The Single-Molecule Approach to Membrane Protein Stoichiometry.
The advent of techniques for imaging solitary fluorescent molecules has made possible many new kinds of biological experiments. Here, we describe the application of single-molecule imaging to the problem of subunit stoichiometry in membrane proteins. A membrane protein of unknown stoichiometry, prestin, is coupled to the fluorescent enhanced green fluorescent protein (eGFP) and synthesized in the human embryonic kidney (HEK) cell line. We prepare adherent membrane fragments containing prestin-eGFP by osmotic lysis. The molecules are then exposed to continuous low-level excitation until their fluorescence reaches background levels. Their fluorescence decreases in discrete equal-amplitude steps, consistent with the photobleaching of single fluorophores. We count the number of steps required to photobleach each molecule. The molecular stoichiometry is then deduced using a binomial model.
Department of Physics, Creighton University, Omaha, NE, USA.
Michael G
MG
Nichols
Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE, USA.
Richard
R
Hallworth
D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D015233:Models, Statistical; D038761:Photobleaching; D011994:Recombinant Proteins / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods*; D000076988:Sulfate Transporters
D027321:Anion Transport Proteins; D011994:Recombinant Proteins; C498719:SLC26A5 protein, human; D000076988:Sulfate Transporters; D049452:Green Fluorescent Proteins
Green fluorescent protein; Prestin; Single-molecule; Stoichiometry