Microtubules (MTs) are one of the major components of the cytoskeleton. They are involved in many key functions of eukaryotic cells, including cell division, intracellular transport, cell motility, and cell shape. MTs are hollow tubules made of parallel filaments, formed by active (non-equilibrium) self-organization of tubulin dimers. The dynamic self-organization of tubulin is facilitated by the GTPase activity of tubulin. Tubulin self-assembles with microtubule-associated proteins (MAPs) and other factors into a wide range of morphologies, including tubulin rings, MT bundles, and the spindle apparatus, segregating chromosomes during cell division. In this review, we shall discuss recent insight into the intimate link between tubulin -biochemistry, -structure, -interactions, -dynamics, -stability, -assembly, -disassembly, and -mechanical properties. We shall then focus on recent time-resolved solution X-ray scattering analysis of tubulin self-organization below and above the critical conditions for microtubule assembly. Finally, we shall discuss some of the challenging multiscale unsolved problems requiring the integration of different experimental and theoretical methods. Microtubule formation is an important target for drugs to treat conditions like gout and a wide range of cancers. Understanding the polymerization mechanism could help in the design of future drugs and in the development of active biomaterials that promote the remodeling or regeneration of tissue after disease or injury.

Virus-like particles (VLPs) are noninfectious nanocapsules that can be used for drug delivery or vaccine applications. VLPs can be assembled from virus capsid proteins around a condensing agent, such as RNA, DNA, or a charged polymer. Electrostatic interactions play an important role in the assembly reaction. VLPs assemble from many copies of capsid protein, with a combinatorial number of intermediates. Hence, the mechanism of the reaction is poorly understood. In this paper, we combined solution small-angle X-ray scattering (SAXS), cryo-transmission electron microscopy (TEM), and computational modeling to determine the effect of ionic strength on the assembly of Simian Vacuolating Virus 40 (SV40)-like particles. We mixed poly(styrene sulfonate) with SV40 capsid protein pentamers at different ionic strengths. We then characterized the assembly product by SAXS and cryo-TEM. To analyze the data, we performed Langevin dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures consistent with the experimental data. We found that close to physiological ionic strength, $$T=1$$VLPs coexisted with VP1 pentamers. At lower or higher ionic strengths, incomplete particles coexisted with pentamers and $$T=1$$particles. Including the simulated structures was essential to explain the SAXS data in a manner that is consistent with the cryo-TEM images.

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.

The current challenges of structural biophysics include determining the structure of large self-assembled complexes, resolving the structure of ensembles of complex structures and their mass fraction, and unraveling the dynamic pathways and mechanisms leading to the formation of complex structures from their subunits. Modern synchrotron solution X-ray scattering data enable simultaneous high-spatial and high-temporal structural data required to address the current challenges of structural biophysics. These data are complementary to crystallography, NMR, and cryo-TEM data. However, the analysis of solution scattering data is challenging; hence many different analysis tools, listed in the SAS Portal (http://smallangle.org/), were developed. In this review, we start by briefly summarizing classical X-ray scattering analyses providing insight into fundamental structural and interaction parameters. We then describe recent developments, integrating simulations, theory, and advanced X-ray scattering modeling, providing unique insights into the structure, energetics, and dynamics of self-assembled complexes. The structural information is essential for understanding the underlying physical chemistry principles leading to self-assembled supramolecular architectures and computational structural refinement.

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