Fluorescence-Detection Size-Exclusion Chromatography: A Technique to Identify the Integrity of Fluorescent Membrane Proteins upon Detergent Solubilization
DEŞİFRE METNİ
Bacterial integral membrane proteins, anchored to lipid bilayer membranes, facilitate transport of molecules across membranes and transduction of signals. For structural studies, specific integral membrane recombinant proteins are overexpressed with fused green fluorescent protein, GFP.
To evaluate recombinant membrane protein stability following detergent solubilization, obtain bacterial membrane fraction containing GFP-tagged integral membrane proteins. Add a non-ionic detergent. Beyond the critical concentration, the detergent forms micelles.
Micelles interact with lipid membranes and membrane proteins, solubilizing the membranes and forming protein-detergent complexes. Additionally, membrane proteins' hydrophobic transmembrane regions may remain unmasked, causing protein aggregation. Further, free GFPs may result due to degradation of membrane proteins.
Centrifuge the mixture. Collect the detergent-solubilized fraction-containing supernatant.
Assemble a size-exclusion chromatography column connected to a fluorescence detector. The column matrix comprises agarose beads with pore sizes defining a broad fractionation range.
Equilibrate the column with detergent-containing buffer to minimize nonspecific interactions and maintain protein solubilization.
Load the solubilized membrane fraction into the column. Protein aggregates evade the biggest pores and move through the interparticle spaces between beads, eluting first. Smaller protein-detergent complexes enter a few pores in the beads, traversing a longer path, and elute later. Free GFPs enter all the bead pores and elute last. The fluorescence detector analyzes the eluted fractions, detecting GFP-emitted fluorescence.
Symmetrical fluorescence peaks of protein-detergent complexes with minimal aggregates and free GFP indicate the detergent's suitability for membrane protein solubilization.
