ABSTRACT
Kinesins are cytoskeletal motor proteins that share a common mechanochemical motor domain, and are responsible for trafficking macromolecules. Here we report the cloning and characterization of a monomeric, kinesin-3 (TKIN) from Thermomyces lanuginosus. TKIN displayed a maximum rate of ATP hydrolysis at approximately 55 degrees C; the K(m)(ATP) was also significantly greater at 50 degrees C. Gliding motility rates reached a maximum of 5.5 microms(-1) at 45 degrees C, which is among the highest rates reported for kinesin. Arrhenius energy barriers were calculated to be approximately 103 kJmol(-1), nearly twofold greater than other mesophilic kinesin motors. The enthalpy of activation and entropy activation of TKIN were also significantly greater when compared to other mesophilic kinesins. A thermally induced aggregation of TKIN, which could be moderated by the addition of ATP, was observed at temperatures above 45 degrees C. Together, these results illustrate the kinetic response and stability of this unique motor protein at elevated temperatures.
Subject(s)
Ascomycota/physiology , Kinesins/metabolism , Microtubules/metabolism , Molecular Motor Proteins/metabolism , Adenosine Triphosphatases/metabolism , Ascomycota/genetics , Hot Temperature , Kinetics , TemperatureSubject(s)
Antibodies/immunology , Kinesins/chemistry , Microfluidic Analytical Techniques/instrumentation , Microtubules/chemistry , Molecular Motor Proteins/chemistry , Nanotechnology/instrumentation , Virion/isolation & purification , Equipment Design , Equipment Failure Analysis , Flow Injection Analysis/instrumentation , Flow Injection Analysis/methods , Immunoassay/instrumentation , Immunoassay/methods , Microfluidic Analytical Techniques/methods , Motion , Nanotechnology/methods , Virion/chemistry , Virion/immunologyABSTRACT
A variety of bifunctional crosslinking agents have been explored for stabilizing microtubule shuttles used for the active transport of nanomaterials in artificial environments. Crosslinking agents that target amine residues form intertubulin crosslinks that produce crosslinked microtubules (CLMTs) with structural and functional lifetimes that can be up to four times as long as those achieved with taxol stabilization. Such CLMTs are stable at temperatures down to -10 degrees C, are resistant to depolymerization induced by metal ions such as Ca2+, and yet continue to be adsorbed and transported by self-assembled monolayers containing the motor protein kinesin. However, crosslinkers that target cysteine residues depolymerize the MTs, probably by interfering with the guanosine triphosphate binding site. The impact of crosslink attributes, including terminal group chemistry, chain length, crosslink density, and specific location on the tubulin surface, on microtubule stability and functionality are discussed.
Subject(s)
Crystallization/methods , Microtubules/chemistry , Microtubules/ultrastructure , Nanostructures/chemistry , Nanostructures/ultrastructure , Nanotechnology/methods , Cross-Linking Reagents/chemistry , Drug Stability , Macromolecular Substances/chemistry , Materials Testing , Models, Chemical , Models, Molecular , Molecular Conformation , Particle Size , Surface PropertiesABSTRACT
Short practical syntheses for five deuterium-labeled derivatives of dimethylallyl diphosphate (DMAPP) useful for enzymological studies are reported. These include the preparation of the C1-labeled derivatives (R)-[1-2H]3-methylbut-2-enyl diphosphate ((R)-[1-2H]1-OPP) and (S)-[1-2H]3-methylbut-2-enyl diphosphate ((S)-[1-2H]1-OPP), the C2-labeled derivative [2-2H]3-methylbut-2-enyl diphosphate ([2-2H]1-OPP), and the methyl-labeled derivatives (E)-[4,4,4-2H3]3-methylbut-2-enyl diphosphate ((E)-[4,4,4-2H3]1-OPP) and (Z)-[4,4,4-2H3]3-methyl-but-2-enyl diphosphate ((Z)-[4,4,4-2H3]1-OPP).
Subject(s)
Deuterium , Hemiterpenes/chemical synthesis , Isotope Labeling/methods , Organophosphorus Compounds/chemical synthesis , Enzymes/metabolismABSTRACT
Farnesyl diphosphate synthase catalyzes the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate. In plants the presence of farnesyl diphosphate synthase isozymes offers the possibility of differential regulation. Three full-length cDNAs encoding putative isoprenoid synthases, FDS-1, FDS-2, and FDS-5, with greater than 89% similarity were isolated from a Big Sagebrush Artemisia tridentata cDNA library using a three-step polymerase chain reaction protocol. One of the open reading frames, FDS-5, encoded a protein with an N-terminal amino acid extension that was identified as a plastidial targeting peptide. Recombinant histidine-tagged versions of three proteins were purified, and their enzymatic properties were characterized. FDS-1 and FDS-2 synthesized farnesyl diphosphate as the final chain elongation product, but their kinetic behavior varied. FDS-1 prefers geranyl diphosphate over dimethylallyl diphosphate as an allylic substrate and is active at acidic pH values compared with FDS-2. In contrast, FDS-5 synthesized two irregular monoterpenoids, chrysanthemyl diphosphate and lavandulyl diphosphate, when incubated with dimethylallyl diphosphate and an additional product, the regular monoterpene geranyl diphosphate, when incubated with isopentenyl diphosphate and dimethylallyl diphosphate. Specific cellular functions are proposed for each of the three enzymes, and a scenario for evolution of isoprenyl synthases in plants is presented.
