Balken E, Ben-Nun I, Fellig A, Khaykelson D, Raviv U. Upgrade of ıt D+ software for hierarchical modeling of X-ray scattering data from complex structures in solution, fibers and single orientations}. Journal of Applied Crystallography [Internet]. 2023;56 (4) :1295–1303. Publisher's VersionAbstract

This article presents an upgrade of the ıt D}+ software [Ginsburg ıt et al.} (2019). ıt J. Appl. Cryst.} \bf 52, 219–242], expanding its hierarchical solution X-ray scattering modeling capabilities for fiber diffraction and single crystallographic orientations. This upgrade was carried out using the reciprocal grid algorithm [Ginsburg ıt et al.} (2016). ıt J. Chem. Inf. Model.} \bf 56, 1518–1527], providing ıt D}+ its computational strength. Furthermore, the extensive modifications made to the Python API of ıt D}+ are described, broadening the X-ray analysis performed with ıt D}+ to account for the effects of the instrument-resolution function and polydispersity. In addition, structure-factor and radial-distribution-function modules were added, taking into account the effects of thermal fluctuations and intermolecular interactions. Finally, numerical examples demonstrate the usage and potential of the added features.

Kozell A, Eliaz D, Solomonov A, Benyamin D, Shmul G, Brookstein O, Rosenhek-Goldian I, Raviv U, Shimanovich U. Modulating amyloids’ formation path with sound energy. Proceedings of the National Academy of Sciences [Internet]. 2023;120 (3) :e2212849120. Publisher's Version
Shemesh A, Ghareeb H, Dharan R, Levi-Kalisman Y, Metanis N, Ringel I, Raviv U. Effect of tubulin self-association on GTP hydrolysis and nucleotide exchange reactions. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics [Internet]. 2023;1871 (2) :140869. Publisher's VersionAbstract

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.

Eliaz D, Paul S, Benyamin D, Cernescu A, Cohen SR, Rosenhek-Goldian I, Brookstein O, Miali ME, Solomonov A, Greenblatt M, et al. Micro and nano-scale compartments guide the structural transition of silk protein monomers into silk fibers. Nature Communications [Internet]. 2022;13 (1) :7856. Publisher's VersionAbstract

Silk is a unique, remarkably strong biomaterial made of simple protein building blocks. To date, no synthetic method has come close to reproducing the properties of natural silk, due to the complexity and insufficient understanding of the mechanism of the silk fiber formation. Here, we use a combination of bulk analytical techniques and nanoscale analytical methods, including nano-infrared spectroscopy coupled with atomic force microscopy, to probe the structural characteristics directly, transitions, and evolution of the associated mechanical properties of silk protein species corresponding to the supramolecular phase states inside the silkworm’s silk gland. We found that the key step in silk-fiber production is the formation of nanoscale compartments that guide the structural transition of proteins from their native fold into crystalline β-sheets. Remarkably, this process is reversible. Such reversibility enables the remodeling of the final mechanical characteristics of silk materials. These results open a new route for tailoring silk processing for a wide range of new material formats by controlling the structural transitions and self-assembly of the silk protein’s supramolecular phases.

Dong Y, Kampf N, Schilt Y, Cao W, Raviv U, Klein J. Dehydration does not affect lipid-based hydration lubrication. Nanoscale. 2022.
Shemesh A, Dharan N, Ginsburg A, Dharan R, Levi-Kalisman Y, Ringel I, Raviv U. Mechanism of the Initial Tubulin Nucleation Phase. The Journal of Physical Chemistry Letters [Internet]. 2022;13 (41) :9725-9735. Publisher's Version
Shemesh A, Ginsburg A, Dharan R, Levi-Kalisman Y, Ringel I, Raviv U. Mechanism of Tubulin Oligomers and Single-Ring Disassembly Catastrophe. The Journal of Physical Chemistry Letters [Internet]. 2022;13 (23) :5246-5252. Publisher's Version
Shemesh A, Ginsburg A, Dharan R, Kalisman-Levi Y, Ringel I, Raviv U. Mechanism of Tubulin Oligomers and Single-Rings Disassembly Catastrophe. ChemRxiv [Internet]. 2022. Publisher's Version
Asor R, Singaram SW, Levi-Kalisman Y, Hagan MF, Raviv U. Effect of Ionic Strength on the Assembly of Simian Vacuolating Virus Capsid Protein Around Poly(Styrene Sulfonate). bioRxiv [Internet]. 2022. Publisher's VersionAbstract

{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 like 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 combinatorial intermediates, and therefore the mechanism of the reaction is poorly understood. In this paper, we determined 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 solution small-angle X-ray scattering (SAXS) and cryo-TEM. To analyze the data, we performed Brownian dynamics simulations using a coarse-grained model that revealed incomplete, asymmetric VLP structures that were consistent with the experimental data. We found that close to physiological ionic strength

Sadeh AS, Dharan R, Ghareeb H, Metanis N, Ringel I, Raviv U. Effect of Tubulin Self-Association on GTP Hydrolysis and Nucleotide Exchange Reactions. ChemRxiv [Internet]. 2021. Publisher's Version
Shemesh A, Ginsburg A, Dharan R, Levi-Kalisman Y, Ringel I, Raviv U. Structure and Energetics of GTP- and GDP-Tubulin Isodesmic Self-Association. ACS Chemical Biology [Internet]. 2021;16 (11) :2212–2227. Publisher's Version
Dharan R, Shemesh A, Millgram A, Zalk R, Frank GA, Levi-Kalisman Y, Ringel I, Raviv U. Hierarchical Assembly Pathways of Spermine-Induced Tubulin Conical-Spiral Architectures. ACS Nano [Internet]. 2021;15 (5) :8836–8847. Publisher's Version
Schilt Y, Berman T, Wei X, Nativ-Roth E, Barenholz Y, Raviv U. Effect of the ammonium salt anion on the structure of doxorubicin complex and PEGylated liposomal doxorubicin nanodrugs. Biochimica et Biophysica Acta (BBA) - General Subjects [Internet]. 2021;1865 (5) :129849. Publisher's VersionAbstract

Background In Doxil®, PEGylated nanoliposomes are created by hydration of the lipids in ammonium sulfate, and are remotely loaded with doxorubicin by a transmembrane ammonium gradient. The ammonium sulfate is then removed from the external aqueous phase, surrounding the liposomes, and replaced by an isoosmotic sucrose solution in 10 mM histidine buffer at pH 6.5. Methods We prepared PEGylated liposomal doxorubicin (PLD) with a series of ammonium monovalent salts that after remote loading became the intraliposome doxorubicin counteranions. We analyzed the liposomes by solution X-ray scattering, differential scanning calorimetry, and electron micropscopy. Results PLDs prepared with sulfonic acid derivatives as counteranion exhibited chemical and physical stabilities. We determined the effect of these ammonium salt counteranions on the structure, morphology, and thermotropic behavior of the PEGylated nanoliposomes, formed before and after doxorubicin loading, and the bulk properties of the doxorubicin-counteranion complexes. By comparing the structure of the doxorubicin complexes in the bulk and inside the nanoliposomes, we revealed the effect of confinement on the structure and doxorubicin release rate for each of the derivatives of the ammonium sulfonic acid counteranions. Conclusions We found that the extent and direction of the doxorubicin confinement effect and its release rate were strongly dependent on the type of counteranion. The counteranions, however, neither affected the structure and thermotropic behavior of the liposome membrane, nor the thickness and density of the liposome PEG layers. In an additional study, it was demonstrated that PLD made with ammonium-methane sulfonate exhibit a much lower Hand and Foot syndrome. General significance The structure, physical state, and pharmacokinetics of doxorubicin in PEGylated nanoliposomes, prepared by transmembrane remote loading using gradients of ammonium salts, strongly depend on the counteranions.

Fink L, Allolio C, Feitelson J, Tamburu C, Harries D, Raviv U. Bridges of Calcium Bicarbonate Tightly Couple Dipolar Lipid Membranes. Langmuir [Internet]. 2020;36 (36) :10715–10724. Publisher's Version
Shukrun Farrell E, Schilt Y, Moshkovitz MY, Levi-Kalisman Y, Raviv U, Magdassi S. 3D Printing of ordered mesoporous silica complex structures. Nano Letters [Internet]. 2020;20 (9) :6598–6605. Publisher's Version
Waltmann C, Asor R, Raviv U, Olvera de la Cruz M. Assembly and Stability of Simian Virus 40 Polymorphs. ACS Nano [Internet]. 2020;14 (4) :4430-4443. Publisher's Version
Asor R, Schlicksup CJ, Zhao Z, Zlotnick A, Raviv U. Rapidly Forming Early Intermediate Structures Dictate the Pathway of Capsid Assembly. Journal of the American Chemical Society [Internet]. 2020; 142 (17) :7868–7882. Publisher's Version
Asor R, Khaykelson D, Ben-nun-Shaul O, Levi-Kalisman Y, Oppenheim A, Raviv U. pH Stability and Disassembly Mechanism of Wild-Type Simian Virus 40. Soft Matter [Internet]. 2020;16 (11) :2803-2814. Publisher's VersionAbstract

Virus 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 viral-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 had been 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, and 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.

Khaykelson D, Raviv U. Studying viruses using solution X-ray scattering. Biophysical Reviews [Internet]. 2020;12 :41–48. Publisher's VersionAbstract

Viruses have been of interest to mankind since their discovery as small infectious agents in the nineteenth century. Because many viruses cause diseases to humans and agriculture, they were rigorously studied for biological and medical purposes. Viruses have remarkable properties such as the symmetry and self-assembly of their protein envelope, maturation into infectious virions, structural stability, and disassembly. Solution X-ray scattering can probe structures and reactions in solutions, down to subnanometer spatial resolution and millisecond temporal resolution. It probes the bulk solution and reveals the average shape and average mass of particles in solution and can be used to study kinetics and thermodynamics of viruses at different stages of their life cycle. Here we review recent work that demonstrates the capabilities of solution X-ray scattering to study in vitro the viral life cycle.

Dharan R, Shemesh A, Millgram A, Levi-Kalisman Y, Ringel I, Raviv U. Hierarchical Assembly Pathways of Spermine Induced Tubulin Conical-Spiral Architectures. [Internet]. 2019. Publisher's Version