Synthesis and Labeling of Syntaxin Transmembrane Domains

Syntaxin 1 TMD (residues 266–288; sx-1TM) from Rattus norvegicus, syntaxin 4 TMD (residues 262–297; sx-4TM) from Homo sapiens and syntaxin 1 TMD mutant (sx-1TM with the following mutations: M267A, C271A and I279A) were synthesized using Fmoc solid phase synthesis. The fluorescent dyes Atto647N NHS-ester (Atto-Tec) and Rodamine red succinimidyl ester (Life Technologies) were coupled to the N-termini of sx-TM. The detailed synthesis is described in ref. 10 (link).
C18:1 (1,2-dioleoyl-sn-glycero-3-phosphocholine), C14:1 (1,2-dimyristoleoyl-sn-glycero-3-phosphocholine), C16:1 (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine), C20:1 (1,2-dieicosenoyl-sn-glycero-3-phosphocholine), brain PI(4,5)P₂, doPI(4,5)P₂ (1,2-dioleoyl-sn-glycero-3-phosphatidyl-(1′-myo-inositol-4′,5′-bisphosphate)) and cholesterol were purchased from Avanti Polar Lipids. Bodipy-labelled PI(4,5)P₂ (bodipy-FL-PI(4,5)P₂, C16) was from Echelon Biosciences, and Top-Fluor labelled PI(4,5)P₂ was from Avanti Polar Lipids. The lipophilic fluorescent probe DiO was from Life Technologies.