Session T40: Physics of Proteins V: Protein-Protein Interaction, and Protein Aggregation

Early aggregation studies of diabetic amyloid in solution

Islet amyloid polypeptide (IAPP, also known as amylin) is responsible for pancreatic amyloid deposits in type II diabetes. The deposits, as well as intermediates in their assembly, are cytotoxic to pancreatic $\beta $-cells and contribute to the loss of $\beta $-cell mass associated with type II diabetes. To better understand the mechanism and cause of such aggregation, molecular simulations with explicit solvent models were used to compare monomer structure and early aggregation mechanism. Using free-energy maps generated~through~a variety of novel, enhanced sampling free-energy calculation techniques, we have found that, in water, the peptide adopts three major structures. One has a small $\alpha $-helix at the N-terminus and a small $\beta $-hairpin at the other end. The second and the most stable one, is a complete $\beta $-hairpin, and the third is a random coil structure. Transition Path Sampling simulations along with reaction coordinate analysis reveal that the peptide follows a ``zipping mechanism'' in folding from $\alpha $-helical to $\beta $-hairpin state. From studies of the dimerization of monomers in water, we have found that the early aggregation proceeds by conversion of all $\alpha $-helical configurations to $\beta $-hairpins, and by two $\beta $-hairpins coming together to form a parallel $\beta $-sheet. Several aspects of the proposed mechanism have been verified by concerted 2D IR experimental measurements, thereby adding credence to the validity of our predictions.   

Applied Electric Fields and the Aggregation of Highly Charged Proteins

The abnormal aggregation of misfolded proteins is associated with the onset of Alzheimer's disease, along with other neurodegenerative disorders, and there is increasing evidence that prefibrillar clusters, rather than fully-formed amyloid plaques, are primarily responsible. Therefore, weakly invasive methods, such as dynamic light scattering, which can probe the size distribution and structure factor of early nuclei and proto-aggregate clusters, can serve an important role in understanding this process, and may lead to insights regarding future therapeutic interventions. Here we study a highly charged model protein, lysozyme, under the influence of applied AC and DC fields in an effort to evaluate general models of protein aggregation, including the coarse-grained ``patchy protein'' method of visualizing charge heterogeneity. This anisotropy in the interprotein interaction can lead to frustrated crystalline order, resulting in low density phases. Dynamic measurements of the size distribution and structure factor can reveal local ordering, hierarchical clustering, and fractal properties of the aggregates. Early results show that applied fields affect early cluster growth by modulating local protein and counterion concentrations, in addition to their influence on protein alignment.   

Ion Specificity in Protein Aggregation Predicted from Diffusivity Measurements in Stable Protein Solutions

The aggregation of therapeutic proteins in solution represents a major challenge in pharmaceutical development, as the mid- and long-term stability of these proteins is crucial for their efficacy and for compliance with FDA requirements. Monitoring slow aggregation experimentally is notoriously time-consuming, yet often unavoidable, since no theory with predictive power is currently available. In the present work, diffusion and aggregation kinetics of the globular model proteins lysozyme and BSA were studied in sodium-salt solutions of different composition and ionic strength using dynamic light scattering. We find a strong correlation between the concentration dependent protein diffusivity in stable solutions and the kinetics of protein aggregation in unstable solutions of similar composition but higher salt content. Our findings suggest a fast and convenient new way to assess a protein's specific tendency to aggregate in different types of electrolytes and buffer solutions.   

Controlling Protein Oligomerization with Surface Curvature on the Nanoscale

We investigate the effect of surface curvature on the conformation of beta-lactoglobulin ($\beta$LG) using Single Molecule Force Spectroscopy. $\beta$LG is a model interfacial protein which stabilizes oil droplets in milk and is known to undergo structural rearrangement when adsorbed onto a surface. We reliably control nanoscale surface curvature by creating close-packed monolayers of monodisperse polystyrene (PS) nanoparticles with diameters of 20, 40, 60, 80 and 140 nm, which are stable in aqueous buffer. By adsorbing $\beta$LG onto these hydrophobic surfaces and collecting force-extension curves in the fluid phase we can compare the conformation of $\beta$LG on 5 different surface curvatures with that on a flat PS film. We demonstrate a transition from oligomeric to monomeric $\beta$LG as the surface curvature is increased. Histograms of contour length from fits to peaks in the force-extension curves show a single maximum near 30 nm for $\beta$LG adsorbed onto nanoparticles with diameters less than 80 nm. For the larger nanoparticles, the histogram approaches that observed for $\beta$LG adsorbed onto a flat PS film, with maxima indicative of $\beta$LG dimers and trimers.   

Computational Analysis of $\beta $-Peptide Self-Assembly

$\beta -$peptides are a class of synthetic oligomers that are capable of folding in precise patterns. The wide variety of side chains that are available for insertion into $\beta $-peptide sequences along with the stability of these folded secondary structures allow precise control over the nanoscale presentation of various chemical functional groups in three dimensional space. Some $\beta $-peptides have been shown to spontaneously fold into complex supramolecular structures, and others have been shown to be effective antimicrobial agents that are believed to act by aggregating in certain types of cell membranes. However, more work is needed to understand what drives this assembly in order to design $\beta $-peptides that assemble in particular ways. Using molecular simulations, the process of $\beta $-peptide aggregation is examined in a variety of environments that allow for direct comparison to experiment. Using new simulation techniques, the structure of the aggregates formed by several $\beta $-peptides are predicted in both bulk solutions, and at interfaces. Free energy surfaces are generated using multiple geometric parameters to directly compare the favorability of different modes of aggregation. By analyzing these results, we gain an understanding of the factors that drive self-assembly and aggregation.   

Electrospun Synthetic Polypeptide Nanofibrous Biomaterials

Water-insoluble nanofiber mats of synthetic polypeptides of defined composition have been prepared from fibers electrospun from aqueous solution in the absence of organic co-solvents. 20-50 kDa poly(L-glutamate, L-tyrosine) 4:1 (PLGY) but not 15-50 kDa or 50-100 kDa poly(L-glutamate) was spinnable at 20-55{\%} (w/v) polymer in water. Applied voltage and needle-collector distance were crucial for spinnability. Attractive fibers were obtained at 50{\%} polymer. Fiber diameter and mat morphology have been characterized by electron microscopy. Exposure of spun fiber mats to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), which reacts with carboxylate, decreased fiber solubility. Fluorescein-conjugated poly(L-lysine) (FITC-PLL) but not the fluorophore alone was able bind PLGY fiber mats electrostatically, judging by fluorescence microscopy. Key advances of this work are the avoidance of an animal source of peptides and of an inorganic co-solvent to achieve polypeptide spinnability. Polypeptide fiber mats are a promising type of nano-structured biomaterial for applications in biomedicine and biotechnology.   

Exploring the dewetting transition in the hydrophobic collapse of melittin

We present our recent results on understanding the hydrophobic collapse of melittin dimers. Melittin dimers have large, complementary hydrophobic patches, and the dimer collapse mechanism involves a dewetting transition [Liu, Huang, Zhou and Berne, \textsl{Nature} \textbf{437}, 159--162 (2005)]. As a result, melittin has become a model system for studying dewetting transitions in proteins. We apply our recently- developed tools for probing density fluctuations in water [Patel, Varilly and Chandler, \textsl{JPCB} \textbf{114}, 1632--1637 (2010)] to understand this dewetting transition in terms of free energy surfaces, their bistability and their barrier heights. We show how the hydrophobic character of melittin's tetramerization surface results in an enhanced probability of density depletion next to that surface. When two dimers come together, the density depletion is further enhanced, so that even at large separations, there is a metastable dry phase in the region between the dimers. As the dimers come together, the dry phase is stabilized and eventually the wet phase is destabilized, leading to the collapse of the dimers. We explore how mutations that have been observed to suppress the dewetting transition affect the corresponding free energy surfaces and discuss our ongoing efforts to fully map out the reaction coordinate of melittin collapse.   

Effect of the ordered interfacial water layer in protein complex formation: a non-local electrostatic approach

Using a non-local electrostatic approach that incorporates the short-range structure of the contacting media, we evaluated the electrostatic contribution to the energy of the complex formation of two model proteins. In this study, we have demonstrated that the existence of an low-dielectric interfacial water layer at the protein-solvent interface [1] reduces the charging energy of the proteins in the aqueous solvent, and consequently increases the electrostatic contribution to the protein binding (change in free energy upon the complex formation of two proteins). This is in contrast with the finding of the continuum electrostatic model, which suggests that electrostatic interactions are not strong enough to compensate for the unfavorable desolvation effects [2]. \\[4pt] [1] Rubinstein and Sherman, Biophys. J. 87, 1544, 2004 \\[0pt] [2] Rubinstein et al., Phys. Rev. E 82, 021915, 2010).   

Modeling virus capsids and their protein binding -- the search for weak regions within the HIV capsid

Viruses remain a threat to the health of humans worldwide with 33 million infected with HIV. Viruses are ubiquitous, infecting animals, plants, and bacteria. Each virus infects in its own unique manner making the problem seem intractable. However, some general physical steps apply to many viruses and the application of basic physical modeling can potentially have great impact. The aim of this theoretical study is to investigate the stability of the HIV viral capsid (protein shell). The structural shell can be compromised by physical probes such as pulsed laser light [1,2]. But, what are the weakest regions of the capsid so that we can begin to understand vulnerabilities of these deadly materials? The atomic structure of HIV capsids is not precisely known and we begin by describing our work to model the capsid structure. We have constructed three representative viral capsids of different CA protein number -- HIV-900, HIV-1260 and HIV-1740. The complexity of the assembly requires a course grained model to investigate protein interactions within the capsid which we will describe. \\[0pt] [1] K-T. Tsen, WS.-D. Tsen, O.F. Sankey, J.G. Kiang, Journal of Physics -- Condensed Matter, 19 472201 (2007). \\[0pt] [2] E.C. Dykeman, D.Benson, K.-T. Tsen, and O.F. Sankey, Physical Review E 80, 041909 (2009).   

Low-Frequency Raman Spectroscopy of Trialanine.

The effect of sample conditions on the structural conformation of trialanine has been investigated with visible Raman spectroscopy. Trialanine is used here as a simple protein-mimetic system in order to more easily isolate the backbone amide vibrational modes, low-frequency tortional modes and modes from hydrogen bonding. Crystalline trialanine is known to exist with both parallel ($p$-Ala$_{3})$ and antiparallel (\textit{ap}-Ala$_{3})$ \textit{$\beta $}-sheet crystal structures, depending on the solvent composition during crystallization. The \textit{ap}-sheet form of trialanine co-crystallizes with water, which is easily removed under vacuum, offering a further opportunity to examine the effect of solvation on the vibrational spectra, especially when also compared with trialanine in solution. By collecting Raman spectra in different sample phases, and at different concentrations, pH and temperatures, the vibrational modes most sensitive to the secondary structure can be identified. The collected data will be compared to the literature, including other vibrational spectroscopic data and high-level simulations.   

Fast X-ray Photon Correlation Spectroscopy measurements from the diffusion of concentrated Alpha Crystallin suspensions

Alpha Crystallin constitute up to half of the total protein found in the mammalian eye lens. It has chaperone like behavior and may play a key role in maintaining lens transparency by preventing condensation of other lens proteins. We report here Fast X-ray Photon Correlation Spectroscopy (XPCS) measurements of protein diffusion within concentrated suspensions of Alpha Crystallin. Bovine calf eye lens cortices were homogenized, centrifuged and ultra-filtered to obtain concentrated Alpha Crystallin suspensions. Diffusion of proteins within these suspensions was measured as a function of temperature. The overall observed diffusion rates imply that the proteins exist in a glassy or gel phase, even at concentrations where equivalent hard sphere system would still be liquid. We interpret these results within the context of strongly interacting proteins, with protein-protein interactions possibly mediated by subunit exchange among Alpha Crystallin oligormers.   

Ultrasound and Hypersound Speeds in Lysozyme Solutions

Ultrasound velocimetry and Brillouin spectroscopy provide information on the compressibility of proteins and the surrounding hydration layer. Employing both techniques we investigate the sound speeds at GHz (hypersound) and MHz (ultrasound) frequencies in lysozyme solutions (250 mg / ml, pH 7) and pure water over the temperature range from 275 K to 335 K. Compared to water the Brillouin peaks in the lysozyme solutions are shifted by about 400 MHz towards higher frequencies. This shift reflects the change in sound speed and is attributed to the influence of the compressibility of the protein and bound water in the hydration shell. In addition, we measure a dispersion of the sound velocity in the lysozyme solution. The higher sound speed at GHz frequencies, as measured by Brillouin scattering, may indicate additional relaxation processes as compared to pure bulk water, where no sound dispersion between ultrasound speed and hypersound speed is observed.