The role of casein in supporting the operation of surface bound kinesin. Export

@article{citeulike:4381177, abstract = {ABSTRACT: Microtubules and associated motor proteins such as kinesin are envisioned for applications such as bioseparation and molecular sorting to powering hybrid synthetic mechanical devices. One of the challenges in realizing such systems is retaining motor functionality on device surfaces. Kinesin motors adsorbed onto glass surfaces lose their functionality or ability to interact with microtubules if not adsorbed with other supporting proteins. Casein, a milk protein, is commonly used in microtubule motility assays to preserve kinesin functionality. However, the mechanism responsible for this preservation of motor function is unknown. To study casein and kinesin interaction, a series of microtubule motility assays were performed where whole milk casein, or its alphas1 and alphas2, beta or kappa subunits, were introduced or omitted at various steps of the motility assay. In addition, a series of epifluorescence and total internal reflection microscopy (TIRF) experiments were conducted where fluorescently labeled casein was introduced at various steps of the motility assay to assess casein-casein and casein-glass binding dynamics. From these experiments it is concluded that casein forms a bi-layer which supports the operation of kinesin. The first tightly bound layer of casein mainly performs the function of anchoring the kinesin while the second more loosely bound layer of casein positions the head domain of the kinesin to more optimally interact with microtubules. Studies on individual casein subunits indicate that beta casein was most effective in supporting kinesin functionality while kappa casein was found to be least effective.}, author = {Verma, V. and Hancock, W. O. and Catchmark, J. M.}, citeulike-article-id = {4381177}, citeulike-linkout-0 = {http://dx.doi.org/10.1186/1754-1611-2-14}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/18937863}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=18937863}, comment = {Andy Maloney showed me this paper today and summarized it for me. They did a really nice study of the effects of whole casein, and individual casein components (alpha, beta, kappa) on gliding motility assays. The article is open access. Particularly interesting is Figure 3b, which shows very nice, long microtubules in the kappa-casein preparation. This looks like a promising way to run the assay. We couldn't find in the article where they purchased their kappa casein. We found a >70\% pure version at Sigma that was about \$100 for 100 mg. Sorta pricey, but manageable.}, doi = {10.1186/1754-1611-2-14}, issn = {1754-1611}, journal = {Journal of biological engineering}, keywords = {casein, kinesin, microtubules}, posted-at = {2009-04-23 00:50:09}, priority = {0}, title = {The role of casein in supporting the operation of surface bound kinesin.}, url = {http://dx.doi.org/10.1186/1754-1611-2-14}, volume = {2}, year = {2008} }

TY - JOUR ID - citeulike:4381177 L3 - citeulike-article-id:4381177 N2 - ABSTRACT: Microtubules and associated motor proteins such as kinesin are envisioned for applications such as bioseparation and molecular sorting to powering hybrid synthetic mechanical devices. One of the challenges in realizing such systems is retaining motor functionality on device surfaces. Kinesin motors adsorbed onto glass surfaces lose their functionality or ability to interact with microtubules if not adsorbed with other supporting proteins. Casein, a milk protein, is commonly used in microtubule motility assays to preserve kinesin functionality. However, the mechanism responsible for this preservation of motor function is unknown. To study casein and kinesin interaction, a series of microtubule motility assays were performed where whole milk casein, or its alphas1 and alphas2, beta or kappa subunits, were introduced or omitted at various steps of the motility assay. In addition, a series of epifluorescence and total internal reflection microscopy (TIRF) experiments were conducted where fluorescently labeled casein was introduced at various steps of the motility assay to assess casein-casein and casein-glass binding dynamics. From these experiments it is concluded that casein forms a bi-layer which supports the operation of kinesin. The first tightly bound layer of casein mainly performs the function of anchoring the kinesin while the second more loosely bound layer of casein positions the head domain of the kinesin to more optimally interact with microtubules. Studies on individual casein subunits indicate that beta casein was most effective in supporting kinesin functionality while kappa casein was found to be least effective. JF - Journal of biological engineering SN - 1754-1611 TI - The role of casein in supporting the operation of surface bound kinesin. VL - 2 KW - casein KW - kinesin KW - microtubules N1 - Andy Maloney showed me this paper today and summarized it for me. They did a really nice study of the effects of whole casein, and individual casein components (alpha, beta, kappa) on gliding motility assays. The article is open access. Particularly interesting is Figure 3b, which shows very nice, long microtubules in the kappa-casein preparation. This looks like a promising way to run the assay. We couldn't find in the article where they purchased their kappa casein. We found a >70% pure version at Sigma that was about $100 for 100 mg. Sorta pricey, but manageable. AU - Verma, V AU - Hancock, WO AU - Catchmark, JM PY - 2008/// UR - http://dx.doi.org/10.1186/1754-1611-2-14 ER -