Tryptophan H33 plays an important role in pyrimidine (6-4) pyrimidone photoproduct binding by a high-affinity antibody.
The importance of Trp H33 in antibody recognition of DNA containing a central pyrimidine (6-4) pyrimidone photoproduct was investigated. This residue was replaced by Tyr, Phe and Ala and the binding abilities of these mutants were determined by surface plasmon resonance and fluorescence spectroscopy. Conservative substitution of Trp H33 by Tyr or Phe resulted in moderate losses of binding affinity; however, replacement by Ala had a significantly larger impact. The fluorescence properties of DNA containing a (6-4) photoproduct were strongly affected by the identity of the H33 residue. DNA binding by both the wild-type and the W-H33-Y mutant was accompanied by a small degree of fluorescence quenching; by contrast, binding by the W-H33-F and W-H33-A mutants produced large fluorescence increases. Taken together, these variations in binding and fluorescence properties with the identity of the H33 residue are consistent with a role in photoproduct recognition by Trp H33 in the high-affinity antibody 64M5.
Kobayashi
H
H
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan and the University of Florida, Gainesville, FL 32611, USA.
Kato
J
J
Morioka
H
H
Stewart
J D
JD
Ohtsuka
E
E
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000915:Antibody Affinity / Q000235:genetics*; D000918:Antibody Specificity / Q000235:genetics*; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011740:Pyrimidine Dimers / Q000276:immunology* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011740:Pyrimidine Dimers; C058406:pyrimidine-pyrimidone dimer; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D004247:DNA; D000409:Alanine