Abstract
Membrane proteins play key roles in many fundamental functions in cells including ATP synthesis, ion and molecule transporter, cell signalling and enzymatic reactions, accounting for ~30% genes of whole genomes. However, the hydrophobic nature of membrane proteins frequently hampers the progress of structure determination. Detergent screening is the critical step in obtaining stable detergent-solubilized membrane proteins and well-diffracting protein crystals. Fluorescence Detection Size Exclusion Chromatography (FSEC) has been developed to monitor the extraction efficiency and monodispersity of membrane proteins in detergent micelles. By tracing the FSEC profiles of GFP-fused membrane proteins, this method significantly enhances the throughput of detergent screening. However, current methods to acquire FSEC profiles require either an in-line fluorescence detector with the SEC equipment or an off-line spectrofluorometer microplate reader. Here, we introduce an alternative method detecting the absorption of GFP (FA-SEC) at 485 nm, thus making this methodology possible on conventional SEC equipment through the in-line absorbance spectrometer. The results demonstrate that absorption is in great correlation with fluorescence of GFP. The comparably weaker absorption signal can be improved by using a longer path-length flow cell. The FA-SEC profiles were congruent with the ones plotted by FSEC, suggesting FA-SEC could be a comparable and economical setup for detergent screening of membrane proteins.
Highlights
Membrane proteins are abundant in cells and play pivotal roles in solute homeostasis, signal transduction and energy production
The correlation demonstrates that FA-size-exclusion chromatography (SEC) traces monitored at 485 nm delivers comparable results to the fluorescence profiles measured in traditional Fluorescence Detection Size Exclusion Chromatography (FSEC)
The A485 peaks observed in the ASBTNM-His8 or HiTehA-His8 Fluorophore Absorption SEC (FA-SEC) profiles are presumably originate from the free heme molecules associated with the overexpressed proteins via heme iron coordinated to the octa-histidine tag [20, 21]
Summary
Membrane proteins are abundant in cells and play pivotal roles in solute homeostasis, signal transduction and energy production. The major obstacles of obtaining membrane protein structures at atomic level are their low heterologous expression level, poor protein stability in detergent micelles and difficulty in crystallization. As a result, it requires a time-consuming and laborious process to perform a series of empirical screenings, such as expression conditions, homolog proteins, detergents, additives and conformationally sensitive antibodies, to eventually obtain crystals of membrane proteins with reasonable diffraction qualities. Injecting detergent-solubilized membranes containing EGFP-deleted ASBTNM-His or HiTehA-His revealed FA-SEC peaks with moderate monodispersity (S2 Fig). We plotted the FA-SEC profile of purified ASBTNM, of which the EGFP and His-tag were cleaved by TEV protease and the background peak was almost invisible (S2 Fig), implicating the chromophores in the ASBTNM-His or HiTehA-His membranes are associated with the overexpressed membrane proteins via the C-terminal His-tag. The A485 peaks observed in the ASBTNM-His or HiTehA-His FA-SEC profiles are presumably originate from the free heme molecules associated with the overexpressed proteins via heme iron coordinated to the octa-histidine tag [20, 21]
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