Internal motion of lysozyme studied by time-resolved fluorescence depolarization of tryptophan residues.
The internal motion of lysozyme was described by the steady-state and time-resolved fluorescence anisotropy of its tryptophan residues. The fluorescence of mutant lysozymes W62Y- and W108Y-lysozyme, in which Trp62 or Trp108 of hen egg white lysozyme was replaced with a tyrosine residue, could be respectively assigned to Trp108 or Trp62 at the longer wavelength region of the total fluorescence spectrum. The segmental motion of Trp62 as shown by its fluorescence anisotropy decay was described with two components originating from the fluctuational rotation of an indole moiety about the Calpha-Cbeta bond and rotational wobble of the peptide segment adjacent to Trp62. Although Trp62 showed a high degree of motional freedom, its motion was significantly suppressed by the interaction of the mutant protein with a trimer of N-acetyl-D-glucosamine. By contrast, the segmental motion of Trp108 is hindered by the local cage structure at temperatures below 30 degreesC, but Relief from restricted motion occurred on the formation of ligand complex or by thermal agitation. Because of overlaps of the fluorescence spectrum, it is difficult to assign the segmental motion of Trp28 or Trp111, the other two tryptophan residues in lysozyme. However, a careful analysis of the fluorescence anisotropy decay of W62Y- and W108Y-lysozyme showed that the fluctuation of the hydrophobic matrix box was greater than that expected from lysozyme's crystal structure, although it was suppressed by the binding of the ligand to the active site of lysozyme.
Nishimoto
E
E
Kyushu National Industrial Research Institute, Saga, Japan.
Yamashita
S
S
Szabo
A G
AG
Imoto
T
T
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002645:Chickens; D005454:Fluorescence Polarization / Q000379:methods; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
D008024:Ligands; D014364:Tryptophan; D009113:Muramidase