Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties.
The gene for the mature human mitochondrial single-stranded-DNA binding protein (HsmtSSB) has been transferred into a protein-overproducing vector and expressed in Escherichia coli. The protein was purified to homogeneity and its physicochemical properties were investigated. From sequence comparison, HsmtSSB shows some similarities to the N-terminal part of the single-stranded DNA-binding protein (SSB) from E. coli (EcoSSB). Hydrodynamic measurements show the protein to be tetrameric and give a sedimentation coefficient of 4.1 S corresponding to a C-terminally shortened EcoSSB. Electron-microscopic images of the free protein show a globular tetrahedral structure. Binding of poly(desoxythymidylic acid) [poly(dT)] leads to a reduction of the tryptophan fluorescence of the protein up to 96%. Fluorescence titrations with poly(dT) show apparent binding-site sizes of 50-70 nucleotides/tetramer between 0.05 M and 2 M NaCl. Binding to poly(dT) proceeds in a nearly diffusion-controlled reaction with an association-rate constant kass of 4 x 10(8) M-1s-1. The rate-limiting step is the formation of a transient complex where less than four binding sites on the protein are involved and the reshuffling of the protein on the linear matrix is fast. Electron microscopy of the complex with poly(dT) using negative staining shows a nearly random distribution of the protein between the individual poly(dT) strands. This leads to the conclusion that the binding cooperativity is low (omega < 150). The two tryptophans of HsmtSSB were replaced by threonine and tyrosine. The environment of both residues is influenced by nucleic acid binding with mutations of Trp68 strongly reducing the DNA-binding affinity of the protein.
Curth
U
U
Medizinische Hochschule, Zentrum Biochemie, Hannover, Germany.
Urbanke
C
C
Greipel
J
J
Gerberding
H
H
Tiranti
V
V
Zeviani
M
M
D001665:Binding Sites; D002498:Centrifugation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure; D004926:Escherichia coli / Q000737:chemistry*; D006801:Humans; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008928:Mitochondria / Q000737:chemistry*; D016296:Mutagenesis; D011071:Poly T / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D011071:Poly T