Movements of truncated kinesin fragments with a short or an artificial flexible neck

To investigate the role of the neck domain of kinesin, we used optical trapping nanometry to perform high-resolution measurements of the movements and forces produced by recombinant kinesin fragments in which the neck domains were shortened or replaced by an artificial random coil. Truncated kinesin fragments (K351) that contain a motor domain consisting of ≈340 aa and a short neck domain consisting of ≈11 aa showed fast movement (800 nm/s) and 8-nm steps. Such behavior was similar to that of recombinant fragments containing the full-length neck domain (K411) and to that of native kinesin. Kinesin fragments lacking the short neck domain (K340), however, showed very slow movement (<50 nm/s), as previously reported. Joining an artificial 11-aa sequence that was expected to form a flexible random chain to the motor domain (K340–chain) produced normal fast (≈700 nm/s) and stepwise movement. The results suggest that the neck domain does not act as a rigid lever arm to magnify the structural change at the catalytic domain as has been believed for myosin, but it does act as a flexible joint to guarantee the mobility of the motor domain.

Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).

Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.