Intermolecular cross-linking of a novel rice Kinesin k16 motor domain with a photoreactive ATP derivative.
A fluorescent photoreactive ATP derivative, 2'(3')-O-(4-benzoylbenzoyl)-1,N(6)-etheno-ATP (Bz(2)-epsilonATP), was synthesized and reacted with the rice kinesin K16 motor domain (K16MD). In the presence of ADP or ATP, UV irradiation of the K16MD solution containing Bz(2)-epsilonATP resulted in a new 100 kDa band, which was an intermolecular cross-linked product of motor domains. In contrast, no cross-linking was observed in the absence of nucleotides. For the motor domain of mouse brain kinesin and skeletal muscle myosin subfragment-1, no such intermolecular photo cross-linking by Bz(2)-epsilonATP was observed. Our results indicate that Bz(2)-epsilonATP acts unusually as a photoreactive crosslinker to detect conformational changes in K16MD induced by nucleotide binding resulting in the formation of dimers.
Umeki
Nobuhisa
N
Laboratories of Plant and Microbial Genome Control, Graduate School of Science and Technology, Niigata University, Niigata 950-2181.
Mitsui
Toshiaki
T
Koike
Yutaka
Y
Maruta
Shinsaku
S
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D000345:Affinity Labels; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D020409:Molecular Motor Proteins; D009711:Nucleotides; D011506:Proteins; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins