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 rule where arginine lysine enriched cytosolic loops aids orientation. Modern algorithms TMHMM Phobius TOPCONS integrate Hidden Markov Models with charge bias improving accuracy to about ninety percent. Experimental confirmation uses reporter fusions PhoA active periplasmic versus LacZ cytoplasmic, substituted cysteine accessibility method SCAM labeling membrane-impermeant reagents, and glycosylation mapping using acceptor sites inserted. SDS-PAGE separates by molecular weight, Western blotting detects immunoreactivity, FRAP measures lateral mobility but only hydropathy plot directly estimates number and positions of transmembrane hydrophobic segments guiding cloning and mutagenesis.
Ref: Kyte and Doolittle, A Simple Method for Displaying Hydropathic Character, J Mol Biol 1982.