Probing the Regulatory and Transport Mechanism of Mitochondrial Carriers with Thermostability Shift Assays

Orateur : Edmund KUNJI (MRC, Mitochondrial Biology Unit, Cambridge, United Kingdom)

Mitochondrial carriers are membrane proteins that transport keto acids, amino acids, fatty acids, nucleotides, vitamins and inorganic ions across the inner membrane of mitochondria1. Here we discuss the use of thermostability shift assays to probe the transport and regulatory mechanism of mitochondrial carriers. In these assays, protein unfolding is monitored with the fluorescent dye 7-diethylamino- 3-(4-maleimidophenyl)-4-methylcoumarin (CPM) that emits a fluorescent signal upon reaction with cysteine residues. A controlled temperature ramp is used to denature the population of membrane proteins, exposing hidden cysteine residues to generate a melting curve. Here we show that thermostability assays can be used to study the nature of the interactions of lipids, detergents, substrates and inhibitors with mitochondrial carriers and uncoupling proteins. In combination with mutagenesis they can also be used to show state-dependent amino acid interactions as part of the transport cycle. Recently, these assays have been used to show interactions between protein domains as part of a calcium-dependent regulatory mechanism.

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