Ni2+ binds to active site of hen egg-white lysozyme and quenches fluorescence of Trp62 and Trp108.
We found that the maximum emission of the tryptophyl fluorescence of hen egg-white lysozyme is shifted from 337 to 323 nm and quenched to the extent of 55% with an increase in concentrations of NiCl2 from 0 to 2M in 50 mM Na acetate buffer (pH 4.7). In contrast, NaCl does not influence the fluorescence of lysozyme up to 2M. To elucidate the particular effects of Ni2+ on the tryptophyl fluorescence of lysozyme, we have measured the assembly behavior and secondary structure of lysozyme in various concentrations of NiCl2, and determined the structures of lysozyme crystals grown in 0.3, 0.5, and 1.0M NiCl2, respectively. The results of analytical centrifugation and circular dichroism experiments show that lysozyme keeps a monomer state and has an identical secondary structure, irrespective of NiCl2 concentrations. The crystal structures show that all crystals grown in different concentrations of NiCl2 have an identical main chain and side chain conformation. And one Ni2+ binding with Odelta atom of Asp52 in the active site and coordinating with five water molecules to form hexagonal coordination has been determined for each crystal structure. Based on these results, we have proposed that Ni2+ quenches the fluorescence of Trp62 and Trp108 due to the binding of Ni2+ to the active site of lysozyme.
Division of Protein Crystallography, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita 565-0871, Japan.
Shu Jie
SJ
Li
Atsushi
A
Nakagawa
Tomitake
T
Tsukihara
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004583:Electrons; D005583:Fourier Analysis; D007477:Ions; D008958:Models, Molecular; D008968:Molecular Conformation; D009113:Muramidase / Q000737:chemistry*; D009532:Nickel / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012965:Sodium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry*; D014461:Ultracentrifugation; D014867:Water / Q000737:chemistry
D007477:Ions; D014867:Water; D001224:Aspartic Acid; D012965:Sodium Chloride; D009532:Nickel; D014364:Tryptophan; D009113:Muramidase