Smita Mukherjee

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.

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.

An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates

The red shift in the fluorescence excitation spectra of thioflavin dyes upon binding to fibrils has been a boon to the amyloid field, offering simple and effective methods for the qualitative detection of amyloid in tissue samples and for quantitation of particular fibril preparations with gravimetric linearity. The quantitative aspect of the thioflavin T (ThT) response, however, comes with an important caveat that bestows both significant limitations and great untapped power. It is now well established that amyloid fibrils of different proteins, as well as polymorphic fibrils of the same protein, can exhibit vastly different ThT fluorescence intensities for the same weight concentration of aggregates. Furthermore, the aggregated intermediates commonly observed in amyloid assembly reactions can exhibit aggregate weight-normalized (AWN) ThT fluorescence intensities that vary from essentially zero through a wide range of intermediate values before reaching the intensity of homogeneous, mature amyloid. These features make it very difficult to quantitatively interpret, without additional data, the time-dependent development of ThT fluorescence intensity in an assembly reaction. In this chapter, we describe a method for coupling ex situ ThT fluorescence determinations with an analytical HPLC supported sedimentation assay (also described in detail) that can provide significant new insights into amyloid assembly reactions. The time dependent aggregation data provided by the sedimentation assay reveals a time course of aggregation that is largely independent of aggregate properties. In addition, the combination of these data with ThT measurements of the same reaction time points reveals important aspects of average aggregate structure at each time point. Examples of the use and potential value of AWN-ThT measurements during amyloid assembly Aβ and polyglutamine peptides are provided.

Surfactant assisted Au nanoparticle layering in titanium oxide thin films

Au Nanoparticle (NP) decorated TiO2 thin films, prepared by a unique surfactant assisted 2D self-assembling technique with molecular level control, showed significant decrease in optical band gap as well as enhanced crystallinity compared to its sol-gel prepared pristine counterpart. Spin coated Au NP overlayers on titania in absence of surfactant, on the other hand, had no appreciable effect on either band gap or crystal structure compared to undoped TiO2 films. Apart from exhibiting band gap tuning of TiO2, this cheap, scalable technique of surfactant aided deposition of 2D layers of Au NPs on semiconducting oxides, may be used for development of multilayered structures with promising light harvesting and unidirectional energy transfer (LUET) applications.