Stabilization of tubulin by deuterium oxide. Export

@article{citeulike:2230447, abstract = {Tubulin is an unstable protein when stored in solution and loses its ability to form microtubules rapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 degrees C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 degrees C, but in buffer prepared in D2O, no activity was lost after 54 h. Tubulin was completely inactivated at 37 degrees C in 8 h in H2O buffer, but only 20\% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slower rate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at both temperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer; however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobic sites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowed somewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involve the disruption of hydrophobic forces. The conformational change is followed by an aggregation process that cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys. Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We also observed that the products of assembly in D2O/8\% DMSO consisted of a high percentage of ribbon structures and incompletely folded microtubules. When these polymers were disassembled and reassembled in H2O/8\% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO, both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation of ribbon structures rather than microtubules.}, address = {Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, and Department of Chemistry, Washburn University, Topeka, Kansas 66621}, author = {Chakrabarti, G. and Kim, S. and Gupta, M. L. and Barton, J. S. and Himes, R. H.}, citeulike-article-id = {2230447}, citeulike-linkout-0 = {http://dx.doi.org/10.1021/bi982461r}, citeulike-linkout-1 = {http://pubs.acs.org/doi/abs/10.1021/bi982461r}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/10074359}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=10074359}, comment = {This is a really great paper, lots of evidence from various methods showing that tubulin is more stable in D2O. Plus that D2O dramatically changes the polymerization properties. A couple things they show: * D2O stabilizes tubulin at 4C. In H2O, tubuilin in H2O at 4C is very unstable and appears to partially unfold and aggregate into inactive forms (as far as polymerization goes). In D2O, this process is much slower. * Microtubules can polymerize in D2O without need for DMSO (or glycerol is what we use). At the same tubulin concentration in H2O, no polymerization occurs. * In D2O + The "normal" concentration of DMSO, they get ribbon structures as opposed to microtubules (as assessed by TEM). Results of this paper are consistent with many of Andy's recent motility assay results, including the fact that microtubules seem much more stable. Plus the fact that he saw really bright structures when polymerizing in D2O (and glycerol) could indicate that he made the same kind of ribbon structures, though that's still unknown. Also, I really want to try storing kinesin in D2O to increase stability. Plus it would be interesting to repeat the kinesin heat-induced aggregation that I did a few years ago. I bet D2O would increase the transition temperature substantially. Finally another thought is that D2O should be investigated in all of the protein aggregation studies related to disease (amyloid stuff). Also there was this surprising sentence: "tudies of the effects of D2O on microtubules in vivo have been reported since 1935 (27)." I was pretty surprised to find out that people were studying D2O effects on the mitotic apparatus in 1935. On the other hand, maybe this isn't surprising}, day = {9}, doi = {10.1021/bi982461r}, issn = {0006-2960}, journal = {Biochemistry}, keywords = {d2o, dtra, heavywater, microtubules, tubulin}, month = {March}, number = {10}, pages = {3067--3072}, posted-at = {2009-11-04 22:17:41}, priority = {0}, title = {Stabilization of tubulin by deuterium oxide.}, url = {http://dx.doi.org/10.1021/bi982461r}, volume = {38}, year = {1999} }

TY - JOUR ID - citeulike:2230447 L3 - citeulike-article-id:2230447 N2 - Tubulin is an unstable protein when stored in solution and loses its ability to form microtubules rapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 degrees C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 degrees C, but in buffer prepared in D2O, no activity was lost after 54 h. Tubulin was completely inactivated at 37 degrees C in 8 h in H2O buffer, but only 20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slower rate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at both temperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer; however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobic sites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowed somewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involve the disruption of hydrophobic forces. The conformational change is followed by an aggregation process that cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys. Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We also observed that the products of assembly in D2O/8% DMSO consisted of a high percentage of ribbon structures and incompletely folded microtubules. When these polymers were disassembled and reassembled in H2O/8% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO, both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation of ribbon structures rather than microtubules. IS - 10 JF - Biochemistry SN - 0006-2960 AD - Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, and Department of Chemistry, Washburn University, Topeka, Kansas 66621 EP - 3072 TI - Stabilization of tubulin by deuterium oxide. VL - 38 SP - 3067 KW - d2o KW - dtra KW - heavywater KW - microtubules KW - tubulin N1 - This is a really great paper, lots of evidence from various methods showing that tubulin is more stable in D2O. Plus that D2O dramatically changes the polymerization properties. A couple things they show: * D2O stabilizes tubulin at 4C. In H2O, tubuilin in H2O at 4C is very unstable and appears to partially unfold and aggregate into inactive forms (as far as polymerization goes). In D2O, this process is much slower. * Microtubules can polymerize in D2O without need for DMSO (or glycerol is what we use). At the same tubulin concentration in H2O, no polymerization occurs. * In D2O + The "normal" concentration of DMSO, they get ribbon structures as opposed to microtubules (as assessed by TEM). Results of this paper are consistent with many of Andy's recent motility assay results, including the fact that microtubules seem much more stable. Plus the fact that he saw really bright structures when polymerizing in D2O (and glycerol) could indicate that he made the same kind of ribbon structures, though that's still unknown. Also, I really want to try storing kinesin in D2O to increase stability. Plus it would be interesting to repeat the kinesin heat-induced aggregation that I did a few years ago. I bet D2O would increase the transition temperature substantially. Finally another thought is that D2O should be investigated in all of the protein aggregation studies related to disease (amyloid stuff). Also there was this surprising sentence: "tudies of the effects of D2O on microtubules in vivo have been reported since 1935 (27)." I was pretty surprised to find out that people were studying D2O effects on the mitotic apparatus in 1935. On the other hand, maybe this isn't surprising AU - Chakrabarti, G AU - Kim, S AU - Gupta, ML AU - Barton, JS AU - Himes, RH PY - 1999/03/09/ UR - http://dx.doi.org/10.1021/bi982461r ER -