The use of amphipathic maleimides to study membrane-associated proteins

Abstract

A series of amphiphilic polymethlyenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus sitting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site of D-beta-hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in the D-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.

Identification of the Site in the Substance P (NK-1) Receptor for Modulation of Peptide Binding by Sulfhydryl Reagents

“…The chemical and physical properties of the amphiphilic maleimide derivatives which we used to localize the cysteine residue on the receptor have been described by Griffiths et al (34,37). As indicated by their partition coefficients between octanol and phosphate buffer at pH 8, the maleimide derivatives with a polymethylene chain of up to 10 carbons are more soluble in the aqueous phase than in the lipid phase and their respective carboxyl groups are ionized and will not penetrate the membrane.…”

“…They also share a similar reactivity toward ␤-mercaptoethanol. Furthermore, it has been demonstrated that all the maleimide derivatives have equal potency in inhibiting the enzyme, D-3-phosphoglyceraldehyde dehydrogenase in the soluble state, but varied in potency when the enzyme was embedded in the membrane (34). These amphiphilic maleimide derivatives have been used to probe the depth of the sulfhydryl groups within the plane of the membrane in several membrane-bound enzymes and transporter proteins (34,38,39).…”

“…Furthermore, it has been demonstrated that all the maleimide derivatives have equal potency in inhibiting the enzyme, D-3-phosphoglyceraldehyde dehydrogenase in the soluble state, but varied in potency when the enzyme was embedded in the membrane (34). These amphiphilic maleimide derivatives have been used to probe the depth of the sulfhydryl groups within the plane of the membrane in several membrane-bound enzymes and transporter proteins (34,38,39). The results of these studies provided strong support for a mechanism whereby the carboxyl group anchors the maleimide derivatives at the membrane and the depth of the maleimide group is determined by the length of their polymethylene chains.…”

“…Since NEM can readily traverse the cellular membrane, whereas AM n molecules for n up to 10 with charged carboxylate anion cannot (34), the cysteine residue that is sensitive to AM5, AM7, and NEM is located within the plane of the membrane. Griffiths et al (34) have provided data that support a model for the mechanism of action of these AM molecules, in which the carboxylate group anchors the reagents at the lipid/water interface and the depths of the reactive maleimides can reach within the plane of the membrane is dependent on the length of their polymethylene chains. Thus, the cysteine residue sensitive to AM5, AM7, and submillimolar NEM appears to be located within the TM-spanning domain of the SP receptor close to the extracellular border.…”