Expression, purification and ligand binding properties of the recombinant translation initiation factor (PeIF5B) from Pisum sativum.
Gene encoding a novel translation initiation factor PeIF5B from Pisum sativum with sequence similarity to eIF5B from H. sapiens, D. melanogaster, S. cerevisiae as well as archaeal aIF5B from M. thermoautotrophicum was earlier reported by us. We now describe the expression and purification of 96 kDa recombinant PeIF5B (rPeIF5B) protein. Using fluorescence and circular dichroism spectra analyses, we show that Mg(2+) binding does not lead to any change in PeIF5B aromatic amino acid micro-environment, whereas GTP binding induces significant changes in the local environment of the aromatic amino acids. However, the protein undergoes changes in secondary structure upon metal ion and nucleotide binding. Charged initiator tRNA binding to PeIF5B is found to be cofactor dependent. PeIF5B binds to GTP in vitro as evident from autoradiography. Based on homology modeling of the catalytic domain of PeIF5B, we could confirm the conformational changes in PeIF5B following ligand binding.
Rasheedi
Sheeba
S
Department of Biochemistry, University of Hyderabad, Hyderabad, India.
Suragani
Madhuri
M
Haq
Soghra K
SK
Sachchidanand
Bhardwaj
Rajesh
R
Hasnain
Seyed E
SE
Ehtesham
Nasreen Z
NZ
D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D012343:RNA, Transfer / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D039642:Eukaryotic Initiation Factors; D008024:Ligands; D011994:Recombinant Proteins; C451459:eukaryotic initiation factor-5B; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D008274:Magnesium