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#protein orientation

2 public questions tagged with this topic.

Plasma Membrane Proteins

Which technique can be used to determine if a membrane protein is inside-out or right-side-out?

Determining orientation of membrane proteins after reconstitution into artificial vesicles essential for establishing vectorial transport competence. Proteoliposomes generated by removing detergent via dialysis may incorporate proteins randomly right-side-out exposing original extracellular domains outward or inside-out exposing cytoplasmic domains outward leading to mixed activity. Sidedness assay exploits membrane impermeability of antibodies one hundred fifty kilodaltons IgG unable to cross sealed bilayer. Intact vesicles incubated with monoclonal antibody against known extracellular epitope such as glycophorin N-terminus or Band 3 loop three undergo binding exclusively when epitope faces external medium detectable by secondary gold-conjugated antibody electron microscopy, flow cytometry, or after pelleting by immunoblotting pellet. Permeabilization with low concentration Triton X-100 zero point one percent exposes total epitopes providing total protein control. Alternative protease protection where extracellular trypsin cleaves only outward loops. Hydropathy plots predict number of spans not orientation in liposomes, SDS-PAGE separates subunits irrespective of sidedness, FRAP measures lateral mobility. Thus antibody labeling using impermeant probe provides definitive sidedness determination for inside-out versus right-side-out vesicles.

Ref: Rohde et al., Determination of Membrane Protein Orientation by Antibody Labeling, J Mol Biol Methods.

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Protein Sorting and Trafficking

The topology of membrane proteins is determined by:

Topological orientation of membrane proteins, defined by which loops face cytosolic versus exoplasmic or lumenal space, is encoded within polypeptide itself as a set of topogenic signals rather than imposed solely by external receptors. As nascent chain enters Sec61 translocon, hydrophobic segments can function either as start-transfer signal-anchor that opens lateral gate and initiates translocation of downstream region, or as stop-transfer sequence that halts translocation and partitions laterally into lipid bilayer through same gate formed by helices 2b and 7. Positive-inside rule, first documented in bacteria and conserved in eukaryotes, shows distribution of positively charged lysine and arginine residues flanking hydrophobic core strongly influences orientation because moving positive charge across membrane costs more energy and because Sec61 retains basic flanks cytosolically via acidic residues lining pore and anionic phospholipid attraction. Order of successive signal-anchor and stop-transfer elements determines multi-spanning topology polytopic proteins. SRP receptor presence or ATP hydrolysis modulates efficiency of targeting but does not rewrite inherent topological information encoded by charge bias and hydrophobicity profile of signal-anchor and stop-transfer sequences.

Ref: von Heijne G, Annu Rev Biophys 36: 2007, Membrane Protein Topology Determinants.

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