(1) Dept. of Biochemical Sciences, University of Rome "La Sapienza", P.le A.Moro 5, 00185 Rome Italy
(2) CNR Centre for Nucleic Acids, P.le A.Moro 5, 00185 Rome Italy
Contact surfaces between partners in oligomeric proteins display similarity and complementarity of shapes and chemical features, as shown from the 3D structures deposited in the PDB. The weak interactions involved in protein-protein recognition mediate a number of fundamental biological events. Thus trying to describe the energetics of specific interactions at the contact regions is very important.
We have employed hemoglobin as a model because of the rich functional and structural information available at high resolution, and focussed our interest on the a1b2 interface, which is cleaved upon dimerization of the tetramer. Based on molecular modelling (1), we produced, purified and characterized (2) eight site directed mutants, five single (i.e. Ta38W, Ta41R, Ha103V, Wb37T and Rb40T) and three double (i.e. Ta38W + Ta41R, Ta38W+Ha103V, and Ta38W+Wb37T). The 3D structure of some of them has been solved by X-Ray crystallography.
Five of these mutants retain full cooperativity, though displaying differences in oxygen affinity. Oxygen equilibria were confirmed by CO binding kinetics, which showed that mutation Ta41R alone is capable of preventing the R to T transition upon deoxygenation. Additional data on deoxy tetramers dissociation kinetics obtained on the mutants provided an insight into the stability of the interface. Results in the presence of 0.5M NaI made clearer the mode of action of this powerful anion and let a hidden binding site be discovered in the mutant Rb40T.
Dimerization equilibrium constants of the CO derivative of all the mutants were calculated by means of analytical ultracentrifuge. From these data we calculated energetic parameters and correlated them with structural changes in intersubunit contacts and with differences in the buried hydrophobic and polar surfaces.
This study sheds light on specific interactions which stabilize the a1b2 contact region in oxy and deoxy Hb, leads to some estimate of the energetics involved in single-site mutations at this interface, and also proved to be useful to investigate general aspects regarding contact surfaces between protein subunits.
A.E.M. has been supported by a grant from "A.Villa Rusconi" foundation.
(1) O.Schaad, B.Vallone, S.Edelstein, C.R.Acad.Sci.Paris
(1993) 316, 564-71.
(2) B.Vallone, A.Bellelli, A.E.Miele, M.Brunori and G.Fermi
J.Biol.Chem. (1996) 271, 12472-80.