Research

Amyloid-beta peptides are the main culprit behind the neurodegenerative disorder, Alzheimer's (AD). A series of experimental and theoretical research has been carried out since the past two decades to unravel the mechanistic aspect of the aggregation process and also shed light on the possible mode of inhibition. Currently, I am working with clinically demanding small molecules, and laboratory synthesized nanomaterials to understand the nucleation, on-pathway and off-pathway aggregation and early-stage oliogomeric intermediates.

Mechanism and Inhibition

The amyloid-beta peptides have been treated in the laboratory following the standard protocol, to begin with, monomeric non-aggregated peptides. Peptides were allowed to self-assemble under physiological conditions followed by treatment with molecules under trials. The aggregation process results in the formation of amyloid fibrils in the absence of the inhibitors and converted to unstructured or amorphous aggregates. The aggregation pathway has been investigated using combined time-dependent spectroscopic and microscopic techniques.

Kinetics and Imaging

Structure and function of HspA

A small heat shock protein (HspA) from a thermophilic cyanobacteria T. vulcanus was expressed heterologously in E. coli. HspA was then purified using Ni-affinity column chromatography and size-exclusion chromatography. Purity of the protein was examined using SDS-PAGE and Mass-spectrometry. Purified HspA was then screened for crystallography and crystals were formed in hanging drop and sitting drop method. The x-ray crystallographic analysis reveals that the unit cell of the HspA crystal contains two monomers, space group I4 (1) and resolution 2.3 A. The structure of HspA demonstrates the evidence of the onset of denaturation. Urea mediated denaturation was observed in the x-ray crystal structure where both direct and indirect interactions have been proposed responsible for the commencement of denaturation.

Structure of Phycobilins

The phycobilins that are phycocyanin and allophycocyanin from the cyanobacteria are one of the major pigmented proteins. The phycobilisome (PBS), which is the primary antenna protein complex, is responsible for absorption of light energy and transfer it to the reaction center. The phycocyanin (PC) and allophycocyanin (APC) from T. vulcanus and T. elongatus were purified using anion-exchange chromatography and sucrose-gradient based methods. The purified proteins were screened for crystallographic trials, and crystals were formed majorly in HD conditions. Data collection were carried out using Synchrotron X-ray beamline and in-house X-ray source. This project is still under investigation at Technion (Israel), which I have started during my postdoctoral research.

Misfolding of proteins leads to aberrant aggregation, which finally results in the formation of aggregates, typically known as amyloid fibrils. The in-vitro aggregation of the hen-egg white lysozyme was carried out in the presence of various additives (Cu(II), sugars, polyphenols, crowding agents, nucleic acids). We have identified aggregates of varying morphology over a period of time. The binding of Cu(II) occurs with distinct coordination pattern which leads to no-effect to effective inhibition of amyloid formation of lysozyme. The aggregation of lysozyme creates a novel reducing environment during the aggregation process. Polyphenols inhibit aggregation of lysozyme, where epicatechin gallate was found to be the efficient one. High-molecular weight PEGs also inhibits the amyloid formation of lysozyme, retaining its native character and lysis property. Glycation of lysozyme leads to the formation of non-amyloidal amorphus aggregates, which can poorly bind with ThT. Binding of nucleic acids occurs with lysozyme fibrils in a non-specific manner and at higher concentration of fibrils as compared with the native species. The investigation of lysozyme aggregation with a combined spectroscopic and microscopic approach provides critical insight of amyloid fibrillation pathway, which is beneficial for screening effective inhibitor molecules against AD.

Insight into the Amyloidogenic pathway of Lysozyme

Google Sites

Report abuse