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  • Title: A single mutation disrupts the pH-dependent dimerization of glycinamide ribonucleotide transformylase.
    Author: Mullen CA, Jennings PA.
    Journal: J Mol Biol; 1998 Mar 06; 276(4):819-27. PubMed ID: 9500916.
    Abstract:
    Monomeric GART reversibly associates into a dimeric form as a function of decreasing solution pH. The transition is consistent with a three-proton transfer reaction with an apparent pKa near 7. We now report that a single mutation, which replaces a glutamic acid at position 70 in the dimer interface with alanine (E70A), disrupts the pH-dependent dimerization of GART based on dynamic light scattering and gel filtration studies. A comparison of data obtained from UV-absorbance difference spectroscopy for both the wild-type and mutant forms of GART indicates that a tyrosine residue(s) undergoes a change in solvent exposure over the pH range 6.55 to 8.19. A conformational change in tertiary structure that accompanies dimerization accounts for 60% of the observed optical difference, while the remaining 40% can be attributed to a pH-dependent process unrelated to dimerization. In addition, fluorescence studies of the mutant protein indicate that a pH-dependent change in tryptophan fluorescence exhibited by the wild-type protein is unrelated to quaternary structural changes and is likely a result of simple fluorescence quenching by nearby protonated histidine side-chains. Taken together, our results indicate that a single amino acid change at the dimer interface is sufficient to interrupt the highly specific, pH-dependent assembly reaction of GART, although pH-dependent conformational changes present in the wild-type protein also occur in E70A GART. This work is a first application of structure-based site-directed mutagenesis to the analysis of this pH-dependent assembly reaction.
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