Impact of Interfacial Chemical Modifications on the Assembly of an Allosteric Protein - Isothermal Calorimetry and Oxygenation Measurements

Abstract

Most allosteric proteins -if not all- present in living organisms tend to associate naturally to form macromolecular assemblies. The classical tenet of allostery invokes that structural changes at quaternary level are responsible for triggering functional changes in the assembly. That is to say, functional changes are not expected to occur in macromolecular systems that lack quaternary structure (with a few exceptions).In the present work, we have investigated the role of the α1β1 interface in α2β2 tetrameric hemoglobin (Hb), long considered inert, and studied the effects of chemical modifications on this interface on the dissociation of tetramers into dimers, as well as oxygenation properties of tetrameric Hb.Chemical modifications of sulfhydryl groups of α104Cys and β112Cys present in the α1β1 interface allow the study of intradimeric communication. Reactions of these Cys residues were carried out with dithiopyridine. Disulfide bonded thiopyridyl groups thus produced faced the central cavity of the tetramer.Tetramers were reassembled and dimer association equilibrium constants and oxygenation properties were measured. We found that any modification on the α1β1 interface produced a pronounced decrease in oxygen affinity. Surprisingly, the dimerization of the ligated derivatives was enhanced rather than impaired. These results suggest that the chemically modified α1β1 interface of the dimer produced such a striking effect. In other words, the quaternary effect originated from the dimer rather than the tetramer.