86] Some approaches to determining the primary structure of membrane proteins

Abstract

Publisher Summary This chapter discusses various approaches for determining the primary structure of membrane proteins. Either gel chromatography in detergent solutions or extraction with various mixtures of water–organic solvents is the method employed for delipidation of membrane proteins. The important step in the determination of the protein primary structure is the fragmentation of their polypeptide chains by various chemical or enzymatic methods. The high specificity—as well as the easiness of removal from the reaction medium—makes cyanogen bromide one of the powerful tools in the quantitative fragmentation of proteins and peptides. The interest in this reagent has increased rapidly, because under controlled conditions it can quantitatively cleave proteins at both the methionine and tryptophan residues. Cyanogen bromide cleavage is regularly employed for fragmentation of bacteriorhodopsin and visual rhodopsin. An aqueous suspension of the protein delipidated with sodium dodecyl sulfate is centrifuged and dry guanidinium hydrochloride is added to the resulting pellet. The mixture is carefully triturated with a glass rod until a fine suspension is obtained, and distilled water is added gradually in drops until the precipitate is dissolved completely. The concentrated formic acid and 500-fold excess of cyanogen bromide are added to the protein solution in guanidinium hydrochloride. The reaction is carried out for 24 hr at room temperature in the dark. Then the peptide mixture is evaporated on a rotor evaporator and vacuum-dried over sodium hydroxide. Analysis showed that this method allowed not only carrying out of the quantitative cleavage of the protein but also avoidance of the aggregation characteristic of cyanogen bromide peptides, thus considerably facilitating their subsequent separation.