Barun Kumar Maity

Major Reaction Coordinates Linking Transient Amyloid-β Oligomers to Fibrils Measured at Atomic Level

The structural underpinnings for the higher toxicity of the oligomeric intermediates of amyloidogenic peptides, compared to the mature fibrils, remain unknown at present. The transient nature and heterogeneity of the oligomers make it difficult to follow their structure. Here, using vibrational and solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, we show that freely aggregating Aβ40 oligomers in physiological solutions have an intramolecular antiparallel configuration that is distinct from the intermolecular parallel β-sheet structure observed in mature fibrils. The intramolecular hydrogen-bonding network flips nearly 90°, and the two β-strands of each monomeric unit move apart, to give rise to the well-known intermolecular in-register parallel β-sheet structure in the mature fibrils. Solid-state nuclear magnetic resonance distance measurements capture the interstrand separation within monomer units during the transition from the oligomer to the fibril form. We further find that the D23-K28 salt-bridge, a major feature of the Aβ40 fibrils and a focal point of mutations linked to early onset Alzheimers disease, is not detectable in the small oligomers. Molecular dynamics simulations capture the correlation between changes in the D23-K28 distance and the flipping of the monomer secondary structure between antiparallel and parallel β-sheet architectures. Overall, we propose interstrand separation and salt-bridge formation as key reaction coordinates describing the structural transition of the small Aβ40 oligomers to fibrils.

Label-Free Ratiometric Imaging of Serotonin in Live Cells

Ratiometric imaging can quantitatively measure changes in cellular analyte concentrations using specially designed fluorescent labels. We describe a label-free ratiometric imaging technique for direct detection of changes in intravesicular serotonin concentration in live cells. At higher concentrations, serotonin forms transient oligomers whose ultraviolet emission is shifted to longer wavelengths. We access the ultraviolet/blue emission using relatively benign three-photon excitation and split it into two imaging channels, whose ratio reports the concentration. The technique is sensitive at a physiologically relevant concentration range (10-150 mM serotonin). As a proof of principle, we measure the increase of intravesicular serotonin concentration with the addition of external serotonin. In general, since emission spectra of molecules are often sensitive to concentration, our method may be applicable to other natively fluorescent intracellular molecules which are present at high concentrations.

Fluorogenic Detection of Monoamine Neurotransmitters in Live Cells

Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g., serotonin, dopamine, and norepinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λex = 401 nm and λem = 490 nm for serotonin; λex = 446 nm and λem = 557 nm for dopamine; and λex = 446 nm and λem = 544 nm for norepinephrine, respectively) are accessible to most modern confocal imaging instruments. The identity of monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation microscopy. Mass spectrometry of cell extracts and of in vitro solutions helps us identify the chemical nature of the adducts and establishes the reaction mechanisms. Our method has low toxicity, high selectivity, and the ability to directly report the location and concentration of monoamines in live cells.

Amyloid Aggregation of Amylin: Gain of Function along Aggregation Pathway?

Abstract: Aggregation of human amylin, a 37 amino acid residue neuropeptide of pancreatic origin, into amyloid aggregates is implicated in the etiology of diabetes mellitus type II. Despite its clinical significance, details of progression of nontoxic amylin monomers into cytotoxic oligomers are not very clear. Studies based on Amyloid-β (Aβ), a peptide with similar size, have suggested that a conformational transition along the aggregation pathway makes the oligomers of Aβ membrane-binding competent (1). We have explored the possibility of amylin undergoing a similar transition during aggregation as it could shed light on potential commonalities in the conformation and function of different toxic amyloid species. Using fluorescence correlation spectroscopy, we identify two distinct oligomers of amylin along the aggregation pathway having hydrodynamic radius of 0.90 nm and 1.6 nm respectively. The membrane affinity of amylin increases remarkably from ∼15 % in smaller species to ∼ 85% in the larger one as assessed by an in vitro membrane-binding assay developed in our lab (2). We observe similar difference in the cell membrane attachment ability of these two species in RIN5mf cell lines using confocal microscopy. A preliminary conformational study in artificial lipid bilayers using a SERS based methodology (3) suggests a temporal conformational reorganization in the peptide backbone. Our data suggest that amylin might acquire toxic function by a mechanism which depends on similar conformational features as Aβ in presence of membranes. Further studies aimed at obtaining high-resolution structural details of amylin oligomers in solution and in membranes are currently in progress.References:1. Nag S, et al. (2013) Phys. Chem. Chem. Phys. 15:19129-33.2. Bhowmik D, et al. (2015) Langmuir. 31(14):4049-53.3. Bhowmik D, et al. (2015) ACS Nano. 9(9):9070-7.