A method for determining transmembrane protein structure.
A simple and rapid protein chemical approach for determining the transmembrane structure of membrane proteins is described. The method involves single substitutions of consecutive amino acid residues, within putative transmembrane segments, to cysteine. This is followed by the analysis of their susceptibility to modification by maleimides with different physico-chemical properties. Fluorescein-5-maleimide (FM), being hydrophilic, modified only residues located in the aqueous environment, while the hydrophobic reagent, benzophenone-4-maleimide (BM) modified residues exposed to the lipid phase. These probes are large enough to cause an increase in the molecular weight of relatively small membrane proteins or polypeptide fragments, which is detectable by SDS-PAGE. Modification by much smaller probes, such as N-ethylmaleimide (NEM), could also be monitored indirectly by the ability to prevent SDS-solubilized protein from being modified with fluorescein-5-maleimide. The approach is demonstrated with the proteolipid complex of the vacuolar H(+)-ATPase expressed in yeast and with the putative Isk K(+)-channel expressed and radiolabelled in E. coli. The advantages of this approach are: (1)it is rapid, easy and inexpensive, (2) detection of the modification of engineered cysteines is simple, (3) it requires only minute quantities of the protein, (4) the protein does not require purification, (5) a broad range of maleimides with different physico-chemical properties can be used, (6) the structure can be investigated under native conditions and does not require protein reconstitution into artificial bilayers.
Jones
P C
PC
Department of Biochemistry & Molecular Biology, The University of Leeds, UK.
Sivaprasadarao
A
A
Wray
D
D
Findlay
J B
JB
D000595:Amino Acid Sequence; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005033:Ethylmaleimide / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013462:Sulfur Radioisotopes
D005452:Fluoresceins; D008563:Membrane Lipids; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011506:Proteins; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; C059983:potassium channel protein I(sk); C037041:fluorescein 5-maleimide; D003545:Cysteine; D005033:Ethylmaleimide