Peter G. Vekilov - US grants

The objective of the proposed research is to obtain quantitative understanding of protein nucleation and post-nucleation growth of small protein crystals (crystallites) in order to: (a) improve crystallization protocols yielding crystals of a structural perfection that matches state-of-the-art X-ray diffraction techniques used in protein structure/function studies and rational drug design; (b) achieve narrow crystallite size distributions, which are essential for steady sustained release of pharmaceutical protein preparations, such as insulin; and (c) find means to regulate protein crystallization in vivo, e.g., the formation of hemoglobin C crystals in red blood cells. The specific aims to be pursued using four model globular proteins are: 1) Obtain insight into the fundamental mechanisms of protein crystal nucleation through studies of the dependencies of the nucleation rate on protein concentration and solution supersaturation. 2) Define and quantify the effects of: (i) soluble additives, biospecific for each protein, in concentrations representative of those commonly occurring in crystallizing protein solutions; and (ii) particulates with known size and concentration. 3) Based on the insight obtained under Aims 1) and 2), demonstrate that protein crystal nucleation can be controlled to achieve improvements in the areas (a)-(c) above. The basic hypothesis underlying this work is that nucleation concepts derived for inorganic systems, subject to modifications for differences in solution interactions and molecular kinetics, can provide guidance in protein nucleation. A key element of the proposed investigation is a novel automated multi-cell microscopy technique for protein nucleation studies. This technique allows direct measurements of the nucleation rates and quantitative correlations to the nucleation conditions. Unlike previous protein nucleation studies, these nucleation rate determinations are not based on any assumptions about the molecular interactions in the crystallization solution.

0609387
Vekilov
This project will synthesize tailored block copolymers with varying length and ratios between components, test droplet generation with several proteins of interest. The polymer creates concentrated protein phases and then is cross-linked and localized onto electrodes. The solvent can be removed and hence a pre-concentration follows. The authors show precise localization on electrodes, amounting to signal amplification. Together, the components will form novel 2D protein microchips. The planned work requires the synthesis of a polymer that will phase separate regardless of the proteins present in the solution and entrap sufficient quantities of each protein in the formed droplets. The second specific aim is to develope procedures for localized generation and deposition of the droplets over microelectrodes.

To perform their function, proteins must operate in the crowded environment of a living cell, thus requiring mechanisms that prevent protein aggregation. When these mechanisms fail, pathological conditions, such as sickle cell anemia or plaque formation, take place. In some cases, on the other hand, specific types of aggregation are actually desirable; examples including storage of insulin in the pancreas and protein in grains, in the form of crystals. Making protein crystals remains the single most important tool for protein structure determination, which is crucial for understanding protein function. It comes as a surprise that for those protein aggregates to form, folded protein molecules must first organize into long-lived clusters of a protein-rich liquid that are about a micron in size: According to the existing paradigms of phase equilibrium, such mesoscopic clusters should not exist, nor have they been seen in other similar systems such as colloids. The project aims to elucidate the molecular mechanism and the thermodynamic basis of how the puzzling mesoscopic clusters form. To accomplish this goal, the project will combine the theoretical and experimental efforts of the Lubchenko and Vekilov labs using four proteins as model systems: lysozyme, hemoglobin, insulin, and lumazine synthase. The roles of water structuring at the protein-solvent interface and the formation of transient protein oligomers in the stabilization of the protein-rich phase will be investigated by molecular modeling and tested by means of dynamic/static light scattering and thermodynamic and rheological characterizations. The rich kinetics of the formation/decay of clusters resulting from the interplay between protein transport and oligomer formation will be worked out by solving non-linear kinetic schemes coupled with diffusion/advection and tested against measured life-times and sizes of the clusters.

The solution of the important problem of mesoscopic aggregates in concentrated protein solutions lies at the interface of biology, physics, chemistry, and materials science. In addition, this research will be a close collaboration between a theoretical and experimental group from departments of chemistry and chemical engineering. These factors will combine to create a unique multidisciplinary research environment for participating students and research infrastructure. Considering the ethnic diversity at the University of Houston and in the greater Houston area, the research will enhance the educational opportunities in several underrepresented groups and promote their participation in advanced research. Existing collaborations with local writers and radio personalities will be utilized to publicize the societal benefits of the research. In addition to the fundamental and clinical significance of the research, its benefits include new potential routs for manufacturing novel materials and improving the nutritional value of crops. This project is jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences and by the Physics of Living Systems program in the Physics Division.

It is generally accepted that a protein's amino acid sequence is optimized so that the molecule consistently folds into a unique, stable 3D shape while avoiding aggregation with other biomolecules. Yet this view is overly simplistic. On the one hand, many proteins are not monolithic structures; for instance, myoglobin must reliably deform to let in oxygen. On the other hand, under certain conditions, proteins may aggregate into amyloid fibers while in a misfolded conformation. Although very complex, these phenomena can still be understood thermodynamically. Proteins are deformable given a sufficiently large native state entropy, while amyloid fibers are stabilized by multiple amino acid contacts within the fiber. Much less understood are the mesoscopic liquid-like aggregates recently found in several protein solutions, including lysozyme, hemoglobin, and gamma-crystallin. These clusters are important as nucleation sites for protein crystals. The objective of this project is to test a microscopic hypothesis for the formation of the mesoscopic clusters by which the clusters result from the formation of long-lived protein complexes stabilized at high densities. The unusually large cluster size stems from the long lifetime of the complexes, which in turn owe their stability to the conformational freedom of constituent protein molecules. A key component of the proposed mechanism is that proteins partially unfold prior to binding and may even undergo domain swapping, whereby protein segments bind to complimentary segments on a different protein. The PIs will employ a combination of dynamic light scattering, Brownian microscopy, amide exchange NMR, and theoretical modeling to test this mechanism in several systems, including proteins barnase, RNase A, hemoglobin, insulin, and lysozyme.

This research is a multidisciplinary effort by a theoretical physical chemist and experimental chemical engineer. A synthesis of a wide range of physicochemical and biochemical measurements and physical modeling will be undertaken to tackle this difficult problem; the outcome is expected to produce fundamental understanding of biological processes and provide new avenues for making new materials. Graduate, undergraduate, and high school students will be trained at the interface between physics, chemistry, and biology. The home institution of the PIs, the University of Houston, is one of the most ethnically diverse research universities in the nation, and is one of only three Hispanic-Serving Institutions in the US. Existing collaborations with local writers and radio personalities will be utilized to publicize the societal benefits of the research. This project is jointly supported by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems Program in the Physics Division.

DESCRIPTION (provided by applicant): The objective of this project is to develop a validated multiscale modeling methodology for quantifying the biophysical characteristics of sickle cell disease (SCD) -- a hematological disorder that affects tens of thousands of people in US with one in every 500 African-American births resulting in a child with SCD. The pathogenesis of SCD results from (1) irregular red blood cell (RBC) shapes due to hemoglobin polymerization inside the RBCs; (2) stiffening of the RBC membrane; and (3) adhesion of sickle RBCs to the endothelium and the other blood cells. The combination of these phenomena results in vaso-occlusive events or crises responsible for the majority of morbidity and mortality associated with SCD but little is certain about the proximal causes or the circumstances in which they occur. The spatio-temporal scales involved in accurately modeling SCD blood flow and vaso-occlusion span at least four orders of magnitude, hence new numerical methods are needed to simulate such multiscale phenomena. We present a general methodology based on 3D dissipative particle dynamics (DPD) to model flow and soft matter seamlessly, i.e., RBCs and other blood cells, blood plasma, cytosol, hemoglobin polymerization, and adhesive dynamics. DPD can be interfaced with molecular dynamics (MD) and with continuum-based description (e.g. Navier-Stokes) based on the triple-decker algorithm we have developed in order to capture molecular details or for computational efficiency in simulating large arteries or networks, respectively. We adopt the same approach here that has proven very effective in our previous work on malaria, namely that models for single RBCs (healthy or sickled), informed and validated from comprehensive single-cell measurements, will be used to predict the collective dynamics and rheology of SCD blood flow. We also present a systematic experimental plan, using microfluidics, nanomechanics and advanced optical techniques, to validate the various stages of the development of our models by targeting individual scales as well as interactions between scales. We will extend the first generation of models to study different modalities of existing and experimental therapeutic interventions for SCD, including simple transfusion, fetal hemoglobin (HbF) induction by hydroxyurea, and RBC hydration. Predictability of multiscale models requires quantifying uncertainty, and, to this end, we will incorporate polynomial chaos methods to model and propagate parametric uncertainties through the multiscale system. We plan to disseminate our models, software tools, and experimental data including the general-purpose triple-decker algorithm, via web-based repositories, existing public open-ware sites, tutorials and through the MSM consortium.

Title: Opportunistic complexation and mesoscopic aggregates in protein solution

Protein aggregation is a central problem of biophysics and other life sciences. This investigation focuses on a particularly puzzling type of protein aggregation during which protein-rich inclusions form in protein solutions. These inclusions are a micron or less in size and have been called the "mesoscopic clusters". Despite their small volume, the clusters are essential nucleation sites for ordered protein solids such as crystals and sickle cell anemia fibers. The mesoscopic clusters are also at odds with standard notions of thermodynamics, which dictates that such clusters should be either much larger or should not exist at all. Understanding the molecular origin of the mesoscopic clusters will resolve a major fundamental question of thermodynamics. This research will test the hypothesis that the mesoscopic clusters are caused by formation of long-lived complexes made up of individual protein molecules. A combination of advanced experimental techniques, theoretical modeling, and computer simulations will be employed to test this hypothesis. Understanding protein aggregation has implications in all facets of biology as well as on biotechnology and health. At the core of the broader impact activities is training of graduate, undergraduate, and, in particular, high school students. The project is a multidisciplinary study that spans many topics in physics, chemistry, and biology and represents a great platform for further academic endeavors of the involved students.

The microscopic hypothesis underlying the proposed work is that the mesoscopic clusters stem from the formation of transient protein-containing complexes. The complexes are stabilized at high protein densities. In contrast with the bulk solution, the complexes are the dominant protein-containing species inside the clusters. In a steady-state cluster, the influx of protein in the form of monomers is exactly balanced by the outflow of protein in the form of complexes. The nature of the complex depends on whether the protein is monomeric, as is lysozyme, or oligomeric, as is hemoglobin, ordinarily a tetrameric protein. The investigators in this study hypothesize that for typically monomeric proteins, complex formation is accompanied by partial protein unfolding and, possibly, domain swapping. In the case of oligomeric proteins, the complexes are oligomers that contain an untypical number of individual monomers. A core aspect of the hypothesis is that the complexes are opportunistic; they represent untypical ways to transiently bind individual protein molecules together. The research team will establish the identity and mechanisms of the complexation using a combination of physicochemical and biochemical experimental techniques (dynamic light scattering, Brownian microscopy, mass spectroscopy, amide exchange NMR, sheer flow) and theoretical tools (molecular modeling employing coarse-grained energy functions and the classical density functional theory). They will test whether the clusters are truly steady-state objects and explore the possibility that cluster formation has a slow, irreversible component. This part of the proposed study may answer the question whether clusters ripen according to an Ostwald-like scenario, or a different mechanism is involved.

Summary The main mechanism of heme detoxification implemented by Plasmodium parasites is the sequestration of heme as non-toxic, crystalline hemozoin. Heme sequestration has been the most successful molecular target for antimalarial drugs. Despite many years of effort, fundamental questions regarding the mechanism of heme detoxification remain elusive. It is not clear whether the drugs inhibit crystallization by forming soluble complexes with hematin, or interact with the hemozoin surface. Other open questions relate to the detailed molecular mechanism of inhibition and the existence of specific sites on the hemozoin surface that are active in drug binding, to whether different antimalarials utilize similar or different mechanisms and whether artemisinin derivatives interfere with heme detoxification. We propose, for the first time in antimalarial research, to elucidate the molecular mechanisms of inhibition of crystal growth by antimalarials and provide atomic-level detail of the relevant active sites on hemozoin crystal surfaces. We will pursue two specific aims: 1. Establish the mechanisms of action in blocking hematin crystallization of several classes of antimalarial drugs and related compounds. 2. Provide an atomic-level view of the active sites for hematin incorporation into crystals and association of the antimalarials and monitor the dynamics of antimalarial drug association with these sites in real time. Our main method of investigation is time-resolved in situ atomic force microscopy (AFM), including atomic resolution AFM, pioneered for studies of hematin crystallization by our group. Completion of the work proposed here will guide us to additional fundamental issues of heme detoxification and its inhibition. Achieving aim 1 will allow us to rank the drugs according to their potency in crystallization inhibition and explore how the crystals respond to the increased supersaturation due to the accumulation of hematin. Achieving aim 2 will provide the basis for state-of-the-art molecular dynamics modeling (using quantum mechanical potentials and explicit solvent) to address drug-hematin interactions in solution and elucidate drug binding modes on crystal surfaces. Such modeling will evolve into a platform for rational design of new antimalarials, to be developed in collaboration with medicinal chemists and parasitologists to study parasite suppression, drug bioavailability, and efficacy.

Non-technical Abstract:
Solution crystallization underlies a broad range of industrial, laboratory, and physiological processes. In recent years, organic and mixed organic-aqueous liquids have garnered increased interest as an alternative solvent for the preparation of crystalline materials and separation (or purification) by crystallization, in particular for high-value materials such as pharmaceuticals and fine chemicals. In contrast to crystallization from aqueous solvents, the level of understanding of the fundamental processes of crystal growth from organic solvents is severely limited. The lack of insight into the relevant fundamental mechanisms and pathways has emerged as the main obstacle to a rational approach to the optimization and control of crystallization in organic and mixed organic-aqueous media. The objective of this project is to provide understanding of the molecular processes comprising crystallization from mixed solvents focusing on the thermodynamic and kinetic consequences of potential solvent structuring.

Technical Abstract:
The roles that organic and mixed solvents play in crystallization are the open question addressed in this project. The solvent structures at the crystal-solution interface are characterized by advanced atomic force microscopy imaging and correlated with their effects on the thermodynamics of crystallization. How the chemical properties of the solvent affect the crystallization mechanism is elucidated by determining the steps that a solute molecule takes en route to a crystal growth site. The nature of the activation barrier for incorporation of molecules into the crystal is elucidated to establish whether solvent structuring and interactions with solvent molecules are the primary mechanisms underlying the existence of this barrier. Determining the steps in the molecular mechanism and their respective governing parameters provides tools to optimize and control crystallization from organic and mixed solvents and suppress defect-inducing instabilities. Comparing data for several porphyrin-solvent pairs elucidates the roles of the chemical moieties in the solvents and their abilities to form hydrogen and van der Waals bonds in the formation of solvent structures. Untangling the interlaced chemical and physical processes comprising crystallization and identifying the relevant governing parameters represents a new paradigm in the rational design of organic crystals.

Summary The broad long-term goal is to optimize critical artemisinin and quinoline malaria drug combinations for maximum killing of the P. falciparum parasite by defeating resistance. In the setting of ongoing drug resistance to currently deployed drugs, this work will quantify a novel quinoline-like mechanism of action for the heme-artemisinin adducts, define reversible or irreversible heme crystal inhibition correlated to level of drug resistance, and explore optimum heme crystal inhibition related to parasite killing with quinoline-artemisinin combinations. Preliminary data validate an additional mechanism of action for the artemisinins based on formation of abundant heme-artemisinin adduct, which inhibits heme crystallization with irreversible action. Exogenous heme-artemisinin adducts inhibit artemisinin ring-resistant mutant Kelch13 P. falciparum parasites with low nM IC50s. The experimental approach employs the synergy between experimental investigations with P. falciparum drug-sensitive and resistant parasites in vitro and physicochemical insights obtained by time-resolved in situ observations of crystal growth by atomic force microscopy in the presence of different drug combinations. The hypothesis is that the inhibition of heme crystal formation by the heme adduct of dihydroartemisinin (DHA, the product of most artemisinin-class drugs in vivo) renders trophozoites of any Plasmodium isolate sensitive, which defeats the artemisinin ring-stage resistance. We also hypothesize that certain combinations of quinolines and heme-dihydroartemisinin adduct (H-DHA) are superior in killing of parasites correlated to heme crystal inhibition as well as separately to the degree of reversible/irreversible heme crystal inhibition. Towards these objectives, we will pursue three specific aims: Aim 1. Establish the inhibition concentrations and mechanism of action of exogenous as well as bio-activated H-DHA in drug sensitive and resistant Plasmodium. Aim 2. Establish reversibility or irreversibility of H-DHA heme crystal inhibition in vivo and in vitro. Aim 3. Establish if double combinations of antimalarial quinolines and H-DHA adducts enhance, weaken, or are indifferent to their partner?s action on parasite killing and the rate of hematin crystallization. This proposed research will quantify the amount of parasite killing by artemisinin adduct metabolites which renders trophozoite stages sensitive to the artemisinin drug class. The work will also inform fundamental knowledge regarding mechanisms to defeat artemisinin resistance, degree of reversibility of hematin crystal growth, and optimum combinations of malaria drugs based upon interaction effects.

NON-TECHNICAL SUMMARY
Solution-grown single crystals serve as semiconductor, optoelectronic, and photovoltaic devices and detectors for high-energy radiation. These studies, supported by the Solid State and Materials Chemistry Program in the Division of Materials Research, fill a gap in understanding crystallization of organic materials that carry promising optical and electronic properties for use as semiconductors, solar cells, and field-effect transistors. Additionally, the research can provide valuable information about crystallization processes, which are essential for a myriad of industrial, natural, and physiological processes. Researchers at the University of Houston take on the grand fundamental science challenge to control crystallization by designing robust control strategies that rest on understanding the fundamental thermodynamic and kinetic mechanisms, and in particular the role of foreign compounds. In industry, soluble foreign compounds that interact with the solution or the crystal-solution interface are deployed to promote or inhibit crystallization. Nature achieves remarkable diversity of shapes, patterns, compositions, and functions of the arising crystalline structures by applying ingredients that control the number of formed crystals and their rates of growth. Insights gained from this project advance the science of organic crystallization in general, and the influence of foreign compounds on the synthesis of solid state organic materials in particular. The researchers also involve a diverse cohort of high school, undergraduate, and graduate students in carrying out this research, which provides them with training in advanced science and engineering concepts and methods. This in turn contributes to narrowing the gap between the demand and availability of educated workforce in Houston, which is among the widest in large U.S. cities.

TECHNICAL SUMMARY
As part of this project, which is supported by the Solid State and Materials Chemistry Program in the Division of Materials Research, the PI and this team design novel strategies to control the nucleation and growth of crystals from organic solvents that employ foreign compounds to regulate nonclassical crystallization behaviors and the nucleation and growth precursors. The accepted models of modifier activity presume that crystal nucleation and growth advance along classical pathways. Recent experiments have accumulated significant discrepancies with the classical theories. The highlighted nonclassical features involve mesoscopic crystallization precursors, ordered or disordered, which assemble in the solution independently of crystallization and may both facilitate nucleation and feed a fast mode of crystal growth. How additives impact the properties of the crystallization precursors to enhance or suppress crystal nucleation and growth has not been examined. The researchers bring complementary expertise in molecular thermodynamics and kinetics of crystallization, crystal design and advanced characterization, and molecular simulations to pursue three specific aims: 1. Design strategies to control crystal nucleation by manipulating precursors involved in nonclassical nucleation modes. 2. Elucidate molecular and mesoscopic crystallization mechanisms that persist after removal of the modifier from the growth medium by exploiting the interactions of modifiers with crystal growth precursors and with step bunches on the crystal surface. 3. Characterize interactions between pairs of modifiers mediated by the step structures and dynamics that lead to antagonistic, additive, or synergistic cooperativities between modifiers; these interactions have been disregarded by classical inhibition models. To cover a diverse array of nucleation and crystallization behaviors, the researchers employ organic crystals that carry promising optical and electronic properties for use as semiconductors, solar cells, and field-effect transistors.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.