Sudipta Maiti

Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a sensitive and widely used technique for measuring diffusion. FCS data are conventionally modeled with a finite number of diffusing components and fit with a least-square fitting algorithm. This approach is inadequate for analyzing data obtained from highly heterogeneous systems. We introduce a Maximum Entropy Method based fitting routine (MEMFCS) that analyzes FCS data in terms of a quasicontinuous distribution of diffusing components, and also guarantees a maximally wide distribution that is consistent with the data. We verify that for a homogeneous specimen (green fluorescent protein in dilute aqueous solution), both MEMFCS and conventional fitting yield similar results. Further, we incorporate an appropriate goodness of fit criterion in MEMFCS. We show that for errors estimated from a large number of repeated measurements, the reduced chi(2) value in MEMFCS analysis does approach unity. We find that the theoretical prediction for errors in FCS experiments overestimates the actual error, but can be empirically modified to serve as a guide for estimating the goodness of the fit where reliable error estimates are unavailable. Finally, we compare the performance of MEMFCS with that of a conventional fitting routine for analyzing simulated data describing a highly heterogeneous distribution containing 41 diffusing species. Both methods fit the data well. However, the conventional fit fails to reproduce the essential features of the input distribution, whereas MEMFCS yields a distribution close to the actual input.

Nature of the Amyloid-β Monomer and the Monomer-Oligomer Equilibrium

The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-β (Aβ) peptide. However, the monomeric state of this aggregation-prone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ± 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both Aβ40 and Aβ42 remain predominantly monomeric up to 3 μm, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If Aβ oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers.

Measuring diffusion in cell membranes by fluorescence correlation spectroscopy

Fluorescence Correlation Spectroscopy (FCS) can measure diffusion on the cell surface with unparalleled sensitivity. In appropriate situations, this can be the most sensitive and accurate method for measuring receptor interaction and oligomerization. Here we attempt to describe FCS in sufficient detail so that the reader is able to judge when there is a compelling reason to choose this technique, understand the basic theory behind it, construct a FCS spectrometer in the laboratory, and analyze the data to obtain a meaningful estimate of the physical parameters.

Significant Structural Differences between Transient Amyloid‐β Oligomers and Less‐Toxic Fibrils in Regions Known To Harbor Familial Alzheimer′s Mutations

Small oligomers of the amyloid β (Aβ) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimers disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solid-state NMR methods. The smallest Aβ40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Aβ mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.

Measurement of the Attachment and Assembly of Small Amyloid-β Oligomers on Live Cell Membranes at Physiological Concentrations Using Single-Molecule Tools

It is thought that the pathological cascade in Alzheimers disease is initiated by the formation of amyloid-β (Aβ) peptide complexes on cell membranes. However, there is considerable debate about the nature of these complexes and the type of solution-phase Aβ aggregates that may contribute to their formation. Also, it is yet to be shown that Aβ attaches strongly to living cell membranes, and that this can happen at low, physiologically relevant Aβ concentrations. Here, we simultaneously measure the aggregate size and fluorescence lifetime of fluorescently labeled Aβ(1-40) on and above the membrane of cultured PC12 cells at near-physiological concentrations. We find that at 350 nM Aβ concentration, large (>>10 nm average hydrodynamic radius) assemblies of codiffusing, membrane-attached Aβ molecules appear on the cell membrane together with a near-monomeric species. When the extracellular concentration is 150 nM, the membrane contains only the smaller species, but with a similar degree of attachment. At both concentrations, the extracellular solution contains only small (∼2.3 nm average hydrodynamic radius) Aβ oligomers or monomers. We conclude that at near-physiological concentrations only the small oligomeric Aβ species are relevant, they are capable of attaching to the cell membrane, and they assemble in situ to form much larger complexes.

Effect of loop length variation on quadruplex-Watson Crick duplex competition

The effect of loop length on quadruplex stability has been studied when the G-rich strand is present along with its complementary C-rich strand, thereby resulting in competition between quadruplex and duplex structures. Using model sequences with loop lengths varying from T to T5, we carried out extensive FRET to discover the influence of loop length on the quadruplex-Watson Crick duplex competition. The binding data show an increase in the binding affinity of quadruplexes towards their complementary strands upon increasing the loop length. Our kinetic data reveal that unfolding of the quadruplex in presence of a complementary strand involves a contribution from a predominant slow and a small population of fast opening conformer. The contribution from the fast opening conformer increases upon increasing the loop length leading to faster duplex formation. FCS data show an increase in the interconversion between the quadruplex conformers in presence of the complementary strand, which shifts the equilibrium towards the fast opening conformer with an increase in loop length. The relative free-energy difference (ΔΔG°) between the duplex and quadruplex indicates that an increase in loop length favors duplex formation and out competes the quadruplex.

Quasihomogeneous nucleation of amyloid beta yields numerical bounds for the critical radius, the surface tension, and the free energy barrier for nucleus formation

Amyloid aggregates are believed to grow through a nucleation mediated pathway, but important aggregation parameters, such as the nucleation radius, the surface tension of the aggregate, and the free energy barrier toward aggregation, have remained difficult to measure. Homogeneous nucleation theory, if applicable, can directly relate these parameters to measurable quantities. We employ fluorescence correlation spectroscopy to measure the particle size distribution in an aggregating solution of Alzheimers amyloid beta molecule (Abeta(1-40)) and analyze the data from a homogeneous nucleation theory perspective. We observe a reproducible saturation concentration and a critical dependence of various aspects of the aggregation process on this saturation concentration, which supports the applicability of the nucleation theory to Abeta aggregation. The measured size distributions show a valley between two peaks ranging from 5 to 50 nm, which defines a boundary for the value of the nucleation radius. By carefully controlling the conditions to inhibit heterogeneous nucleation, we can hold off nucleation in a 25 times supersaturated solution for at least up to 3 h at room temperature. This quasi-homogeneous kinetics implies that at room temperature, the surface energy of the Abeta/water interface is > or =4.8 mJ/m(2), the free energy barrier to nucleation (at 25 times supersaturation) is > or =1.93x10(-19) J, and the number of monomers in the nucleus is > or =29.

Protein aggregation probed by two-photon fluorescence correlation spectroscopy of native tryptophan.

Fluorescence correlation spectroscopy (FCS) has proven to be a powerful tool for the study of a range of biophysical problems including protein aggregation. However, the requirement of fluorescent labeling has been a major drawback of this approach. Here we show that the intrinsic tryptophan fluorescence, excited via a two-photon mechanism, can be effectively used to study the aggregation of tryptophan containing proteins by FCS. This method can also yield the tryptophan fluorescence lifetime in parallel, which provides a complementary parameter to understand the aggregation process. We demonstrate that the formation of soluble aggregates of barstar at pH 3.5 shows clear signatures both in the two-photon tryptophan FCS data and in the tryptophan lifetime analysis. The ability to probe the soluble aggregates of unmodified proteins is significant, given the major role played by this species in amyloid toxicity.

On the Stability of the Soluble Amyloid Aggregates

Many amyloid proteins form metastable soluble aggregates (or protofibrils, or protein nanoparticles, with characteristic sizes from approximately 10 to a few hundred nm). These can coexist with protein monomers and amyloid precipitates. These soluble aggregates are key determinants of the toxicity of these proteins. It is therefore imperative to understand the physical basis underlying their stability. Simple nucleation theory, typically applied to explain the kinetics of amyloid precipitation, fails to predict such intermediate stable states. We examine stable nanoparticles formed by the Alzheimers amyloid-beta peptide (40 and 42 residues), and by the protein barstar. These molecules have different hydrophobicities, and therefore have different short-range attractive interactions between the molecules. We also vary the pH and the ionic strength of the solution to tune the long-range electrostatic repulsion between them. In all the cases, we find that increased long-range repulsion results in smaller stable nanoparticles, whereas increased hydrophobicity produces the opposite result. Our results agree with a charged-colloid type of model for these particles, which asserts that growth-arrested colloid particles can result from a competition between short-range attraction and long-range repulsion. The nanoparticle size varies superlinearly with the ionic strength, possibly indicating a transition from an isotropic to a linear mode of growth. Our results provide a framework for understanding the stability and growth of toxic amyloid nanoparticles, and provide cues for designing effective destabilizing agents.

Three-photon microscopy shows that somatic release can be a quantitatively significant component of serotonergic neurotransmission in the mammalian brain

Recent experiments on monoaminergic neurons have shown that neurotransmission can originate from somatic release. However, little is known about the quantity of monoamine available to be released through this extrasynaptic pathway or about the intracellular dynamics that mediate such release. Using three‐photon microscopy, we directly imaged serotonin autofluorescence and investigated the total serotonin content, release competence, and release kinetics of somatic serotonergic vesicles in the dorsal raphe neurons of the rat. We found that the somata of primary cultured neurons contain a large number of serotonin‐filled vesicles arranged in a perinuclear fashion. A similar distribution is also observed in fresh tissue slice preparations obtained from the rat dorsal raphe. We estimate that the soma of a cultured neuron on an average contains about 9 fmoles of serotonin in about 450 vesicles (or vesicle clusters) of ≤370 nm average diameter. A substantial fraction (>30%) of this serotonin is released with a time scale of several minutes by K+‐induced depolarization or by para‐chloroamphetamine treatment. The amount of releasable serotonin stored in the somatic vesicles is comparable to the total serotonin content of all the synaptic vesicles in a raphe neuron, indicating that somatic release can potentially play a major role in serotonergic neurotransmission in the mammalian brain.