Purification and characterization of YihA, an essential GTP-binding protein from Escherichia coli.
YihA has previously been characterized as an essential gene of unknown function in both Escherichia coli and Bacillus subtilis. It is conserved in bacteria and represents an attractive target for the discovery of new antibiotics. YihA encodes a putative GTP-binding protein. We have cloned and overexpressed the gene encoding E. coli YihA and initiated biochemical studies as a first step towards understanding its biological function. We showed by circular dichroism that the purified protein has a secondary structure typical of most GTP-binding proteins. It binds guanine nucleotides specifically, as demonstrated by fluorescence resonance energy transfer between 2'-(or-3')-O-(N-methylanthraniloyl) nucleotides (mant-nucleotides) and the tryptophans of YihA. The K(d) values for GDP and GTP were determined by competition with 2'-(or-3')-O-(N-methylanthraniloyl) GDP to be 3 and 27 microM, respectively. Using mutants of YihA we show that nucleotide binding occurs at the putative GTP-binding domain predicted from the primary sequence.
Lehoux
Isabelle E
IE
GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, UP4430, P.O. Box 5089, PA 19426-0989, Collegeville, USA.
Mazzulla
Marie J
MJ
Baker
Audrey
A
Petit
Chantal M
CM
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D016415:Sequence Alignment
D029968:Escherichia coli Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; C485968:YihA protein, E coli