Structure, Assembly, and Disassembly of Tubulin Single Rings

Single and double tubulin rings were studied under a range of conditions and during microtubule (MT) assembly and disassembly. Here, tubulin was purified from porcine brain and used without any further modifications or additives that promote ring assembly. The structure of single GDP-rich tubulin rings was determined by cryo-transmission electron microscopy and synchrotron solution X-ray scattering. The scattering curves were fitted to atomic models, using our state-of-the-art analysis software, D+. We found that there is a critical concentration for ring formation, which increased with GTP concentration with temperature. MT assembly or disassembly, induced by changes in temperature, was analyzed by time-resolved small-angle X-ray scattering. During MT assembly, the fraction of rings and unassembled dimers simultaneously decreased. During MT disassembly, the mass fraction of dimers increased. The increase in the concentration of rings was delayed until the fraction of dimers was sufficiently high. We verified that pure dimers, eluted via size-exclusion chromatography, could also form rings. Interestingly, X-ray radiation triggered tubulin ring disassembly. The concentration of disassembled rings versus exposure time followed a first-order kinetics. The disassembly rate constant and initial concentration were determined. X-ray radiation-triggered disassembly was used to determine the concentration of rings. We confirmed that following a temperature jump, the mass fraction of rings decreased and then stabilized at a constant value during the first stage of the MT assembly kinetics. This study sheds light on the most basic assembly and disassembly conditions for in vitro single GDP-rich tubulin rings and their relation to MT kinetics

Effects of Non-Ionic Micelles on the Acid-Base Equilibria of a Weak Polyelectrolyte

Weak polyelectrolytes (WPEs) are widely used as pH-responsive materials, pH modulators and charge regulators in biomedical and technological applications that involve multi-component fluid environments. In these complex fluids, coupling between (often weak) interactions induced by micelles, nanoparticles and molecular aggregates modify the pKa as compared to that measured in single component solutions. Here we investigated the effect of coupling between hydrogen bonding and excluded volume interactions on the titration curves and pKa of polyacrylic acid (PAA) in solutions comprising PEO-based micelles (Pluronics and Brij-S20) of different size and volume fraction. Titration experiments of dilute, salt-free solutions of PAA (5 kDa, 30 kDa and 100 kDa) at low degree of polymer ionization (α < 0.25) drive spatial re-organization of the system, reduce the degree of ionization and consequentially increase the pKa by up to ~0.7 units. These findings indicate that the actual degree of ionization of WPEs measured in complex fluids is significantly lower (at a given pH) than that measured in single-component solutions

Mechanism of Tubulin Oligomers and Single-Rings Disassembly Catastrophe

Cold tubulin dimers coexist with tubulin oligomers and single-rings. These structures are involved in microtubule assembly, however, their dynamics are poorly understood. Using state-of-the-art solution synchrotron time-resolved small-angle X-ray scattering we discovered a disassembly catastrophe (half-life of about 0.1 sec) of tubulin rings and oligomers upon dilution or addition of guanosine triphosphate. A slower disassembly (half-life of about 38 sec) was observed following a temperature increase. Our analysis showed that the assembly and disassembly processes were consistent with an isodesmic mechanism, involving a sequence of reversible reactions at which dimers were rapidly added/removed one at a time, terminated by a two orders-of-magnitude slower ring-closing/opening step. We revealed how assembly conditions varied the mass fraction of tubulin in each of the coexisting structures, the rate constants, and the standard Helmholtz free energies for closing a ring and for longitudinal dimer-dimer associations

Mechanism of Tubulin Oligomers and Single-Ring Disassembly Catastrophe

Cold tubulin dimers coexist with tubulin oligomers and single rings. These structures are involved in microtubule assembly; however, their dynamics are poorly understood. Using state-of-the-art solution synchrotron time-resolved small-angle X-ray scattering, we discovered a disassembly catastrophe (half-life of ∼0.1 s) of tubulin rings and oligomers upon dilution or addition of guanosine triphosphate. A slower disassembly (half-life of ∼38 s) was observed following an increase in temperature. Our analysis showed that the assembly and disassembly processes were consistent with an isodesmic mechanism, involving a sequence of reversible reactions in which dimers were rapidly added or removed one at a time, terminated by a 2 order-of-magnitude slower ring-closing/opening step. We revealed how assembly conditions varied the mass fraction of tubulin in each of the coexisting structures, the rate constants, and the standard Helmholtz free energies for closing a ring and for longitudinal dimer–dimer associations

Hierarchical Assembly Pathways of Spermine Induced Tubulin Conical-Spiral Architectures

Tubulin, an essential cytoskeletal protein, assembles into various morphologies by interacting with cellular factors. Spermine, an endogenous polyamine, promotes and stabilizes tubulin assemblies. Yet, the assembled structures and their formation pathways are poorly known. Here we show that spermine induced tubulin to assemble in vitro into hierarchical architectures, based on a tubulin conical-spiral (TCS) subunit. Using solution X-ray scattering and cryo-TEM, we showed that with progressive increase of spermine concentration, tubulin-dimers assembled into a tubulin helical-pitch (or a short TCS), TCSs, TCS that stacked into tubes through base-to-top packing, antiparallel bundles of TCS tubes in a quasi-hexagonal symmetry, and eventually twisted hexagonal bundles of inverted tubulin tubules. Time-resolved experiments revealed that tubulin assemblies formed at low spermine concentrations were precursors of the assemblies formed at higher spermine concentrations. The results provide insight into the variety of morphologies that tubulin can form, and contribute to our understanding of the fundamental interactions that control the composition and construction of protein-based biomaterials.</p

Structure and Energetics of GTP- and GDP-Tubulin Isodesmic Self-Association

Tubulin self-association is a critical process inmicrotubule dynamics. The early intermediate structures, energetics,and their regulation by fluxes of chemical energy, associatedwith guanosine triphosphate (GTP) hydrolysis, are poorlyunderstood. We reconstituted an in vitro minimal model system,mimicking the key elements of the nontemplated tubulin assembly.To resolve the distribution of GTP- and guanosine diphosphate(GDP)-tubulin structures, at low temperatures (∼10 °C) andbelow the critical concentration for the microtubule assembly, weanalyzed in-line size-exclusion chromatography−small-angle X-rayscattering (SEC-SAXS) chromatograms of GTP- and GDP-tubulinsolutions. Both solutions rapidly attained steady state. The SEC-SAXS data were consistent with an isodesmic thermodynamic modelof longitudinal tubulin self-association into 1D oligomers, terminated by the formation of tubulin single rings. The analysis showedthat free dimers coexisted with tetramers and hexamers. Tubulin monomers and lateral association between dimers were notdetected. The dimer−dimer longitudinal self-association standard Helmholtz free energies were −14.2 ± 0.4 k$_B$T (−8.0 ± 0.2 kcalmol$^{−1}$) and −13.1 ± 0.5 k$_B$T (−7.4 ± 0.3 kcal mol$^{−1}$) for GDP- and GTP-tubulin, respectively. We then determined the massfractions of dimers, tetramers, and hexamers as a function of the total tubulin concentration. A small fraction of stable tubulin singlerings, with a radius of 19.2 ± 0.2 nm, was detected in the GDP-tubulin solution. In the GTP-tubulin solution, this fraction wassignificantly lower. Cryo-TEM images and SEC-multiangle light-scattering analysis corroborated these findings. Our analyses providean accurate structure−stability description of cold tubulin solutions

Mechanism of the Initial Tubulin Nucleation Phase

Tubulin nucleation is a highly frequent event in microtubule (MT) dynamics but is poorly understood. In this work, we characterized the structural changes during the initial nucleation phase of dynamic tubulin. Using size-exclusion chromatography-eluted tubulin dimers in an assembly buffer solution free of glycerol and tubulin aggregates enabled us to start from a well-defined initial thermodynamic ensemble of isolated dynamic tubulin dimers and short oligomers. Following a temperature increase, time-resolved X-ray scattering and cryo-transmission electron microscopy during the initial nucleation phase revealed an isodesmic assembly mechanism of one-dimensional (1D) tubulin oligomers (where dimers were added and/or removed one at a time), leading to sufficiently stable two-dimensional (2D) dynamic nanostructures, required for MT assembly. A substantial amount of tubulin octamers accumulated before two-dimensional lattices appeared. Under subcritical assembly conditions, we observed a slower isodesmic assembly mechanism, but the concentration of 1D oligomers was insufficient to form the multistranded 2D nucleus required for MT formation

Effect of tubulin self-association on GTP hydrolysis and nucleotide exchange reactions

We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. Both reactions are relevant for microtubule (MT) dynamics. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied by determining the concentrations of tubulin-free and tubulin-bound GTP and GDP. We observed no GTP hydrolysis below the critical conditions for MT assembly (either below the critical tubulin concentration and/or at low temperature), despite the assembly of tubulin 1D curved oligomers and single-rings, showing that their assembly did not involve GTP hydrolysis. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Cryo-TEM images showed that GTP-tubulin 1D oligomers were curved also at 36 °C. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to determine the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites. This conclusion may have implications on the dynamics at the tip of the MT plus end

pH stability and disassembly mechanism of wild-type simian virus 40

Viruses are remarkable self-assembled nanobiomaterial-based machines, exposed to a wide range of pH values. Extreme pH values can induce dramatic structural changes, critical for the function of the virus nanoparticles, including assembly and genome uncoating. Tuning cargo–capsid interactions is essential for designing virus-based delivery systems. Here we show how pH controls the structure and activity of wild-type simian virus 40 (wtSV40) and the interplay between its cargo and capsid. Using cryo-TEM and solution X-ray scattering, we found that wtSV40 was stable between pH 5.5 and 9, and only slightly swelled with increasing pH. At pH 3, the particles aggregated, while capsid protein pentamers continued to coat the virus cargo but lost their positional correlations. Infectivity was only partly lost after the particles were returned to pH 7. At pH 10 or higher, the particles were unstable, lost their infectivity, and disassembled. Using time-resolved experiments we discovered that disassembly began by swelling of the particles, poking a hole in the capsid through which the genetic cargo escaped, followed by a slight shrinking of the capsids and complete disassembly. These findings provide insight into the fundamental intermolecular forces, essential for SV40 function, and for designing virus-based nanobiomaterials, including delivery systems and antiviral drugs

Surfactant-Mediated Co-Existence of Single-Walled Carbon Nanotube Networks and Cellulose Nanocrystal Mesophases

Hybrids comprising cellulose nanocrystals (CNCs) and percolated networks of single-walled carbon nanotubes (SWNTs) may serve for the casting of hybrid materials with improved optical, mechanical, electrical, and thermal properties. However, CNC-dispersed SWNTs are depleted from the chiral nematic (N*) phase and enrich the isotropic phase. Herein, we report that SWNTs dispersed by non-ionic surfactant or triblock copolymers are incorporated within the surfactant-mediated CNC mesophases. Small-angle X-ray measurements indicate that the nanostructure of the hybrid phases is only slightly modified by the presence of the surfactants, and the chiral nature of the N* phase is preserved. Cryo-TEM and Raman spectroscopy show that SWNTs networks with typical mesh size from hundreds of nanometers to microns are distributed equally between the two phases. We suggest that the adsorption of the surfactants or polymers mediates the interfacial interaction between the CNCs and SWNTs, enhancing the formation of co-existing meso-structures in the hybrid phases