Molecular modelling, simulations and functional analysis of aquaporin from antarctic Pseudomonas sp. ams3

Aquaporin 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 on water transport mechanism particularly from low temperature microbes remains scarce. Antarctic microbes including psychrophilic aquaporins are known for their adaptation to extreme cold environments, offering insights into extremophile biology and potential biotechnological applications. The limited availability of the structural study of psychrophilic aquaporins is an ongoing issue and obtaining detailed insights into their three-dimensional structure is crucial for understanding their unique properties and optimizing their potential applications such as biomimetic membrane, drug transporter and also in cosmetics. By investigating these, it could potentially uncover novel structural features that are specific to these coldadapted proteins. Therefore, this research was conducted to understand the structural and functional properties of aquaporin Z1 (AqpZ1) from Antarctic Pseudomonas sp. AMS3. In this study, structure analysis and 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 AqpZ1 AMS3 structures are 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 helix’s region throughout the simulated temperatures. The selectivity filter and NPA motifs play a major role in solute selectivity and pore radius of the protein. AqpZ1 AMS3 was successfully subcloned into Pet22b vector 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 aquaporin was in 0.5 Mm IPTG incubated at 25ºC for 20 hours induction time. Upon the surfactants optimization, a zwitterionic mild detergent [(3-cholamidopropyl) dimethylammonio]-1- propanesulfonate (CHAPS) was chosen to be the suitable surfactant for the protein solubilization due to its as it is known to ba a mild detergent for membrane proteins. The protein was then purified via affinity chromatography with a molecular mass of 33 kDa. In the biophysical analysis, Circular Dichroism (CD) spectroscopy analysis of AqpZ1 AMS3 was conducted in different temperatures to investigate its secondary structure conformation across a range of temperatures from 10 ºC to 60 ºC. The findings revealed that this protein exhibits a notably predominant α-helical secondary structure at a temperature of 20ºC. The liposome and proteoliposome reconstitution of aquaporin was conducted to assess the functional characteristics of AqpZ1 AMS3. Dynamic Light Scattering (DLS) allowed for the observation of dynamic changes in vesicle size over time. The study was able to measure the alterations in vesicle size and efficiency of water transport, shedding light on the permeability characteristics of aquaporin within the lipid bilayer environment This information obtained from this psychrophilic aquaporin 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.

Text 122753.pdf Download (1MB) Official URL or Download Paper: http://ethesis.upm.edu.my/id/eprint/18654

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