Using Mutant Cycle Analysis to Elucidate Long-Range Functional Coupling in Allosteric Receptors

摘要

Functional coupling of residues that are far apart in space is the quintessential property of allosteric\udreceptors. Data from functional studies of allosteric receptors, such as whole-cell dose–response relations,\udcan be used to determine if mutation to a receptor significantly impacts agonist potency. However, the\udclassification of perturbations as primarily impacting binding or allosteric function is more challenging,\udoften requiring detailed kinetic studies. This protocol describes a simple strategy, derived from mutant\udcycle analysis, for elucidating long-range functional coupling in allosteric receptors (ELFCAR). Introduction\udof a gain-of-function reporter mutation, followed by a mutant cycle analysis of the readily measured\udmacroscopic EC_(50) values can provide insight into the role of many physically distant targets. This new\udmethod should find broad application in determining the functional roles of residues in allosteric receptors.