Phase state of lipid bilayers modulates ammonia and water permeability through AtTip2;1.

Abstract

Lipid-protein interactions determine the structure, stability and function of transmembrane proteins. Protein conformation can be influenced by local interactions at the lipid-protein interface or global interactions such as the lateral pressure profile of the membrane, which in turn is influenced by the lipid composition of the membrane. However, despite its methodological importance for in vitro protein characterization and its biological implications the influence of lipid bilayer fluidity on membrane protein activity is largely unknown. Reconstituting the aqua-ammonia channel AtTip2;1, from Arabidopsis thaliana, into OSPC containing large unilamellar vesicles we modulate its unitary water and ammonia permeability switching between the liquid-disordered state and the gel phase state of the bilayer. Quantitative activity characterization utilizing fluorescence correlation spectroscopy, stopped-flow methodology, and dynamic light scattering is complemented by molecular dynamics simulations of AtTIP2;1 in OSPC bilayers. Our in vitro experiments revealed a unitary water permeability in the liquid-disordered phase of 2.5·10−13 cm³s−1. On the contrary, selective transport of ammonia is an order of magnitude lower questioning if ammonia transport is the main function of AtTIP2;1. Moreover, results obtained from our MD simulations show, similar to our in vitro efforts, that AtTIP2;1 exhibits lower activity in the gel state as compared to its activity in the liquid-disordered state. This finding highlights the crucial importance of the lipid matrix in in vitro and in silico protein characterization and is expected to trigger a wealth of future investigations on permeability and regulation of AQPs and other membrane proteins.