title: Direct analysis of cooperativity in multisubunit allosteric proteins.
abstract: Allosteric regulation of protein function is a fundamental phenomenon of major importance in many cellular processes. Such regulation is often achieved by ligand-induced conformational changes in multimeric proteins that may give rise to cooperativity in protein function. At the heart of allosteric mechanisms offered to account for such phenomenon, involving either concerted or sequential conformational transitions, lie changes in intersubunit interactions along the ligation pathway of the protein. However, structure-function analysis of such homooligomeric proteins by means of mutagenesis, although it provides valuable indirect information regarding (allosteric) mechanisms of action, it does not define the contribution of individual subunits nor interactions thereof to cooperativity in protein function, because any point mutation introduced into homooligomeric proteins will be present in all subunits. Here, we present a general strategy for the direct analysis of cooperativity in multisubunit proteins that combines measurement of the effects on protein function of all possible combinations of mutated subunits with analysis of the hierarchy of intersubunit interactions, assessed by using high-order double-mutant cycle-coupling analysis. We show that the pattern of high-order intersubunit coupling can serve as a discriminative criterion for defining concerted versus sequential conformational transitions underlying protein function. This strategy was applied to the particular case of the voltage-activated potassium channel protein (Kv) to provide compelling evidence for a concerted all-or-none activation gate opening of the Kv channel pore domain. An direct and detailed analysis of the contribution of high-order intersubunit interactions to cooperativity in the function of an allosteric protein has not previously been presented.
authors.affiliation: Department of Life Sciences and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
authors.forename: Nitzan
authors.initials: N
authors.lastname: Zandany
authors.forename: Maya
authors.initials: M
authors.lastname: Ovadia
authors.forename: Irit
authors.initials: I
authors.lastname: Orr
authors.forename: Ofer
authors.initials: O
authors.lastname: Yifrach
mesh_terms: D000494:Allosteric Regulation; D015640:Ion Channel Gating; D024642:Potassium Channels, Voltage-Gated / Q000032:analysis* / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000032:analysis / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D024642:Potassium Channels, Voltage-Gated; D021122:Protein Subunits