Mily Bhattacharya

Insights into the Mechanism of Aggregation and Fibril Formation from Bovine Serum Albumin

We have investigated the fibrillation propensity of different conformational isomers of an archetypal, all α-helical protein, namely, bovine serum albumin (BSA), under different pH conditions and ionic strengths using fluorescence and circular dichroism (CD) spectroscopy. At low pH and higher protein concentration, the partially folded conformers associate to form oligomers that are converted into ordered amyloid-like fibrils when incubated at elevated temperature. We have elucidated the mechanism of fibril formation, especially the early steps, by monitoring the kinetics of structural changes during the aggregation process. Various structural probes in tandem were utilized to decipher the temporal evolution of both conformational and size changes by measuring the time dependence of fluorescence intensity and anisotropy of intrinsic tryptophans and several extrinsic fluorophores during the aggregation. Additionally, CD spectroscopy was utilized to monitor the changes in protein secondary structural content during fibrillation. Our findings suggest that the conformational conversion occurs in the oligomers that serve as precursors to amyloid fibrils and precedes the overall fibrillar growth.

pH-induced Conformational Isomerization of Bovine Serum Albumin Studied by Extrinsic and Intrinsic Protein Fluorescence

Serum albumins are multi-domain all α-helical proteins that are present in the circulatory system and aid in the transport of a variety of metabolites, endogenous ligands, drugs etc. Earlier observations have indicated that serum albumins adopt a range of reversible conformational isomers depending on the pH of the solution. Herein, we report the concurrent changes in the protein conformation and size that are inherent to the pH-induced conformational isomers of bovine serum albumin (BSA). We have investigated the fluorescence properties of both intrinsic (tryptophan) and extrinsic (ANS, pyrene) fluorophores to shed light into the structural features of the pH-dependent conformers. We have been able to identify a number of conformational isomers using multiple fluorescence observables as a function of pH titration. Our results indicate that at pH 3, a partially-folded, ‘molten-globule-like’ state is populated. Moreover, equilibrium unfolding studies indicated that the ‘molten-globule-like’ state unfolds in a non-cooperative fashion and is thermodynamically less stable than the native state. The fluorescence-based approach described in the present work has implications in the study of pH-induced conformational plasticity of other physiologically relevant proteins.

Quadratic nonlinearity of one- and two-electron oxidized metalloporphyrins and their switching in solution.

We report the quadratic nonlinearity of one- and two-electron oxidation products of the first series of transition metal complexes of meso-tetraphenylporphyrin (TPP). Among many MTPP complexes, only CuTPP and ZnTPP show reversible oxidation/reduction cycles as seen from cyclic voltammetry experiments. While centrosymmetric neutral metalloporphyrins have zero first hyperpolarizability, beta, as expected, the cation radicals and dications of CuTPP and ZnTPP have very high beta values. The one- and two-electron oxidation of the MTPPs leads to symmetry-breaking of the metal-porphyrin core, resulting in a large beta value that is perhaps aided in part by contributions from the two-photon resonance enhancement. The calculated static first hyperpolarizabilities, beta0, which are evaluated in the framework of density functional theory by a coupled perturbed Hartree-Fock method, support the experimental trend. The switching of optical nonlinearity has been achieved between the neutral and the one-electron oxidation products but not between the one- and the two-electron oxidation products since dications that are electrochemically reversible are unstable due to the formation of stable isoporphyrins in the presence of nucleophiles such as halides.

Structural and dynamical insights into the molten-globule form of ovalbumin.

Ovalbumin is a 45 kDa egg-white glycoprotein which belongs to the class of serpin superfamily. We have studied the structural properties of both native and partially unfolded molten-globule forms of ovalbumin using a diverse array of spectroscopic tools. Time-resolved fluorescence measurements provided important structural and dynamical insights into the native and molten-globule states. Fluorescence anisotropy decay analysis indicated that there is a conformational swelling from the native to the molten-globule form of ovalbumin. We have also carried out red-edge excitation shift measurements to probe the dipolar relaxation dynamics around the intrinsic tryptophan residues. Additionally, stopped-flow fluorescence experiments revealed that the conformational transition from the native to the molten-globule form proceeds in a stepwise manner involving a burst-phase with a submillisecond conformational change followed by biphasic slower conformational reorganizations on the millisecond time scale leading to the final molten-globule state.

Kinetics of surfactant-induced aggregation of lysozyme studied by fluorescence spectroscopy.

The study of protein conformational changes in the presence of surfactants and lipids is important in the context of protein folding and misfolding. In the present study, we have investigated the mechanism of the protein conformational change coupled with aggregation leading to size growth of Hen Egg White Lysozyme (HEWL) in the presence of an anionic detergent such as sodium dodecyl sulphate (SDS) in alkaline pH. We have utilized intrinsic protein fluorescence (tryptophan) and extrinsic fluorescent reporters such as 8-anilinonaphthalene-1-sulfonic acid (ANS), dansyl and fluorescein to follow the protein conformational change in real-time. By analyzing the kinetics of fluorescence intensity and anisotropy of multiple fluorescent reporters, we have been able to delineate the mechanism of surfactant-induced aggregation of lysozyme. The kinetic parameters reveal that aggregation proceeds with an initial fast-phase (conformational change) followed by a slow-phase (self-assembly). Our results indicate that SDS, below critical micelle concentration, induces conformational expansion that triggers the aggregation process at a micromolar protein concentration range.

Conformational Switching and Nanoscale Assembly of Human Prion Protein into Polymorphic Amyloids via Structurally Labile Oligomers.

Conformational switching of the prion protein (PrP) from an α-helical normal cellular form (PrP(C)) to an aggregation-prone and self-propagating β-rich scrapie form (PrP(Sc)) underlies the molecular basis of pathogenesis in prion diseases. Anionic lipids play a critical role in the misfolding and conformational conversion of the membrane-anchored PrP into the amyloidogenic pathological form. In this work, we have used a diverse array of techniques to interrogate the early intermediates during amyloid formation from recombinant human PrP in the presence of a membrane mimetic anionic detergent such as sodium dodecyl sulfate. We have been able to detect and characterize two distinct types of interconvertible oligomers. Our results demonstrate that highly ordered large β-oligomers represent benign off-pathway intermediates that lack the ability to mature into amyloid fibrils. On the contrary, structurally labile small oligomers are capable of switching to an ordered amyloid-state that exhibits profound toxicity to mammalian cells. Our fluorescence resonance energy transfer measurements revealed that the partially disordered PrP serves as precursors to small amyloid-competent oligomers. These on-pathway oligomers are eventually sequestered into higher order supramolecular assemblies that conformationally mature into polymorphic amyloids possessing varied nanoscale morphology as evident by the atomic force microscopy imaging. The nanoscale diversity of fibril architecture is attributed to the heterogeneous ensemble of early obligatory oligomers and offers a plausible explanation for the existence of multiple prion strains in vivo.

Nanoscopic Amyloid Pores Formed via Stepwise Protein Assembly

Protein aggregation leading to various nanoscale assemblies is under scrutiny due to its implications in a broad range of human diseases. In the present study, we have used ovalbumin, a model non-inhibitory serpin, to elucidate the molecular events involved in amyloid assembly using a diverse array of spectroscopic and imaging tools such as fluorescence, laser Raman, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images revealed a progressive morphological transition from spherical oligomers to nanoscopic annular pores that further served as templates for higher-order supramolecular assembly into larger amyloid pores. Raman spectroscopic investigations illuminated in-depth molecular details into the secondary structural changes of the protein during amyloid assembly and pore formation. Additionally, Raman measurements indicated the presence of antiparallel β-sheets in the amyloid core. Overall, our studies revealed that the protein conformational switch in the context of the oligomers triggers the hierarchical assembly into nanoscopic amyloid pores. Our results will have broad implications in the structural characterization of amyloid pores derived from a variety of disease-related proteins.

Nanoscale Fluorescence Imaging of Single Amyloid Fibrils.

Amyloid formation is implicated in a variety of human diseases. It is important to perform high-resolution optical imaging of individual amyloid fibrils to delineate the structural basis of supramolecular protein assembly. However, amyloid fibrils do not lend themselves to the conventional microscopic resolution, which is hindered by the diffraction limit. Here we show super-resolution fluorescence imaging of fluorescently stained amyloid fibrils derived from disease-associated human β2-microglobulin using near-field scanning fluorescence microscopy. Using this technique, we were able to resolve the fibrils that were spatially separated by ∼75 nm. We have also been able to interrogate individual fibrils in a fibril-by-fibril manner by simultaneously monitoring both nanoscale topography and fluorescence brightness along the length of the fibrils. This method holds promise to detect conformational distributions and heterogeneity that are believed to correlate with the supramolecular packing of misfolded proteins within the fibrils in a diverse conformationally enciphered prion strains and amyloid polymorphs.

pH-Responsive Mechanistic Switch Regulates the Formation of Dendritic and Fibrillar Nanostructures of a Functional Amyloid

In contrast to pathological amyloids, functional amyloids are involved in crucial physiological functions. For instance, the melanosomal protein comprising a highly amyloidogenic polypeptide repeat domain assembles into amyloid fibrils that act as templates for melanin biosynthesis within acidic melanosomes. However, the mechanism-morphology-function relationship of functional amyloids is poorly understood. Here, we demonstrate that the repeat domain of the melanosomal protein exhibits two distinct types of aggregation pathways that display nanoscale polymorphism in acidic pH. In the pH range of 4.5-6, aggregation proceeds via a typical nucleation-dependent mechanism, resulting in the formation of highly ordered β-rich curvy thread-like fibrils. On the contrary, at pH < 4.5, aggregation occurs through a rapid nucleation-independent isodesmic polymerization process that yields dendritic aggregates having lower degree of internal packing. These dendritic nanostructures can be converted into more stable fibrils by switching the pH. The nanoscale polymorphism associated with the mechanistic switch is likely to be mediated by the altered conformational propensities and intermolecular interactions due to the protonation/deprotonation of critical glutamate residues. We propose that this striking shift in the mechanism that dictates the nanoscale morphology regulates the melanosomal maturation.

Studying Protein Misfolding and Aggregation by Fluorescence Spectroscopy

Protein misfolding leading to aggregation and amyloid fibril formation has been implicated in a variety of neurodegenerative disorders. Under suitably designed in vitro conditions, intermolecular contacts between polypeptide chains mediated by various non-covalent interactions result in the formation of oligomeric species that are eventually sequestered into β-sheet-rich amyloid fibrils. Owing to the inherent heterogeneity and complexity of protein aggregation processes, detection and structural characterization of the early, transiently-populated cytotoxic oligomeric intermediates during the amyloid fibrillation cascade still poses a formidable challenge. Fluorescence spectroscopy is an extremely sensitive multiparametric technique that provides simultaneous information about the conformational- and size changes for both early oligomeric species as well as for the large-sized aggregates. In this review, we emphasize recent and selected examples on the application of various fluorescence spectroscopic techniques in the study of protein aggregation. Additionally, we also summarize the recent results on protein aggregation studies using fluorescence spectroscopy from our laboratory.