Protein Translocation Activity in Surface-Supported Lipid Bilayers
Kanokporn Chattrakun, David Hoogerheide, Chunfeng Mao, Linda L. Randall, Gavin M. King
Surface-supported lipid bilayers are used widely throughout the nanoscience community as robust mimics of cellular membrane. For example, they are frequently employed in single molecule atomic force microscopy (AFM) studies to shed light on membrane protein conformational dynamics and folding in fluid. However, in AFM as well as in other surface-sensing techniques the close proximity of the supporting surface raises questions about the preservation of activity, and hence, biological relevance. Employing the translocase from the general secretory (Sec) system of E. coli as a model, here we quantify activity via two biochemical activity assays in surface-supported lipid bilayers. The first assay assesses ATP hydrolysis, the second, polypeptide translocation across the membrane. Hydrolysis assays revealed multiple distinct levels of activation that were similar in magnitude to traditional solution experiments. Translocation assays revealed turnover numbers that were comparable to solution, but with a 10-fold reduction in apparent rate constant. Despite difference in kinetics, the chemo-mechanical coupling efficiency (ATP hydrolyzed per residue translocated) of the Sec system was only reduced 2-fold on glass compared to solution. Activity varied with the topographic complexity of the underlying surface. Neutron reflectometry corroborated the measurements. Overall, translocation activity was maintained for the surface-adsorbed Sec system, albeit with a slower rate-limiting step. More generally, polypeptide translocation activity measurements yield valuable quantitative metrics to assess the local environment about surface-supported lipid bilayers.
, Hoogerheide, D.
, Mao, C.
, Randall, L.
and King, G.
Protein Translocation Activity in Surface-Supported Lipid Bilayers, Langmuir, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=928333
(Accessed June 7, 2023)