Protein Clusters

Protein misfolding followed by aggregation is the major cause of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, familial amyloid poly neuropathy (FAP), Huntington’s, type-II diabetes, etc. Common aspect of all protein aggregation diseases is the altered protein conformation known as partially unfolded amyloidogenic intermediate that is capable of assembly into amyloid structures. Recently discovered mesoscopic protein-rich clusters may act as crucial precursors for the nucleation of ordered protein solids, such as crystals, sickle hemoglobin polymers, and amyloid fibrils. These clusters challenge settled paradigms of protein condensation as the constituent protein molecules present features characteristic of both partially misfolded and native proteins. Some of their unusual features include the kinetically determined size, thermodynamically controlled number, and their distinct nature from aggregation triggered by reduction of the intramolecular S−S bonds and amyloid aggregates.      We investigated the role of protein structural flexibility on its ability to induce formation of mesoscopic clusters for multiple proteins including the p53, known as guardian of genome, which contains multi dis-ordered and b-sheet rich domains; hemoglobin A, which is the major component of red blood cells and contains a compact structure rich in a-helices; antimicrobial enzyme lysozyme which is a robust model in study of protein aggregation. Whereas lysozyme and hemoglobin A demonstrate mesoscopic clusters at high protein concentrations, p53, whose aggregation is tied to cancer development, exhibits clustering at physiological temperatures for low concentrations of the protein. These findings suggest that the clusters are a product of limited protein structural flexibility. Furthermore, we discovered that the crowding environment of the inside cell significantly promotes clustering of intrinsic disordered proteins (IDPs) such as p53. About half of human cancers are associated with mutations of the tumor suppressor p53. Mutated p53 emerges as a powerful oncogene, which blocks the activity of wild-type p53 and several distinct anticancer pathways. The gained functions of the mutant have been related to the aggregation behaviors of wild-type and mutant p53. Our data reveals that in presence of crowders, the p53 clusters can capture some of the crowder molecules, which causes steric hindrance effects and raises the nucleation barrier of the aggregation. Thus these clusters can potentially act as storage of proteins and protect them from formation of toxic amyloid aggregates by providing sufficient time for the proteomic and chaperonin machinery to clear out or refold the misfolded aggregated species in the cell. The nucleation of p53 fibrils deviates from the accepted mechanism of sequential association of single solute molecule. We find the mesoscopic clusters serve as a pre-assembled precursor of high p53 concentration that facilitate fibril assembly. Fibril nucleation hosted by precursors represents a novel biological pathway, which awards unexplored avenues to suppression of protein fibrillation in aggregation diseases.

Pathological Crystallization

We established the mechanisms of action in blocking hematin crystallization of several classes of antimalarial drugs and related compounds. The results of my colleagues on the mechanism of hematin crystallization, concurrent with studies of parasite physiology, suggest that hemozoin crystals form in organic sub-phases comprised of a blend of several lipids, which may be suspended in the digestive vacuole or lining its walls. They have demonstrated that the formation of hematin crystals follows a classical mechanism whereby new crystal layers are nucleated on top of existing ones and grow by the association of solute molecules that leads to the spreading of the layers until they cover the entire face. They have shown that quinoline-class antimalarials possess an extremely efficient pathway of inhibition of hematin crystallization by binding to specific crystal surface sites and impeding the incorporation of soluble hematin.

 

Polymorphism in Organic Crystallization

Crystals are the most ordered state of matter. The processes by which atoms, molecules, or particles
assemble into translationally symmetric arrays have been subjects of investigation for centuries.The slow crystal nucleation rates predicted by classical theory diverge from experiments revealing a significant knowledge gap that poses challenges for the control of crystallization. Polymorphism is a fundamental property of crystals whereby a single compound may adopt numerous crystal structures. This structure multiplicity leads to materials with distinct properties (e.g., solubility, catalytic activity) that impact their performance in numerous applications. Structure multiplicity may also bring high reformulation costs to a manufacturing process, such as the first HIV/AIDS medication, ritonavir, where a metastable polymorph reverted to a more stable and less soluble form during the manufacturing process, reducing the bioavailability of the formulated dosage. Metastable forms have higher solubility and may exhibit other desired properties, such as higher compressibility. About 75% of the compounds deposited in the Cambridge Structural Database (CSD) crystallize in more than one structure. Crystal structure polymorphism manifests a FEL, which, in contrast to that for protein folding has not been optimized by evolution and contains not one minimum, but multiple local minima with similar free energies. Crystal structure multiplicity is magnified by the propensity of crystals to incorporate solvent molecules, creating solvates. We combine molecular modeling and analytical theory with thermodynamic (enthalpies, entropies, and free energies of formation) and kinetic (nucleation rates and crystallization precursor properties) measurements of polymorphs and solvates to develop (i) fundamental insights into the number of polymorphs of a compound, (ii) their structural stability, and (iii) the sequence in which polymorphs appear. We will design and validate tools based on molecular templates to direct
the nucleation of a desired polymorph or suppress the formation of undesired crystal forms.

Molecular mechanisms of fibrillization and toxicity of amyloid β

Alzheimer’s patients accumulate fibrils and plaques of the protein fragment amyloid-b (Ab) outside neurons in the brain. Recent clinical evidence strongly supports the amyloid hypothesis, according to which Ab aggregates impair the neurons and promote cognitive decline. Our main hypothesis is that Ab fibrils—even if minimally neurotoxic in themselves—represent a crucial element in the generation, through fragmentation and templating (also called secondary nucleation), of Ab soluble oligomers, larger aggregates, or fibril fragments, all of which may be neurotoxic. The assembly of Ab peptides into fibrils is exceedingly complex as it combines the association of peptide chains from the solution with their simultaneous restructuring into a conformation typical of the fibril bulk. How the folding and binding of incoming peptide occurs remains largely unknown yet this step offers new opportunities for the rational design of AD treatments. Aimed at inhibiting amyloidogenesis itself, elucidating the events at the tips of fibrils of different structures (polymorphs) on approach of wild-type and mutant peptides and in the presence of potential fibrillization inhibitors will provide fundamental understanding of fibrillogenesis and suggest strategies to suppress fibrillization. The main novelty of our approach is the synergistic combination of state-of-the-art molecular dynamics simulations with state-of-the-art direct time-resolved imaging using in situ atomic force microscopy (AFM) to examine the mechanisms of Aβ fibril growth by monitoring growth of individual fibrils. The correlations between the measured growth rates, the peptide concentration in the solution, and the effects of foreign molecules will directly illuminate the mechanism of peptide incorporation and yield kinetic and structural parameters that can be quantitatively compared to the computer simulations. We will further characterize the fibril structures by transmission electron microscopy (TEM) and establish how they affect the measured and computer-simulated molecular motions. This three-pronged strategy focuses on the crucial events at the fibril tip, avoiding thereby the confounding processes, such a primary and secondary nucleation, that are involved in bulk aggregation studies. The team of investigators, which has expertise in modeling the dynamics of complex biological structures, in the thermodynamics and kinetics of protein aggregation, and in the structural characterization of protein assemblies, will pursue three specific aims. Aim 1. Establish how fibril structure and conformation as well as peptide sequence impact the dynamics of an incoming peptide at the fibril tip. Aim 2. Identify neurotoxic Ab aggregates, fibrils, self-assembled oligomers, aggregates templated on fibrils, fibril fragments, and others, and elucidate how they form. Aim 3. Establish novel pathways to suppress or otherwise modify fibril growth. We will dissect the conformational motions that enable peptide chain incorporation at the fibril tips and thus determine their growth kinetics, elucidate how deleterious and protective Ab mutations, the bulk fibril structure, and the binding of foreign molecules modulate the events at the tip and the rate of fibril growth, thereby providing novel molecular targets to control fibrillization.