Pramit Chowdhury

Cholesterol-dependent phase separation in cell-derived giant plasma membrane vesicles

Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. The results of the present study extend previous observations in purified lipid systems to the complex environment of the plasma membrane and provide insight into the effect of cholesterol on lipid phase separation and abundance.

Effect of Macromolecular Crowding on Protein Folding Dynamics at the Secondary Structure Level

Macromolecular crowding is one of the key characteristics of the cellular environment and is therefore intimately coupled to the process of protein folding in vivo. While previous studies have provided invaluable insight into the effect of crowding on the stability and folding rate of protein tertiary structures, very little is known about how crowding affects protein folding dynamics at the secondary structure level. In this study, we examined the thermal stability and folding-unfolding kinetics of three small folding motifs (i.e., a 34-residue alpha-helix, a 34-residue cross-linked helix-turn-helix, and a 16-residue beta-hairpin) in the presence of two commonly used crowding agents, Dextran 70 (200 g/L) and Ficoll 70 (200 g/L). We found that these polymers do not induce any appreciable changes in the folding kinetics of the two helical peptides, which is somewhat surprising as the helix-coil transition kinetics have been shown to depend on viscosity. Also to our surprise and in contrast to what has been observed for larger proteins, we found that crowding leads to an appreciable decrease in the folding rate of the shortest beta-hairpin peptide, indicating that besides the excluded volume effect, other factors also need to be considered when evaluating the net effect of crowding on protein folding kinetics. A model considering both the static and the dynamic effects arising from the presence of the crowding agent is proposed to rationalize these results.

Effect of dehydration on the aggregation kinetics of two amyloid peptides.

It is well-known that water plays a crucial role in the folding, dynamics, and function of proteins. Here we provide further evidence showing that the aggregation kinetics of peptides also depend strongly on their hydration status. Using reverse micelles as a tool to modulate the accessible number of water molecules and infrared spectroscopy and transmission electron microscopy as means to monitor aggregate formation, we show that the rate of aggregation of two amyloid forming peptides increases significantly under conditions where limited hydration of the peptide molecule is expected to occur. These results not only are in accord with recent computer simulations indicating that the expulsion of interfacial water molecules is a key event in the dimerization/oligmerization of amyloid beta (Abeta) peptides but also have implications for amyloid formation in vivo where molecular crowding is expected to influence the solvation status of proteins.

Using an Amino Acid Fluorescence Resonance Energy Transfer Pair To Probe Protein Unfolding: Application to the Villin Headpiece Subdomain and the LysM Domain

Previously, we have shown that p-cyanophenylalanine (Phe CN) and tryptophan (Trp) constitute an efficient fluorescence resonance energy transfer (FRET) pair that has several advantages over commonly used dye pairs. Here, we aim to examine the general applicability of this FRET pair in protein folding-unfolding studies by applying it to the urea-induced unfolding transitions of two small proteins, the villin headpiece subdomain (HP35) and the lysin motif (LysM) domain. Depending on whether Phe CN is exposed to solvent, we are able to extract either qualitative information about the folding pathway, as demonstrated by HP35, which has been suggested to unfold in a stepwise manner, or quantitative thermodynamic and structural information, as demonstrated by LysM, which has been shown to be an ideal two-state folder. Our results show that the unfolding transition of HP35 reported by FRET occurs at a denaturant concentration lower than that measured by circular dichroism (CD) and that the loop linking helix 2 and helix 3 remains compact in the denatured state, which are consistent with the notion that HP35 unfolds in discrete steps and that its unfolded state contains residual structures. On the other hand, our FRET results on the LysM domain allow us to develop a model for extracting structural and thermodynamic parameters about its unfolding, and we find that our results are in agreement with those obtained by other methods. Given the fact that Phe CN is a non-natural amino acid and, thus, amenable to incorporation into peptides and proteins via existing peptide synthesis and protein expression methods, we believe that the FRET method demonstrated here is widely applicable to protein conformational studies, especially to the study of relatively small proteins.

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F‐helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F‐helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen‐bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function. Proteins 2002;46:268–277.

Folding Kinetics of a Naturally Occurring Helical Peptide: Implication of the Folding Speed Limit of Helical Proteins

The folding mechanism and dynamics of a helical protein may strongly depend on how quickly its constituent alpha-helices can fold independently. Thus, our understanding of the protein folding problem may be greatly enhanced by a systematic survey of the folding rates of individual alpha-helical segments derived from their parent proteins. As a first step, we have studied the relaxation kinetics of the central helix (L9:41-74) of the ribosomal protein L9 from the bacterium Bacillus stearothermophilus , in response to a temperature-jump ( T-jump) using infrared spectroscopy. L9:41-74 has been shown to exhibit unusually high helicity in aqueous solution due to a series of side chain-side chain interactions, most of which are electrostatic in nature, while still remaining monomeric over a wide concentration range. Thus, this peptide represents an excellent model system not only for examining how the folding rate of naturally occurring helices differs from that of the widely studied alanine-based peptides, but also for estimating the folding speed limit of (small) helical proteins. Our results show that the T-jump induced relaxation rate of L9:41-74 is significantly slower than that of alanine-based peptides. For example, at 11 degrees C its relaxation time constant is about 2 micros, roughly seven times slower than that of SPE(5), an alanine-rich peptide of similar chain length. In addition, our results show that the folding rate of a truncated version of L9:41-74 is even slower. Taken together, these results suggest that individual alpha-helical segments in proteins may fold on a time scale that is significantly slower than the folding time of alanine-based peptides. Furthermore, we argue that the relaxation rate of L9:41-74 measured between 8 and 45 degrees C provides a realistic estimate of the ultimate folding rate of (small) helical proteins over this temperature range.

Effect of pH on the Fluorescence and Absorption Spectra of Hypericin in Reverse Micelles

Abstract The well-characterized, monodispersed nature of reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane and their usefulness in approximating a membrane-like environment have been exploited to investigate the effect of pH and water pool size on the photophysical properties of hypericin (Hyp). Our measurements reveal two titratable groups of pKa ∼1.5 and ∼12.5. These are assigned to the HypH+/Hyp equilibrium (the deprotonation of a carbonyl group) and the Hyp−/Hyp2− equilibrium (the deprotonation of a peri hydroxyl group). The low-energy absorbance maxima of HypH+, of Hyp and Hyp− and of Hyp2− are 583, 594 and 613 nm, respectively. Neither at pH 13 nor at 1 M HCl is the system entirely in the Hyp2− or the HypH+ forms. Ours is the first study of Hyp in reverse micelles as well as the first time-resolved study of Hyp as a function of pH.

Do Macromolecular Crowding Agents Exert Only an Excluded Volume Effect? A Protein Solvation Study

The effect of macromolecular crowding on protein structure and dynamics has mostly been explained on the basis of the excluded volume effect, its origin being entropic. In recent times a progressive shift in this view has been taking place with increasing emphasis on soft interactions that are enthalpic by nature. Using very low concentrations (1-10 g/L) of both synthetic (dextran- and poly(ethylene glycol) (PEG)-based) and protein (α-synuclein and myoglobin)-based crowders, we have shown that the solvation of probe molecule ANS (1-anilinonapthalene-8-sulfonate) bound to serum proteins bovine serum albumin (BSA) and human serum albumin (HSA) is significantly modulated in both a protein- and crowder-dependent fashion. Since under such conditions the effect of excluded volume is appreciably low, we propose that our observations are direct evidence of soft interactions between the macromolecular crowding agents used and the serum proteins. Moreover, our data reveal, that since at these low crowder concentrations major perturbations to the protein structure are unlikely to take place while minor perturbations might not be readily visible, protein solvation provides a unique spectral signature for capturing such local dynamics, thereby allowing one to decouple hard-sphere interactions from soft sphere ones. Furthermore, since fast fluctuations are known to play a major role in determining the functional characteristics of proteins and enzymes, our results suggest that such motions are prone to be modulated even when the cellular crowding conditions are quite relaxed. In other words, by the time the excluded volume effects come into the picture in the physiological milieu, modulations of functionally important protein motions that need a relatively lower activation energy have already taken place as a result of the aforementioned enthalpic (soft) interactions.

Generation of Fluorescent Adducts of Malondialdehyde and Amino Acids: Toward an Understanding of Lipofuscin¶

Lipofuscin is a yellow‐brown, highly fluorescent pigment that undergoes an age‐related progressive accumulation in animal cells, mainly in postmitotic cells. It is a heterogeneous, high‐molecular weight material associated with proteins, lipids and nucleic acids. Lipofuscin is implicated in many aspects of human health, including aging, oxidative stress, macular degeneration, lipid peroxidation, atherosclerosis, dementia (Alzheimers Disease) and diseases associated with prions. Although the fluorescent properties of lipofuscin have long been recognized, neither histologists nor chemists have yet isolated the pigments themselves or characterized their optical properties. We have prepared lipofuscinlike species by reacting malondialdehyde (MDA) with cysteine (Cys). MDA: Cys adducts 3:2 and 2:2 are two of those that have been identified among the many that were present in the reaction. Whereas previous attempts to synthesize lipofuscinlike species resulted in compounds that were either nonfluorescent or emitted principally in the blue, the MDA: Cys adducts reported in this study are not only fluorescent but also emit over a broader range.

Understanding Growth Kinetics of Nanorods in Microemulsion: A Combined Fluorescence Correlation Spectroscopy, Dynamic Light Scattering, and Electron Microscopy Study

Even though nanostructures of various shapes and sizes can be controlled by microemulsions, there is substantial difficulty in understanding their growth mechanism. The evolution of nanostructures from the time of mixing of reactants to their final stage is a heterogeneous process involving a variety of intermediates. To obtain a deeper insight into these kinetic steps, we studied the slow growth kinetics (extending over eight days) of iron oxalate nanorods inside the polar core of water-in-oil microemulsion droplets made of cetyltrimethylammonium bromide/1-butanol/isooctane. Fluorescence correlation spectroscopy (FCS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) have been employed to monitor the nanostructure growth at (near) the single-droplet level and in an ensemble. Analyzing FCS data with suitable kinetic model we obtain transient dimer lifetime (28 μs) and the droplet fusion rates (and fusion tendency) on each day as the reaction proceeds. The droplet fusion rate is found to directly control the nanorod growth in microemulsion solution and attains its maximum value (3.55 × 10(4) s(-1)) on day 6, when long nanorods are found in TEM data, implying that more and more reactants are fed into the growing system at this stage. Combining FCS, DLS, and TEM results, we find three distinct periods in the entire growth process: a long nucleation-dominant nanoparticle growth period which forms nanoparticles of critical (average) size of ∼53 nm, followed by a short period where isotropic nanoparticles switch to anisotropic growth to form nanorods, and finally elongation of nanorods and growth (and shrinking) of nanoparticles.