scholarly journals In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB

2005 ◽  
Vol 169 (2) ◽  
pp. 233-238 ◽  
Author(s):  
Christopher A. Sontag ◽  
James T. Staley ◽  
Harold P. Erickson
Keyword(s):  
Critical Concentration ◽  
Three Dimensional ◽  
Outer Diameter ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Assembly Mechanism ◽  
In Vitro Assembly ◽  
Cooperative Assembly ◽  
Versus Concentration

Arecent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35–36 nm in the presence of GTP or GDP. Mixtures of BtubB and BtubA formed long protofilament bundles, 4–7 protofilaments wide (20–30 protofilaments in the three-dimensional bundle). Regardless of the starting stoichiometry, the polymers always contained equal concentrations of BtubA and BtubB, suggesting that BtubA and B alternate along the protofilament. BtubA showed negligible GTP hydrolysis, whereas BtubB hydrolyzed 0.40 mol GTP per min per mol BtubB. This GTPase activity increased to 1.37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4–1.0 μM was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism.

scholarly journals Bacterial Microtubules Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

2017 ◽  
Author(s):  
César Díaz-Celis ◽  
Viviana I. Risca ◽  
Felipe Hurtado ◽  
Jessica K. Polka ◽  
Scott D. Hansen ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Intracellular Transport ◽  
Complex Dynamics ◽  
Critical Concentration ◽  
Polarized Growth ◽  
Gtp Hydrolysis ◽  
Filament Dynamics ◽  
Mixed Polarity ◽  
Self Association

AbstractBacteria of the genusProsthecobacterexpress homologs of eukaryotic α-and β-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). ThebtubABgenes likely entered theProsthecobacterlineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is GTP-dependent and reversible and that BtubA/B folding does not require chaperones. To better understand bMT behavior and gain insight into the evolution of microtubule dynamics, we characterizedin vitrobMT assembly using a combination of polymerization kinetics assays, and microscopy. Like eukaryotic microtubules, bMTs exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by bMT polymerization drives a stochastic mechanism of bMT disassembly that occurs via polymer breakage. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of bMT fragments. Unlike MTs, polymerization of bMTs requires KCl, which reduces the critical concentration for BtubA/B assembly and induces bMTs to form stable mixed-orientation bundles in the absence of any additional bMT-binding proteins. Our results suggest that at potassium concentrations resembling that inside the cytoplasm ofProsthecobacter, bMT stabilization through self-association may be a default behavior. The complex dynamics we observe in both stabilized and unstabilized bMTs may reflect common properties of an ancestral eukaryotic tubulin polymer.ImportanceMicrotubules are polymers within all eukaryotic cells that perform critical functions: they segregate chromosomes in cell division, organize intracellular transport by serving as tracks for molecular motors, and support the flagella that allow sperm to swim. These functions rely on microtubules remarkable range of tunable dynamic behaviors. Recently discovered bacterial microtubules composed of an evolutionarily related protein are evolved from a missing link in microtubule evolution, the ancestral eukaryotic tubulin polymer. Using microscopy and biochemical approaches to characterize bacterial microtubules, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules, but differ in how they self-associate into bundles and become destabilized. Our results demonstrate the diversity of mechanisms that microtubule-like filaments employ to promote filament dynamics and monomer turnover.

scholarly journals Ubiquitously Expressed Dynamin-II Has a Higher Intrinsic GTPase Activity and a Greater Propensity for Self-assembly Than Neuronal Dynamin-I

1997 ◽  
Vol 8 (12) ◽  
pp. 2553-2562 ◽  
Author(s):  
Dale E. Warnock ◽  
Takeshi Baba ◽  
Sandra L. Schmid
Keyword(s):  
Self Assembly ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Gtpase Activity ◽  
Coated Pits ◽  
Gtp Hydrolysis ◽  
Rich Domain ◽  
Dynamin I ◽  
Intrinsic Gtpase Activity

To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a ∼threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.

Microtubule associated proteins in microtubule preparations made with and without glycerol

1984 ◽  
Vol 62 (9) ◽  
pp. 803-813 ◽  
Author(s):  
Robert A. B. Keates
Keyword(s):  
Binding Assay ◽  
Critical Concentration ◽  
Brain Homogenate ◽  
Microtubule Assembly ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
The Difference

Preparation of microtubule protein in the presence or absence of glycerol results in differences in polymerization properties and content of microtubule associated proteins. The variation in properties appears to result from the reduced proportion of microtubule associated proteins in preparations made with glycerol. I have used the colchicine binding assay to monitor recovery of active tubulin and have found that a single factor can account for the difference. During the in vitro assembly of microtubules from the crude brain homogenate, glycerol promotes polymerization of the bulk of the tubulin, while less than half is incorporated into microtubules in the absence of glycerol. Assembly of partly purified microtubule protein is not enhanced by glycerol however. Microtubule associated proteins present in the crude homogenate are almost completely incorporated into the microtubules regardless of the presence of glycerol, and their high content in glycerol-free preparations appears to be the trivial result of low tubulin recovery. The high affinity of microtubule associated proteins for the assembled microtubules has other consequences for in vitro studies of microtubule assembly, and critical concentration plots to determine the polymerization equilibrium constant can be distorted unless the preparation used has a high content of microtubule associated proteins.

scholarly journals Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate.

1991 ◽  
Vol 114 (1) ◽  
pp. 73-81 ◽  
Author(s):  
R A Walker ◽  
N K Pryer ◽  
E D Salmon
Keyword(s):  
Dynamic Instability ◽  
Elongation Rate ◽  
Gtp Hydrolysis ◽  
Microtubule Stability ◽  
Assembly Mechanism ◽  
High Elongation ◽  
Microtubule Growth ◽  
Hydrolysis Of ◽  
Brain Tubulin

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build-up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.

scholarly journals Assembly of an FtsZ Mutant Deficient in GTPase Activity Has Implications for FtsZ Assembly and the Role of the Z Ring in Cell Division

2001 ◽  
Vol 183 (24) ◽  
pp. 7190-7197 ◽  
Author(s):  
Amit Mukherjee ◽  
Cristian Saez ◽  
Joe Lutkenhaus
Keyword(s):  
Cell Division ◽  
Critical Concentration ◽  
Gtpase Activity ◽  
Support Cell ◽  
Z Ring ◽  
Ftsz Assembly ◽  
The Stability

ABSTRACT FtsZ, the ancestral homologue of eukaryotic tubulins, assembles into the Z ring, which is required for cytokinesis in prokaryotic cells. Both FtsZ and tubulin have a GTPase activity associated with polymerization. Interestingly, the ftsZ2 mutant is viable, although the FtsZ2 mutant protein has dramatically reduced GTPase activity due to a glycine-for-aspartic acid substitution within the synergy loop. In this study, we have examined the properties of FtsZ2 and found that the reduced GTPase activity is not enhanced by DEAE-dextran-induced assembly, indicating it has a defective catalytic site. In the absence of DEAE-dextran, FtsZ2 fails to assemble unless supplemented with wild-type FtsZ. FtsZ has to be at or above the critical concentration for copolymerization to occur, indicating that FtsZ is nucleating the copolymers. The copolymers formed are relatively stable and appear to be stabilized by a GTP-cap. These results indicate that FtsZ2 cannot nucleate assembly in vitro, although it must in vivo. Furthermore, the stability of FtsZ-FtsZ2 copolymers argues that FtsZ2 polymers would be stable, suggesting that stable FtsZ polymers are able to support cell division.

scholarly journals Polymerization of FtsZ, a Bacterial Homolog of Tubulin

2001 ◽  
Vol 276 (15) ◽  
pp. 11743-11753 ◽  
Author(s):  
Laura Romberg ◽  
Martha Simon ◽  
Harold P. Erickson
Keyword(s):  
Steady State ◽  
Critical Concentration ◽  
Gtp Hydrolysis ◽  
Nucleotide Hydrolysis ◽  
High Affinity ◽  
Hydrolysis Rates ◽  
Bacterial Homolog ◽  
Nucleation Phase ◽  
Maximum Steady State

FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis.In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers. Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or “isodesmic”) assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration. We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme. The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates. It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolarKD.

scholarly journals Control by Potassium of the Size Distribution of Escherichia coli FtsZ Polymers Is Independent of GTPase Activity

2013 ◽  
Vol 288 (38) ◽  
pp. 27358-27365 ◽  
Author(s):  
Rubén Ahijado-Guzmán ◽  
Carlos Alfonso ◽  
Belén Reija ◽  
Estefanía Salvarelli ◽  
Jesús Mingorance ◽  
...  
Keyword(s):  
Size Distribution ◽  
Turnover Rate ◽  
Potassium Concentration ◽  
Critical Concentration ◽  
Hydrolysis Rate ◽  
Gtpase Activity ◽  
Narrow Size Distribution ◽  
Gtp Hydrolysis ◽  
Inherent Feature ◽  
Z Ring

The influence of potassium content (at neutral pH and millimolar Mg2+) on the size distribution of FtsZ polymers formed in the presence of constantly replenished GTP under steady-state conditions was studied by a combination of biophysical methods. The size of the GTP-FtsZ polymers decreased with lower potassium concentration, in contrast with the increase in the mass of the GDP-FtsZ oligomers, whereas no effect was observed on FtsZ GTPase activity and critical concentration of polymerization. Remarkably, the concerted formation of a narrow size distribution of GTP-FtsZ polymers previously observed at high salt concentration was maintained in all KCl concentrations tested. Polymers induced with guanosine 5′-(α,β-methylene)triphosphate, a slowly hydrolyzable analog of GTP, became larger and polydisperse as the potassium concentration was decreased. Our results suggest that the potassium dependence of the GTP-FtsZ polymer size may be related to changes in the subunit turnover rate that are independent of the GTP hydrolysis rate. The formation of a narrow size distribution of FtsZ polymers under very different solution conditions indicates that it is an inherent feature of FtsZ, not observed in other filament-forming proteins, with potential implications in the structural organization of the functional Z-ring.

scholarly journals Complete and cooperative in vitro assembly of computationally designed self-assembling protein nanomaterials

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Adam J. Wargacki ◽  
Tobias P. Wörner ◽  
Michiel van de Waterbeemd ◽  
Daniel Ellis ◽  
Albert J. R. Heck ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Building Blocks ◽  
Target Structure ◽  
Design Strategies ◽  
Self Assembling ◽  
Single Target ◽  
In Vitro Assembly ◽  
Predictive Design ◽  
Cooperative Assembly

AbstractRecent advances in computational methods have enabled the predictive design of self-assembling protein nanomaterials with atomic-level accuracy. These design strategies focus exclusively on a single target structure, without consideration of the mechanism or dynamics of assembly. However, understanding the assembly process, and in particular its robustness to perturbation, will be critical for translating this class of materials into useful technologies. Here we investigate the assembly of two computationally designed, 120-subunit icosahedral complexes in detail using several complementary biochemical methods. We found that assembly of each material from its two constituent protein building blocks was highly cooperative and yielded exclusively complete, 120-subunit complexes except in one non-stoichiometric regime for one of the materials. Our results suggest that in vitro assembly provides a robust and controllable route for the manufacture of designed protein nanomaterials and confirm that cooperative assembly can be an intrinsic, rather than evolved, feature of hierarchically structured protein complexes.

scholarly journals The in vitro assembly of flagellar outer doublet tubulin.

1978 ◽  
Vol 79 (2) ◽  
pp. 500-515 ◽  
Author(s):  
L I Binder ◽  
J L Rosenbaum
Keyword(s):  
Gel Electrophoresis ◽  
Critical Concentration ◽  
Mammalian Brain ◽  
Basal Bodies ◽  
Temperature Dependent ◽  
Two Dimensional Gel Electrophoresis ◽  
Microtubule Associated Proteins ◽  
In Vitro Assembly ◽  
Associated Proteins

Flagellar outer doublet microtubules were solubilized by use of sonication, and the tubulin was reassembled in vitro into single microtubules containing 14 and 15 protofilaments. The tubulin assembly was dependent on both the KCl and tubulin concentrations, exhibiting a critical concentration of 0.72 mg/ml at optimum solvent conditions. Flagellar tubulin was purified by cycles of temperature-dependent assembly-disassembly and molecular sieve chromatography, and characterized by two-dimensional gel electrophoresis. Although doublet microtubules were not formed in vitro, outer doublet tubulin assembled onto intact A- and B-subfibers of outer doublet microtubules and basal bodies of Chlamydomonas; the rate of assembly from the distal ends of these structures was greater than that from the proximal ends. Microtubule-associated proteins (MAPs) from mammalian brain stimulated outer doublet tubulin assembly, decorating the microtubules with fine filamentous projections.

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