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Effect of multiple symmetries on the association of R67 DHFR subunits bearing interfacial complementing mutations

¹ Unité de Repliement et Modélisation des Protéines (CNRS URA 2185) and
² Unité de Bio-Informatique Structural (CNRS URA 2185), Institut Pasteur, Paris Cedex, France

Reprint requests to: Arnaud Blondel, Unité de Repliement et Modélisation des Protéines, Institut Pasteur, F-75724, Paris Cedex 15, France; e-mail: ablondel{at}pasteur.fr; fax: 33-1-40-61-30-43.

It was shown previously that complementation could be a powerful mean to probe protein–protein interactions in the normally tetrameric R67 DHFR. Indeed, mixing complementing inactive dimeric mutants produced active heterotetramers. This approach turned a homo-oligomer into a hetero-oligomer and thus allowed the use of combinatorial assays, a subtle analysis of the association forces, and a precise determination of the equilibrium dissociation constants (K_D) by titrimetry. However, for some of the complementing pairs, the experimental data implied multiple equilibria involving heterodimers, although no monomers could be detected. Thus, the reactions involved had to be identified to elaborate a suitable model to determine the K_D of those pairs correctly. That model suggested that homodimers associated rapidly before the protomers could be redistributed in a multiple equilibrium system. Kinetic data confirmed that view. The association data at equilibrium were analyzed by multiple curve fitting with all plausible combinations of parameters. This gave a confidence interval for K_D that is safer than the usual 67% or 90% confidence interval. Finally, the K_D of one specific reaction, the dissociation of a heterotetramer with the relevant symmetry into two homodimers could be determined with the relevant model for each complementing pair, although multiple equilibria were present. These K_D can thus be used as a set of references data to test and improve theoretical methods such as association free energy calculations.

Keywords: Association interface; equilibrium dissociation constant; titrimetry; multiple equilibria; data modeling

Abbreviations: R67 DHFR, dihydrofolate reductase from resistance plasmid R67 • (nX)₂ and (nX)₄, R67 DHFR homodimer or homotetramer bearing point mutation at position n leading to residue X • (nX;mZ)₂ and (nX;mZ)₄, R67 DHFR homodimer or homotetramer bearing two point mutations at position n and m • (WT)₄, wild-type R67 DHFR homotetramer • (nX)(mZ), heterodimers • (nX)₂:(mZ)₂, heterocomplex of homodimers • [(nX)(mZ)]₂, homocomplex of heterodimers • (nX)₂ + (mZ)₂, heterocomplex of unspecified symmetry, or pair of complementing mutants • NADPH, nicotinamide adenine dinucleotide phosphate • THF, 5,6,7,8-tetrahydrofolate • DHF, 7,8-dihydrofolate • Tris, Tris [hydroxymethyl]aminomethane • MES, 2-[N-Morpholino]ethanesulfonic acid • MTA buffer, 100 mM Tris, 50 mM MES, 50 mM acetic acid • DTT, 1,4-dithio-DL-threitol • TCA, trichloro acetic acid • IEF PAGE, isoelectrofocalization polyacrylamide gel electrophoresis • U-Model, complex model of association yielding a U-shaped iso-fluorescence titration curve • U_s-Model, simplified U-model yielding a U-shaped curve • V-Model, simple model of association yielding a V-shaped iso-fluorescence titration curve

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