Peter Vekilov - Academia.edu (original) (raw)

Papers by Peter Vekilov

and the Nucleation Precursors in Protein Crystallization • Experiments to be performed on the LMM... more and the Nucleation Precursors in Protein Crystallization • Experiments to be performed on the LMM. • Experiments explore the effects of an unstudied factor for the nucleation of protein crystals: solution shear flow. • Benchmarking against data obtained at zero flow rates, only possible in space. • Methods of detecting and monitoring protein aggregates leading to nucleation require experiments in solution layers significantly thicker than 100µm.

Methods in Molecular Biology

Amyloid fibrils, highly ordered protein aggregates, play an important role in the onset of severa... more Amyloid fibrils, highly ordered protein aggregates, play an important role in the onset of several neurological disorders. Many studies have assessed amyloid fibril formation under specific solution conditions, but they all lack an important phenomena in biological solutions-buffer specific effects. We have focused on the formation of hen egg-white lysozyme (HEWL) fibrils in aqueous solutions of different buffers in both acidic and basic pH range. By means of UV-Vis spectroscopy, fluorescence measurements and CD spectroscopy, we have managed to show that fibrillization of HEWL is affected by buffer identity (glycine, TRIS, phosphate, KCl-HCl, cacodylate, HEPES, acetate), solution pH, sample incubation (agitated vs. static) and added excipients (NaCl and PEG). HEWL only forms amyloid fibrils at pH = 2.0 under agitated conditions in glycine and KCl-HCl buffers of high enough ionic strength. Phosphate buffer on the other hand stabilizes the HEWL molecules. Similar stabilization effect was achieved by addition of PEG12000 molecules to the solution.

Olanzapine (OZPN) is a BCS class II drug used to treat schizophrenia (bipolar disorder). OZPN exh... more Olanzapine (OZPN) is a BCS class II drug used to treat schizophrenia (bipolar disorder). OZPN exhibits rich solid-state diversity. To date, 60 distinct forms have been identified, including 3 polymorphs (I, II, III), 52 crystalline solvates, 3 dihydrates (DB, DD, DE), a disordered higher hydrate, plus an amorphous form. Atomic Force Microscopy (AFM) results suggest that the nucleation of OZPN DD on the surface of OZPN I in water may follow a non-classical mechanisms that includes formation of solute-rich mesoscopic clusters [1]. Since the solubility of OZPN I in water is very low, the kinetics of transformation are difficult to monitor. To increase the solubility of OZPN I, we added different ratios of a co-solvent, ethanol. AFM observations revealed that clusters similar to those seen in purely aqueous environments are present on the surface of OZPN:EtOH:H2O crystals in contact with both supersaturated and undersaturated EtOH/H2O solutions. To establish the mechanism of cluster for...

Nature Chemistry, 2020

Accepted/In press). Olanzapine crystal symmetry originates in preformed centrosymmetric solute di... more Accepted/In press). Olanzapine crystal symmetry originates in preformed centrosymmetric solute dimers. Nature Chemistry.

ABSTRACTProtein crystallization is central to understanding of molecular structure in biology, a ... more ABSTRACTProtein crystallization is central to understanding of molecular structure in biology, a vital part of processes in the pharmaceutical industry, and a crucial component of numerous disease pathologies. Crystallization starts with nucleation and how nucleation proceeds determines the crystallization rate and essential properties of the resulting crystal population. Recent results with several proteins indicate that crystals nucleate within preformed mesoscopic protein-rich clusters. The origin of the mesoscopic clusters is poorly understood. In the case of lysozyme, a common model of protein biophysics, earlier findings suggest that clusters exist owing to the dynamics of formation and decay of weakly-bound transient dimers. Here we present evidence of a weakly bound lysozyme dimer in solutions of this protein. We employ two electrospray mass spectrometry techniques, a combined ion mobility separation mass spectrometry and a high-resolution implementation. To enhance the weak...

Medical Research Archives, 2015

We present Confocal Depolarized Dynamic Light Scattering as a novel optical method for the charac... more We present Confocal Depolarized Dynamic Light Scattering as a novel optical method for the characterization of nanoparticles in solution. The method is validated in conditions of biological interest where traditional optical methods fail. As a test case we study highly concentrated, strongly scattering, samples of thermosensitive core-shell particles constituted by a spherical PMMA core surrounded by a PNIPAM network and, follow the kinetics of the processes induced by temperature changes. We prove that through the confocal optical scheme, the multiple scattering contribution is reduced by orders of magnitude. Moreover, the method allows the efficient characterization of NPs with a superior accuracy in particle size determination. Low degrees of polydispersity can be easily characterized through an adapted cumulant method.

CrystEngComm, 2015

Hematin crystallization, the primary heme detoxification mechanism of malaria parasites infecting... more Hematin crystallization, the primary heme detoxification mechanism of malaria parasites infecting human erythrocytes, most likely requires the participation of lipid structures.

Methods in enzymology, 2003

Publisher Summary This chapter discusses the molecular mechanisms of defect formation. The goal o... more Publisher Summary This chapter discusses the molecular mechanisms of defect formation. The goal of this chapter is to summarize the recent work on the molecular level on the processes that accompany crystallization, and lead to defects and associated lattice strain, and potential plastic deformations such as mosaicity. While the mechanisms leading to relatively perfect protein crystals have been studied in great detail at both the mesoscopic1-3 and the molecular level 4-8, only a few of the processes leading to defects have been monitored. This chapter demonstrates an argument that diffraction resolution is affected only by short-scale molecular disorder and not by mosaicity, striae, zoning, and block structures. On the other hand, the diffraction resolution is determined by the signal-to-noise ratio of high-index reflections. Finally, this chapter concludes by explaining that if the crystal consists of a few large blocks, the beam in X-ray diffraction experiments can be focused on only one of these blocks, and high-resolution structure determinationscan still be achieved.

The Journal of Physical Chemistry B, 2006

The solvent around protein molecules in solutions is structured and this structuring introduces a... more The solvent around protein molecules in solutions is structured and this structuring introduces a repulsion in the intermolecular interaction potential at intermediate separations. We use Monte Carlo simulations with isotropic, pair-additive systems interacting with such potentials. We test if the liquid-liquid and liquidsolid phase lines in model protein solutions can be predicted from universal curves and a pair of experimentally determined parameters, as done for atomic and colloid materials using several laws of corresponding states. As predictors, we test three properties at the critical point for liquid-liquid separation: temperature, as in the original van der Waals law, the second virial coefficient, and a modified second virial coefficient, all paired with the critical volume fraction. We find that the van der Waals law is best obeyed and appears more general than its original formulation: A single universal curve describes all tested nonconformal isotropic pair-additive systems. Published experimental data for the liquid-liquid equilibrium for several proteins at various conditions follow a single van der Waals curve. For the solid-liquid equilibrium, we find that no single system property serves as its predictor. We go beyond corresponding-states correlations and put forth semiempirical laws, which allow prediction of the critical temperature and volume fraction solely based on the range of attraction of the intermolecular interaction potential.

The Journal of Physical Chemistry B, 2007

Dense liquid phases, metastable with respect to a solid phase, but stable with respect to the sol... more Dense liquid phases, metastable with respect to a solid phase, but stable with respect to the solution, have been known to form in solutions of proteins and small-molecule substances. Here, with the protein lumazine synthase as a test system, using dynamic and static light scattering and atomic force microscopy, we demonstrate submicron size clusters of dense liquid. In contrast to the macroscopic dense liquid, these clusters are metastable not only with respect to the crystals, but also with respect to the low-concentration solution: the characteristic cluster lifetime is limited to approximately 10 s, after which they decay. The cluster population is detectable only if they occupy &amp;amp;amp;amp;amp;amp;amp;gt;10(-6) of the solution volume and have a number density &amp;amp;amp;amp;amp;amp;amp;gt;105 cm-3 for 3 to 11% of the monitored time. The cluster volume fraction varies within wide limits and reaches up to 10(-3). Increasing protein concentration increases the frequency of cluster detection but does not affect the ranges of the cluster sizes, suggesting that a preferred cluster size exists. A simple Monte Carlo model with protein-like potentials reproduces the metastable clusters of dense liquid with limited lifetimes and variable sizes and suggests that the mean cluster size is determined by the kinetics of growth and decay and not by thermodynamics.

Proteins: Structure, Function, and Genetics, 2001

We apply in situ atomic force microscopy to the crystallization of ferritins from solutions conta... more We apply in situ atomic force microscopy to the crystallization of ferritins from solutions containing Ϸ5% (w/w) of their inherent molecular dimers. Molecular resolution imaging shows that the dimers consist of two bound monomers. The constituent monomers are likely partially denatured, resulting in increased hydrophobicity of the dimer surface. Correspondingly, the dimers strongly adsorb on the crystal surface. The adsorbed dimers hinder step growth and on incorporation by the crystal initiate stacks of up to 10 triple and single vacancies in the subsequent crystal layers. The molecules around the vacancies are shifted by Ϸ0.1 molecular dimensions from their crystallographic positions. The shifts strain the lattice and, as a consequence, at crystal sizes > 200 m, the accumulated strain is resolved by a plastic deformation whereupon the crystal breaks into mosaic blocks 20-50 m in size. The critical size for the onset of mosaicity is similar for ferritin and apoferritin and close to the value for a third protein, lysozyme; it also agrees with theoretical predictions. Trapped microcrystals in ferritin and apoferritin induce strain with a characteristic length scale equal to that of a single point defect, and, as a consequence, trapping does not contribute to the mosaicity. The sequence of undesired phenomena that include heterogeneity generation, adsorption, incorporation, and the resulting lattice strain and mosaicity in this and other proteins systems, could be avoided by improved methods to separate similar proteins species (microheterogeneity) or by increasing the biochemical stability of the macromolecules against oligomerization. Proteins 2001;43:343-352.

Nature Materials, 2012

ABSTRACT Real-time transmission electron microscopy shows that the formation of crystal nuclei of... more ABSTRACT Real-time transmission electron microscopy shows that the formation of crystal nuclei of organic molecules in solution occurs inside dense liquid nanoclusters.

Journal of Molecular Biology, 2007

Polymerization of sickle cell hemoglobin (HbS) in deoxy state is one of the basic events in the p... more Polymerization of sickle cell hemoglobin (HbS) in deoxy state is one of the basic events in the pathophysiology of sickle cell anemia. For insight into the polymerization process, we monitor the kinetics of nucleation and growth of the HbS polymer fibers. We define a technique for the determination of the rates J and delay times θ of nucleation and the fiber growth rates R of deoxy-HbS fibers, based on photolysis of CO-HbS by laser illumination. We solve numerically time-dependent equations of heat conductance and CO transport, coupled with respective photo-chemical processes, during kinetics experiments under continuous illumination. After calibration with experimentally determined values, we define a regime of illumination ensuring uniform temperature and deoxy-HbS concentration, and fast (within < 1 s) egress to steady conditions. With these procedures, data on the nucleation and growth kinetics have relative errors of < 5% and are reproducible within 10% in independent experiments. The nucleation rates and delay times have steep, exponential dependencies on temperature. In contrast, the average fiber growth rates only weakly depend on temperature. The individual growth rates vary by up to 40% under identical conditions. These variations are attributed to instability of the coupled kinetics and diffusion towards the growing end of a fiber. The activation energy for incorporation of HbS molecules into a polymer is E A = 50 kJ mol − 1 , a low value indicating the significance of the hydrophobic contacts in the HbS polymer. More importantly, the contrast between the strong θ(T) and weak R(T) dependencies suggests that the homogenous nucleation of HbS polymers occurs within clusters of a precursor phase. This conclusion may have significant consequences for the understanding of the pathophysiology of sickle cell anemia and should be tested in further work.

Journal of Crystal Growth, 2000

Ribonuclease, insulin, cytochrome C, myoglobin and ovalbumin were introduced into solutions from ... more Ribonuclease, insulin, cytochrome C, myoglobin and ovalbumin were introduced into solutions from which ferritin and lysozyme crystals were grown. These measurements were also performed for the ferritin dimers trapped by growing ferritin crystals. The crystals were later dissolved in a pure solvent, the impurity concentrations were measured by high performance liquid chromatography and the e!ective impurity distribution coe$cient, K, was evaluated relative to the initial concentrations of ferritin or lysozyme. The density of impurity species in crystal relative to its density in mother solution were used to calculate volumetric distribution coe$cient, k. These distribution coe$cients were found to exceed unity (k'1) in terrestrial condition for all impurity species, except for insulin and cytochrome C lysozyme. For ferritin dimers, K"4, k"1.8;10. Crystals grown in space under the otherwise identical conditions incorporated lower amounts of all of these impurities, majority of them below the detection limit. The lower impurity incorporation obtained in stagnant solution may be partially due to more di$cult impurity supply through the impurity depletion zone arising around the growing crystals at k'1 in the absence of buoyancy driven convection or stirring. Analytical estimates of the depletion zone show reasonable agreement with measurements for ferritin dimers. Step bunching and other #ow-dependent surface processes may also contribute to lower distribution coe$cient.