Fluorescence studies of pyrene maleimide-labeled translin: excimer fluorescence indicates subunits associate in a tail-to-tail configuration to form octamer.
Translin is an octameric single-stranded DNA binding protein consisting of 228 amino acid residues per monomer. This protein contains two cysteine residues per monomer. Studies of reactions with DTNB show that both cysteines are reactive and exhibit biphasic reaction kinetics. Further studies with two site-directed mutants, C58S and C225S, confirm that Cys-58 reacts slowly while Cys-225 reacts quickly. Pyrene excimer emission was observed for pyrene maleimide-labeled C58S mutant. This was not observed, however, with the pyrene maleimide-labeled C225S mutant. DAS (decay associated spectra) revealed that all excited pyrene labels on C225 residues can form excimers with pyrenes of adjacent subunits within a few nanoseconds. Time-resolved emission anisotropy detects a rotational correlation time appropriate for octameric but not dimeric species. These results indicate proximity for the Cys-225 residues on adjacent monomers and that the subunits must interact in a tail-to-tail orientation. Moreover, disulfide bonds are not required for the formation of an octamer.
Han
Myun K
MK
Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20007, USA.
Lin
Paul
P
Paek
David
D
Harvey
John J
JJ
Fuior
Elena
E
Knutson
Jay R
JR
D001483:Base Sequence; D001704:Biopolymers; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D005454:Fluorescence Polarization; D006801:Humans; D008301:Maleimides / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011721:Pyrenes / Q000737:chemistry*
D001704:Biopolymers; D017931:DNA Primers; D004268:DNA-Binding Proteins; D008301:Maleimides; D011721:Pyrenes; C095563:TSN protein, human; C043592:maleimide; D004228:Dithionitrobenzoic Acid; C030984:pyrene