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Characterization of ppGpp‐Binding Proteins in Escherichia coli
Author(s) -
Nguyen Duc Tien,
Jung Che Hun
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.760.6
Subject(s) - biochemistry , stringent response , escherichia coli , guanosine , chemistry , biology , gene
Guanosine‐5′‐diphosphate 3′‐diphosphate (ppGpp) is a bacterial signal molecule that generates stringent responses under conditions of deprivation and amino acid deficiency. In recent years, substantial progress has been made in our understanding of the molecular mechanisms of ppGpp metabolism and ppGpp‐mediated regulation. Although many studies focus on studying direct and indirect targets of ppGpp regulation, the overall mechanism of ppGpp is still not clear. In this study, ppGpp was immobilized on NHS‐activated Sepharose. ppGpp binding proteins were isolated by affinity chromatography, and further separated by 2‐D gel electrophoresis. The resolved proteins were identified by MALDI‐TOF MS. The tryptic hydrolysates were also analysed by LC‐MS/MS. Among the identified proteins, some proteins of interest were cloned and over‐expressed. The cloned proteins (pepB, iadA, map, dld, ldh, gldA, lon, ycbZ, pyrD, lpdA) were tested for their binding to ppGpp by fluorescence spectroscopy and were indentified as ppGpp‐binding proteins with various Kd s. Interestingly, we found ppGpp inhibit membrane D‐lactate dehydrogenase non‐competitively with Ki 48 mM and fermentative D‐lactate dehydrogenase competitively with Ki 120 mM. These results provide that this method will be very effective for screening proteins which new plausible ppGpp regulated proteins. And further studies on these proteins will expand the scope for the functions of ppGpp. Support or Funding Information BK21 PLUS (Brain Korea 21 Program for Leading Universities & Students), National Research Foundation (NRF) of Korea