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ACS nano 15(5), 8836 - 8847 (2021). doi:10.1021/acsnano.1c01374, Tubulin, an essential cytoskeletal protein, assembles into various morphologies by interacting with an array of cellular factors. One of these factors is the endogenous polyamine spermine, which may promote and stabilize tubulin assemblies. Nevertheless, the assembled structures and their formation pathways are poorly known. Here we show that spermine induced the in vitro assembly of tubulin into several hierarchical architectures based on a tubulin conical-spiral subunit. Using solution X-ray scattering and cryo-TEM, we found that with progressive increase of spermine concentration tubulin dimers assembled into conical-frustum-spirals of increasing length, containing up to three helical turns. The subunits with three helical turns were then assembled into tubules through base-to-top packing and formed antiparallel bundles of tubulin conical-spiral tubules in a distorted hexagonal symmetry. Further increase of the spermine concentration led to inverted tubulin tubules assembled in hexagonal bundles. Time-resolved experiments revealed that tubulin assemblies formed at higher spermine concentrations assembled from intermediates, similar to those formed at low spermine concentrations. These results are distinct from the classical transition between twisted ribbons, helical, and tubular assemblies, and provide insight into the versatile morphologies that tubulin can form. Furthermore, they may contribute to our understanding of the interactions that control the composition and construction of protein-based biomaterials., Published by Soc., Washington, DC

ACS omega 3(11), 16246 - 16252 (2018). doi:10.1021/acsomega.8b01289, Exposure of a protein to cosolutes, like denaturants, changes its folding equilibrium. To determine the ensemble of protein conformations at equilibrium, in the presence of weakly interacting cosolutes, we present a two-stage analysis of solution X-ray scattering data. In the first stage, Guinier analysis and Kratky plot revealed information about the compactness and flexibility of the protein. In the second stage, elastic network contact model and coarse-grained normal mode analysis were used to generate an ensemble of conformations. The scattering curves of the conformations were computed and fitted to the measured scattering curves to get insights into the dominating folding states at equilibrium. Urea and guanidine hydrochloride (GuHCl) behaved as preferentially included weakly interacting cosolutes and induced denaturation of hen egg-white lysozyme, which served as our test case. The computed models adequately fit the data and gave ensembles of conformations that were consistent with our measurements. The analysis suggests that in the presence of urea, lysozyme retained its compactness and assumed molten globule characteristics, whereas in the presence of GuHCl lysozyme adopted random coiled conformations. Interestingly, no equilibrium intermediate states were observed in both urea and GuHCl., Published by ACS Publications, Washington, DC

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.

Dipolar lipid membranes may adsorb multivalent ions. The binding constant depends on the type of lipid and ions. In this paper, we focus on the adsorption of calcium ions onto 1,2-dilauroylphosphatidylcholine (DLPC) membrane. Using small-angle-X-ray scattering we found that at ambient room temperature ca. 0.6 mM CaCl2 is a critical concentration at which calcium ions adsorbed to 30 mg/mL (ca. 48 mM) DLPC membrane. We then determined the structure of the lamellar phases formed at CaCl2 concentrations below and above the critical concentration and characterized the effect of temperature and incubation time on the adsorption process. Our findings suggest that calcium adsorption to DLPC membranes requires an initial nucleation phase.

Cyanobacteria light-harvesting complexes can change their structure to cope with fluctuating environmental conditions. Studying in vivo structural changes is difficult owing to complexities imposed by the cellular environment. Mimicking this system in vitro is challenging, as well. The in vivo system is highly concentrated, and handling similar in vitro concentrated samples optically is difficult because of high absorption. In this research, we mapped the cyanobacteria antennas self-assembly pathways using highly concentrated solutions of phycocyanin (PC) that mimic the in vivo condition. PC was isolated from the thermophilic cyanobacterium Thermosynechococcus vulcanus and measured by several methods. PC has three oligomeric states: hexamer, trimer, and monomer. We showed that the oligomeric state was changed upon increase of PC solution concentration. This oligomerization mechanism may enable photosynthetic organisms to adapt their light-harvesting system to a wide range of environmental conditions.