some event leading normal cell to a malignant phenotype.

Orientador: Luis Arthur Loyola Chardulo / Banca: Antonio Carlos Silveira / Banca: Albino Luchiari Filho / Resumo: O experimento foi realizado no Setor de Confinamento de Gado de Corte da Faculdade de Medicina Veterinária e Zootecnia e no Laboratório de Bioquímica de Proteínas do Instituto de Biociências. Foram utilizados animais da raça Nelore, mestiços u Aberdeen X Nelore e mestiços u Simental X Nelore, abatidos com idade entre 12 e 15 meses conforme estabelecido pelo modelo biológico superprecoce. O experimento foi conduzido em um delineamento inteiramente casualizado. O objetivo do presente estudo foi análise de componentes da maciez de novilhos superprecoces de grupos genéticos distintos. Não foi verificada diferença estatística (p>0,01) entre os grupos genéticos para a força de cisalhamento, Índice de Fragmentação Miofibrilar (MFI) e frações do colágeno, entretanto houve influência (p<0,01) do período postmortem, exceto para o colágeno. A carne de animais abatidos entre 12 e 15 meses de idade apresenta atributos de qualidade independente do grupo genético utilizado e com sete dias de maturação todos os animais apresentaram carne com grau de maciez desejável. / Abstract: The experiment was accomplished in the Section of Feedlot of cattle of Faculdade de Medicina Veterinária e Zootecnia and in the Laboratório de Bioquímica de Proteínas do Instituito de Biociências. Nelore breed, u Aberdeen X Nelore crossbreed and u Simental X Nelore crossbreed were used and slaughtered accordingly with the brazilian system called "superprecoce". The experiment was accomplished in a completely randomized design. The objective of the present study was the evaluation of tenderness components of "superprecoce" of different genetic groups. There was no statistics difference (p>0,01) between genetic groups for the shear force values, Myofibrillar Fragmentation Index (MFI) and collagen, however there was influence (p<0,01) of the ageing, except for the collagen. The meat of animals slaughtered between 12 and 15 months of age showed attributes of quality independent of the genetic group and with seven days of ageing all animals had a desirable tenderness. / Mestre

O Brasil dedica-se em grande parte à criação de animais Bos indicus, principalmente da raça Nelore e seus cruzamentos, já que são animais resistentes a doenças e adaptados ao clima tropical. Entretanto, em relação à característica de maciez da carne, restrições têm sido atribuídas a este tipo de animal. Particularmente neste aspecto, existe grande interesse pela seleção de animais cuja genética seja favorável à carne mais macia e com redução na variação da maciez da carne. Durante o período de maturação da carne, a proteólise é o fator que mais contribui com o aumento da maciez. As proteases neutras ativadas por íons de cálcio, denominadas calpaínas, são parcialmente responsáveis pela proteólise post mortem, conduzindo ao aumento progressivo da maciez da carne. Entretanto, existe dificuldade em obter dados fenotípicos relacionados à maciez. Assim, a Seleção Assistida por Marcadores (MAS) pode ter grande impacto para melhorar estas características de difíc il mensuração. Marcadores do gene da calpaína (CAPN) e da calpastatina (CAST), denominados SNPs (Single Nucleotide Polymorphisms), já foram analisados e polimorfismos foram associados favoravelmente com maciez da carne bovina. Entretanto, pouco se conhece sobre a expressão gênica da calpaína e da calpastatina, particularmente em bovinos castrados da raça Nelore com diferentes combinações genotípicas para os marcadores moleculares SNP associados a essas proteases. Assim, este estudo teve como objetivo avaliar a expressão gênica da calpaína e da calpastatina em animais castrados da raça Nelore, com genótipos contrastantes identificados previamente por marcadores SNPs para um, sete e 14 dias de maturação. Foram genotipados 16 animais castrados da raça Nelore e dois marcadores foram utilizados, sendo um associado ao gene da µ-calpaína e outro associado à calpastatina (CAPN4751 e UOGCAST). Através da eletroforese bidimensional (2DE) as intensidades de expressão dos spots foram determinadas. As análises estatística s foram realizadas com auxílio do programa Statistical Analysis System (SAS), utilizando o procedimento PROC MIXED. A proteólise post mortem entre os tempos determinados de maturação foi avaliada e foram observados que 41 spots apresentaram efeitos significativos entre os tempos de maturação. Seis spots explicaram 57% da variabilidade da maciez aos 14 dias de maturação. Com o objetivo de verificar a existência de possíveis diferenças de expressão gênica entre amostras com genótipos distintos, avaliações proteômicas foram realizadas visando avaliar as diferenças no perfil proteico e foram baseadas no estudo comparativo (match) dos géis inter e intra combinações genotípicas. Dois spots explicaram 69% da variabilidade da maciez aos sete dias de maturação. Para o período de 14 dias de maturação 19 spots apresentaram efeito principal significativo para a fonte de variação de interação CAPN4751(UOGCAST) e, neste caso, a análise de regressão apresentou um spot que explica 70% da variabilidade da maciez aos 14 dias de maturação. / Brazil is largely devoted to the breeding of Bos indicus, especially Nellore and their intersections, because these animals are resist ant to diseases and adapted to tropical climate. However, in relation to beef tenderness, restrictions have been assigned to this breed. Particularly in this respect, there is great interest in the genetic selection of animals with more tender beef and reduced variation in meat tenderness. During the aging of the meat, proteolysis is the major factor that contributes to the increased tenderness. The neutral proteases activated by calcium ions, called calpains, are partially responsible for postmortem proteolysis, leading to a progressive increase in meat tenderness. However, there is difficulty in obtaining phenotypic data related to tenderness, in this way, Marker Assisted Selection (MAS) may have great impact to improve these characteristics. Gene markers of calpain (Capn) and calpastatin (CAST), called SNPs (single nucleotide polymorphisms) have been studied and polymorphisms were associated positively with beef tenderness. However, not much is known about the gene expression of calpain and calpastatin, particularly in Nellore steers with different genotype combinations for the SNP markers associated with these proteases. This study aimed to evaluate the gene expression of calpain and calpastatin in castrated Nellore, with contrasting genotypes previously identified by SNPs marker for one, seven and 14 days of aging. We genotyped 16 castrated Nellore, and two markers were used, one being associated with the µ-calpain gene and the other associated with calpastatin (CAPN4751 and UOGCAST). Through two-dimensional electrophoresis (2DE) the intensities of the spots were determined. Statistical analyzes were performed using the Statistical Analysis System (SAS) using the PROC MIXED. The postmortem proteolysis among the times of aging was evaluated and it was observed that 41 spots showed significant effects between different times of aging. Six spots explained up to 57% of the variability of tenderness at 14 days of aging. In order to verify the existence of possible differences in expression among samples with different genotypes, proteomics aimed to evaluate the differences in protein profile, based on comparative study (match) of the gels inter and intra genotype combinations. Two spots explained up to 69% of the variability of tenderness to seven days of maturation. For the period of 14 days of aging 19 spots showed significant main effect for interaction CAPN4751(UOGCAST), in this case regression analysis showed a spot that explains 70% of the variability of tenderness to 14 days of aging.

As fat is removed from Mozzarella cheese, the resulting increase in protein content causes the cheese to become tough, thus decreasing the desired physical characteristics of meltability and stretch. Low-fat (6% fat) Mozzarella cheese was manufactured with the addition of several levels of a Lactococcus lactis adjunct culture that was proteinase positive and lactose deficient in an attempt to improve these physical properties. During cheese manufacture , milk was acidified to pH 6.0, then inoculated with Lactobacillus helveticus and Streptococcus thermophilus. Experimental vats were also inoculated with either 0.25, 0.50, or 1.0% of the adjunct culture. Cheeses made with the adjunct culture had increased melt properties at d 1. During the first 14 d of storage, cheeses manufactured with 0.50% and 1.0% adjunct culture melted more readily than the control; by 28 d, the meltability of all cheeses was similar. Breakdown of cheese body was more rapid in the experimental cheeses and was particularly apparent during shredding. The increase in softness was presumed to be the result of increased proteolysis in the cheeses. There were no significant differences in melt viscosity between control and experimental cheeses. Storage time, however, was significant, and between d 14 and d 28, melt viscosity decreased for all cheeses. Protein hydrolysis was measured using SDS-PAGE, but no differences were observed in the disappearance of intact caseins. In the second part of this study, part-skim (18% fat) Mozzarella cheese was manufactured from milk standardized to a casein-to-fat ratio of 1.2 and inoculated with L. helveticus strain and S. thermophilus strain. Low-fat (6% fat) Mozzarella cheese was manufactured from milk with a casein-to-fat ratio of 4.2 and inoculated with the same starter culture with (or without) addition of the proteinase positive, lactose deficient adjunct culture. The cheese was molded into 1.5-lb blocks and stored at 4°C. Meltability and melt viscosity of the cheese were measured during 28 d storage. Disappearance of αs1-casein and ß-casein was measured using free solution capillary electrophoresis, which separated intact proteins and large peptides. Micellar electrokinetic capillary chromatography was used to study the appearance of small peptides (<30 >kDa) during storage. After 28 d storage, there were significant decreases in the amount of intact αs1-casein remaining after 28 d, but no measurable change in ß-casein in either the part-skim or low-fat cheeses. In part-skim cheese, 71% αs1-casein remained, but in the low-fat cheeses only 20% intact αs1-casein remained after 28 d. If adjunct culture was used in low-fat cheese, then only 14% a 5 1-casein was found after 28 d. A similar increase in proteolysis in the low-fat cheeses was observed based on the amount of small peptides produced. Part of these differences may be a function of increased moisture content of the low-fat cheese, 61% vs 51% in part-skim cheese. During storage, part-skim Mozzarella showed a typical increase in melt with a corresponding decrease in melt viscosity. Melt increased from 10.6 cm at d 1 to 16.9 cm at d 28; melt viscosity at 80°C decreased from 1.0 x 106 cP at d 1 to 2.1 x 105 cP at d 28. There was less change in melt in the low-fat cheese during storage, 8.9 cm at d 1 and 10.9 cm at d 28. Melt viscosity decreased from 4.8 x 105 cP at d 1 to 1.9 x 105cP at d 28. It appears that adding the adjunct culture increased initial meltability of the low-fat cheese by accelerating proteolysis during the first 14 d but caused an increase in viscosity and decrease in melt after 14 d of refrigerated storage.

No description available.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

No description available.