title: Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase.
abstract: Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.
authors.affiliation: Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.
authors.forename: Sonali
authors.initials: S
authors.lastname: Mehrotra
authors.forename: Mylarappa
authors.initials: M
authors.lastname: B Ningappa
authors.forename: Jayalakshmi
authors.initials: J
authors.lastname: Raman
authors.forename: Ranjith P
authors.initials: RP
authors.lastname: Anand
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Balaram
mesh_terms: D000265:Adenylosuccinate Synthase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D019943:Amino Acid Substitution; D002850:Chromatography, Gel; D003300:Copper / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D004795:Enzyme Stability; D019807:Iodoacetic Acid / Q000737:chemistry; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000201:enzymology*; D011489:Protein Denaturation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D021122:Protein Subunits; D015800:Protozoan Proteins; D008345:Manganese; D003300:Copper; D014364:Tryptophan; D014508:Urea; D000265:Adenylosuccinate Synthase; D003545:Cysteine; C029892:cupric chloride; D019807:Iodoacetic Acid