RESUMO
Tuberculosis, a contagious disease of infectious origin is currently a major cause of deaths worldwide. Mycobacterium indicus pranii (MIP), a saprophytic nonpathogen and a potent immunomodulator is currently being investigated as an intervention against tuberculosis along with many other diseases with positive outcome. The apparent paradox of multiple chaperones in mycobacterial species and enigma about the cellular functions of the client proteins of these chaperones need to be explored. Chaperones are the known immunomodulators; thus, there is need to exploit the proteome of MIP for identification and characterization of putative chaperones. One of the immunogenic proteins, MIP_05962 is a member of heat shock protein (HSP) 20 family due to the presence of α-crystallin domain, and has amino acid similarity with Mycobacterium lepraeHSP18 protein. The diverse functions of M. lepraeHSP18 in stress conditions implicate MIP_05962 as an important protein that needs to be explored. Biophysical and biochemical characterization of the said protein proved it to be a chaperone. The observations of aggregation prevention and refolding of substrate proteins in the presence of MIP_05962 along with interaction with non-native proteins, surface hydrophobicity, formation of large oligomers, in-vivo thermal rescue of Escherichia coli expressing MIP_05962, enhancing solubility of insoluble protein maltodextrin glucosidase (MalZ) under in-vivo conditions, and thermal stability and reversibility confirmed MIP_05962 as a molecular chaperone.
Assuntos
Proteínas de Bactérias/metabolismo , Citrato (si)-Sintase/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas de Choque Térmico HSP20/metabolismo , Chaperonas Moleculares/metabolismo , Complexo Mycobacterium avium/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sobrevivência Celular , Citrato (si)-Sintase/química , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Proteínas de Choque Térmico HSP20/química , Proteínas de Choque Térmico HSP20/genética , Resposta ao Choque Térmico , Temperatura Alta/efeitos adversos , Interações Hidrofóbicas e Hidrofílicas , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Agregados Proteicos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Redobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
Maltodextrin glucosidase (MalZ) hydrolyses short malto-oligosaccharides from the reducing end releasing glucose and maltose in Escherichia coli. MalZ is a highly aggregation prone protein and molecular chaperonins GroEL and GroES assist in the folding of this protein to a substantial level. The N-terminal region of this enzyme appears to be a unique domain as seen in sequence comparison studies with other amylases as well as through homology modelling. The sequence and homology model analysis show a probability of disorder in the N-Terminal region of MalZ. The crystal structure of this enzyme has been reported in the present communication. Based on the crystallographic structure, it has been interpreted that the N-terminal region of the enzyme (Met1-Phe131) might be unstructured or flexible. To understand the role of the N-terminal region of MalZ in its enzymatic activity, and overall stability, a truncated version (Ala111-His616) of MalZ was created. The truncated version failed to fold into an active enzyme both in E. coli cytosol and in vitro even with the assistance of chaperonins GroEL and GroES. Furthermore, the refolding effort of N-truncated MalZ in the presence of isolated N-terminal domain didn't succeed. Our studies suggest that while the structural rigidity or orientation of the N-terminal region of the MalZ protein may not be essential for its stability and function, but the said domain is likely to play an important role in the formation of the native structure of the protein when present as an integral part of the protein.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Glucosídeos/química , Glicosídeo Hidrolases/química , Sequência de Aminoácidos , Sítios de Ligação , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Glucosídeos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Agregados Proteicos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
The industrially important species of corynebacteria viz. Corynebacterium acetoacidophilum appear to be alternative hosts for recombinant protein production; despite many efforts, a strong promoter-based system in corynebacteria has not been established so far. Described here is a T7 promoter-based expression system which was functional in both gram-positive C. acetoacidophilum and gram-negative Escherichia coli in an external inducer independent manner. This is the very first report of a T7 expression system for Corynebacterium sp. Also, it is a useful addition in the existing T7 expression systems of E. coli.
