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#protein topology

3 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 epitop

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

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Plasma Membrane Proteins

Which experimental technique is commonly used to determine the number of transmembrane domains in a protein?

Prediction of membrane topology begins with hydropathy profiling visualizing hydrophobic potential along polypeptide. Kyte-Doolittle scale assigns each amino acid numerical value reflecting water to vapor transfer free energy: isoleucine four point five, valine four point two, leucine three point eight highly hydrophobic, arginine minus four point five highly hydrophilic. Sliding window average typically nineteen to twenty one residues equivalent to hydrophobic thickness smooths profile revealing peaks above threshold about one point six indicating membrane-spanning segments. Positive-inside r

Ref: Kyte and Doolittle, A Simple Method for Displaying Hydropathic Character, J Mol Biol 1982.

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Protein Sorting and Trafficking

What determines the insertion orientation of a Type II membrane protein?

Insertion orientation of Type II single-pass membrane proteins, characterized by single non-cleaved signal-anchor yielding N-cytosolic C-lumenal topology, follows positive-inside rule first quantified in bacterial inner membrane proteins and validated in ER. Energetic penalty for translocating positively charged lysine and arginine across hydrophobic core is substantial, and cytosolic leaflet enriched in anionic phosphatidylserine attracts basic side chains, while Sec61 channel vestibule contains acidic residues influencing positioning. Therefore distribution of basic residues flanking signal-

Ref: Hartmann et al., PNAS 86: 1989, Positive Charges Determine Type II Orientation.

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