| Popis: |
AsystematicsearchforEscherichiacoliproteins with thezinc-binding activity was performed using the assay ofradioactive Zn(II) binding to total E.coliproteins fraction-atedbytwomethodsoftwo-dimensionalgelelectrophoresis.A total of 30–40 radioactive spots were identified, of which14 have been assigned from N-terminal sequencing.Inaddition to five known zinc-binding proteins, nine zinc-binding proteins were newly identified including: acetatekinase (AckA), DnaK, serine hydroxymethyltransferase(GlyA), transketolase isozymes (TktA/TktB), translationelongation factor Ts (Tsf), ribosomal proteins L2 (RplB),L13 (RplM) and one of S15 (RpsO), S16 (RpsP) or S17(RpsQ).Together with about 20 known zinc-binding pro-teins, the total number of zinc-binding proteins in E.coliincreased up to more than 30 species (or more than 3% ofabout 1000 proteins expressed under laboratory cultureconditions).The specificity and affinity of zinc-binding wereanalysed for some of the zinc-binding proteins.Keywords: zinc-binding protein; Escherichiacoli;proteome;two-dimensional gel electrophoresis.Zinc is an essential trace element, but virtually nontoxic, incontrast to iron, copper and mercury.Over 300 enzymes orproteins have been identified that require zinc for function[1,2].Physical and chemical properties of zinc, such as itsstable association with proteins and its co-ordinationflexibility, make it highly adaptable to meeting the needsof proteins and enzymes that carry out diverse biologicalfunctions [3].In zinc-containing enzymes or proteins, zinchas two major functions, i.e. catalytic and structural. Thecatalytic role specifies that zinc participates directly inenzyme catalysis, while structural zinc atoms are requiredfor stabilization of proteins by supporting their folding andoligomerization.Zinc is therefore not simply the cofactorfor enzyme catalytic functions but also the structural factorfor folding of domains involved in protein–protein andprotein–DNA interactions.A large majority of the zinc-containing enzymes have asingle zinc site consisting of a combination of specificamino-acid residues such as Cys, His, Asp and Glu, and asolvent water molecule completing the co-ordination sphere[3].After the finding of a number of zinc-containing DNA-binding proteins in higher eukaryotes, many different typesof the zinc-binding motif have been identified, includingthose tetrahedrally co-ordinated to imidazole nitrogenatoms from His and thiol groups from Cys [2].Thefunctions of protein-bound zinc are beginning to catch upwith the increasing number of zinc-containing proteins.Upto the present time, the structural and functional roles ofzinc have been analysed in detail with zinc-containingproteins from higher eukaryotes, but little is known aboutthe zinc-binding proteins in prokaryotes.Ros homologuesthat exit in plant-associated agrobacteria are the onlybacterial proteins with the typical C2H2-type zinc-fingermotif [4].Various types of zinc-containing protein with thezinc finger or zinc cluster exist in yeast, which, however,lacks proteins with the hormone receptor-type zinc-bindingmotif.This type of zinc-binding proteins appears inCaenorhabditiselegans [5] and the number of this type ofzinc-binding proteins increases in higher animals.The aim of this study is to identify as many zinc-bindingproteins as possible in the model prokaryote, Escherichiacoli.For a systematic and experimental detection of zinc-binding proteins, we employed a conventional method ofradioactive zinc blotting with whole cell extracts fraction-ated by two methods of two-dimensional gel electrophor-esis, the widely used O’Farrell system [6] and the newlydevelopedradical-freeandhighlyreducing(RFHR)method[7].Results indicate that most of the newly identifiedbacterial zinc-binding proteins do not contain the knownzinc-binding motifs, most of which have been identified inhigher eukaryotes.This reportalsodescribes the affinityandspecificity of zinc binding for some of the E.coli zinc-binding proteins. |
