Dimerization properties of a Xenopus laevis kinesin-II carboxy-terminal stalk fragment.
We have analysed the structural and physical properties of the carboxy-terminal stalk region of a kinesin-II, Xenopus kinesin-like protein 3A/B (Xklp3A/B), which we showed to be essential for heterodimerization in a previous work (De Marco et al., 2001). We expressed the corresponding A-stalk and B-stalk fragments and investigated their modes of interaction by analytical ultracentrifugation (AUC), circular dichroism spectroscopy, denaturation assays and electron microscopy. Co-expression of the A-stalk and B-stalk produced the properly folded, hetero-dimeric coiled coil at high yields. The dimeric nature of the complex was confirmed by AUC. We also found that the isolated A-stalk fragment forms a stable helix by itself and shows a significant tendency towards homodimer and higher-order complex formation. In the absence of the corresponding A-stalk fragment, the isolated B-stalk fragment remains partially unfolded, which suggests that the A-stalk provides a template structure for the B-stalk in order to recompose the complete heterodimeric coiled coil.
De Marco
Valeria
V
European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
De Marco
Ario
A
Goldie
Kenneth N
KN
Correia
John J
JJ
Hoenger
Andreas
A
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D019281:Dimerization; D016547:Kinesins; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D014461:Ultracentrifugation; D029867:Xenopus Proteins; D014982:Xenopus laevis
D002135:Calcium-Binding Proteins; C499377:KIF3A protein, Xenopus; D009124:Muscle Proteins; D010446:Peptide Fragments; C499376:XKLP3 protein, Xenopus; D029867:Xenopus Proteins; C074152:kinesin-II; D016547:Kinesins