RESUMEN
Aquaporin (AQP) is a water channel protein from the family of transmembrane proteins which facilitates the movement of water across the cell membrane. It is ubiquitous in nature, however the understanding of the water transport mechanism, especially for AQPs in microbes adapted to low temperatures, remains limited. AQP also has been recognized for its ability to be used for water filtration, but knowledge of the biochemical features necessary for its potential applications in industrial processes has been lacking. Therefore, this research was conducted to express, extract, solubilize, purify, and study the functional adaptations of the aquaporin Z family from Pseudomonas sp. AMS3 via molecular approaches. In this study, AqpZ1 AMS3 was successfully subcloned and expressed in E. coli BL21 (DE3) as a recombinant protein. The AqpZ1 AMS3 gene was expressed under optimized conditions and the best optimized condition for the AQP was in 0.5 mM IPTG incubated at 25°C for 20 h induction time. A zwitterionic mild detergent [(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate was the suitable surfactant for the protein solubilization. The protein was then purified via affinity chromatography. Liposome and proteoliposome was reconstituted to determine the particle size using dynamic light scattering. This information obtained from this psychrophilic AQP identified provides new insights into the structural adaptation of this protein at low temperatures and could be useful for low temperature application and molecular engineering purposes in the future.
Asunto(s)
Acuaporinas , Proteínas Bacterianas , Clonación Molecular , Escherichia coli , Pseudomonas , Proteínas Recombinantes , Pseudomonas/metabolismo , Pseudomonas/genética , Pseudomonas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Acuaporinas/química , Acuaporinas/genética , Acuaporinas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Expresión Génica , Proteolípidos/metabolismo , Proteolípidos/química , Regiones Antárticas , Liposomas/metabolismo , Liposomas/química , Agua/química , Agua/metabolismo , Solubilidad , Secuencia de AminoácidosRESUMEN
Aquaporin is a water channel protein that facilitates the movement of water across the cell membrane. Aquaporin from the Antarctic region has been noted for its psychrophilic properties and its ability to perform at a lower temperature but there remains limited understanding of the water mechanism of Antarctic Pseudomonas sp. strain AMS3 However, studies regarding aquaporin isolated from psychrophilic Pseudomonas sp. are still scattered. Recently, the genome sequence of an Antarctic Pseudomonas sp. strain AMS3 revealed a gene sequence encoding for a putative aquaporin designated as AqpZ1 AMS3. In this study, structure analysis and a molecular dynamics (MD) simulation of a predicted model of a fully hydrated aquaporin tetramer embedded in a lipid bilayer was performed at different temperatures for structural flexibility and stability analysis. The MD simulation results revealed that the structures were able to remain stable at low to medium temperatures. The protein was observed to have high flexibility in the loop region as compared to the helices region throughout the simulated temperatures. The selectivity filter and NPA motifs play a major role in solute selectivity and the pore radius of the protein. The structural and functional characterization of this psychrophilic aquaporin provides new insights for the future applications of this protein.Communicated by Ramaswamy H. Sarma.
