A modified, dual reporter TOXCAT system for monitoring homodimerization of transmembrane segments of proteins.
The TOXCAT assay system developed by Russ and Engelman [TOXCAT: a measure of transmembrane helix association in a biological membrane, Proc. Natl. Acad. Sci. USA 96 (1999) 863-868] provides an in vivo means of selecting for and evaluating the strength of interaction between identical transmembrane alpha-helices. In the course of utilizing TOXCAT to study the architecture of a sodium channel hNa(V)1.5, an apparently strong dimerization of two of its putative transmembrane segments was revealed. Following random mutagenesis of these regions, several amino acids critical for the observed dimerizations were identified. In order to develop a more efficient means of isolating mutations which specifically disrupt dimerization of these transmembrane segments without affecting their membrane-targeting properties, we developed a modification to the original TOXCAT design in which the C-terminal maltose binding protein moiety is replaced by the beta-lactamase. We show that this assay system is capable of simultaneously monitoring the integrity of the chimeric protein, its membrane insertion activity, and the ability of the transmembrane segment under study to dimerize.
Lis
Maciej
M
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, 3435 Main St., Buffalo, NY 14214, USA.
Blumenthal
Kenneth
K
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008320:Maltose / Q000235:genetics; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics
D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D011993:Recombinant Fusion Proteins; C568320:SCN5A protein, human; D015222:Sodium Channels; D008320:Maltose; D001618:beta-Lactamases