Session H40: Focus Session: Protein Association II: Aggregation and Fibril Formation

The Physics of Amyloid Aggregation and Templating in Prions

The problem of self-assembled amyloid aggregation of proteins in structures with beta-strands perpendicular to a one dimensional grown axis is interesting at a fundamental level (is this the most generic end state of proteins?), from a biological level (if the self-assembly can be regulated it is of use in contexts like spider silk and bacterial colony formation), for human public health (aggregation unregulated induces diseases like mad cow and Alzheimer's), and for possible materials applications (e.g., in tissue scaffolding). In this presentation, I will review the work of my group in examining the possibility that the left-handed beta helix (LHBH) structure can be the building block of the aggregates of mammalian prion and yeast prion proteins. I will also discuss our efforts to assess the possibility of a novel pH driven structural switch between LHBH and alpha-helical forms in the ordered half of the mammalian prion protein, and now the possibly pH stabilized LHBH structure can template aggregate growth of the disordered half of the protein, identified in numerous experimental studies as most relevant to disease.   

Early-Aggregation Studies of Polyglutamine in Solution

Several neurodegenerative diseases, notably Huntington's disease, are associated with certain proteins containing extended polyglutamine tracts. In all polyglutamine diseases, the age of onset is inversely correlated with the length of the polyglutamine domain beyond some pathological threshold. Diseased cells are characterized by intranuclear inclusions rich in aggregated polyglutamine. Experimental evidence suggests that oligomeric aggregate species, not mature amyloid fibrils, are the species most toxic to the cell. Little is known about the structures and aggregation dynamics of polyglutamine oligomers due to their short lifetimes. A better understanding of the pathway through which polyglutamine peptides form oligomeric aggregates will aid the design of therapies to inhibit their toxic activity. In this work, we report structural characterization of polyglutamine monomers and dimers from atomistic molecular dynamics simulations in explicit water. Umbrella sampling simulations reveal that the stability of the dimer species with respect to the disassociated monomers is an increasing function of the chain length.   

Search for Length Dependent Stable Structures of Polyglutamaine Proteins with Replica Exchange Molecular Dynamic

Motivated by the need to find beta-structure aggregation nuclei for the polyQ diseases such as Huntington's, we have undertaken a search for length dependent structure in model polyglutamine proteins. We use the Onufriev-Bashford-Case (OBC) generalized Born implicit solvent GPU based AMBER11 molecular dynamics with the parm96 force field coupled with a replica exchange method to characterize monomeric strands of polyglutamine as a function of chain length and temperature. This force field and solvation method has been shown among other methods to accurately reproduce folded metastability in certain small peptides, and to yield accurately de novo folded structures in a millisecond time-scale protein. Using GPU molecular dynamics we can sample out into the microsecond range. Additionally, explicit solvent runs will be used to verify results from the implicit solvent runs. We will assess order using measures of secondary structure and hydrogen bond content.   

Lysozyme Aggregation and Fibrillation Monitored by Dynamic Light Scattering

The aggregation of amyloidogenic proteins provides a rich phase space with significant biomedical implications, including a link with several age-related diseases. We employed dynamic light scattering to monitor the aggregation of lysozyme, a model protein, from a monomeric state until the formation of micron-sized fibrils. For an aqueous lysozyme solution buffered at pH 2, the auto-correlation function of the scattered light intensity was found to be well-fit by a single exponential function with decay time $\tau $ = 1/(2Dq$^{2})$ = 0.25 ms, which corresponds to a mean hydrodynamic radius (R$_{H})$ of 2.2 nm, very likely generated by monomers. Ethanol (4{\%} v/v final concentration) induced a partial unfolding, to R$_{H}$ = 4.6 nm. The subsequent addition of 70 mM KCl was found to shrink the size back to R$_{H}$ = 2.5 nm, as expected when a denatured protein refolds due to partial screening of the intramolecular repulsion. However, further aggregation was not observed. At pH 4, using a low-salt acetate buffer, more ethanol (10{\%} v/v) was required to initiate unfolding, but once it occurred, larger aggregates formed. These results are consistent with the model that partial unfolding, which exposes beta-motif secondary structure, is a prerequisite for aggregation and fibrillation, but the aggregation fate depends on the protein charge state (pH) and screening (salt concentration).   

Primary nucleation and linear aggregation: novel methods take us further

Understanding linear aggregation has long been of interest in the study of proteins, as it is relevant in several human diseases such Alzheimer's Disease. While much work has been carried out experimentally and numerically, relatively little has been done to develop an analytical model for the time evolution and scaling behaviour of such systems. Here, we present a novel mathematical framework, guided by physical insight, for approaching these problems. We then apply this to derive an exact expression for the evolution of the polymer length distribution of an amyloid system, driven by primary nucleation, taking into account both elongation and depolymerisation. This is presented in terms of a perturbative expansion, and is valid for negligible monomer depletion. We then apply a similar framework to solve a similar system that also includes an arbitrary number of conformational intermediates before reaching the final conformation - we uncover the time evolution of the total mass of polymers in the final conformational state, and demonstrate various properties about its time-scaling behaviour. We then apply self-consistent iterative schemes, starting from these solutions, to derive a series of approximate analytical models for these systems, taking monomer depletion into account.   

The Mechanisms of Aberrant Protein Aggregation

We discuss the development of a kinetic theory for understanding the aberrant loss of solubility of proteins. The failure to maintain protein solubility results often in the assembly of organized linear structures, commonly known as amyloid fibrils, the formation of which is associated with over 50 clinical disorders including Alzheimer's and Parkinson's diseases. A true microscopic understanding of the mechanisms that drive these aggregation processes has proved difficult to achieve. To address this challenge, we apply the methodologies of chemical kinetics to the biomolecular self-assembly pathways related to protein aggregation. We discuss the relevant master equation and analytical approaches to studying it. In particular, we derive the underlying rate laws in closed-form using a self-consistent solution scheme; the solutions that we obtain reveal scaling behaviors that are very generally present in systems of growing linear aggregates, and, moreover, provide a general route through which to relate experimental measurements to mechanistic information. We conclude by outlining a study of the aggregation of the Alzheimer's amyloid-beta peptide. The study identifies the dominant microscopic mechanism of aggregation and reveals previously unidentified therapeutic strategies.   

Amyloid growth: combining experiment and kinetic theory

The conversion of proteins from their soluble forms into fibrillar amyloid nanostructures is a general type of behaviour encountered for many different proteins in the context of disease as well as for the generation of a select class of functional materials in nature. This talk focuses on the problem of defining the rates of the individual molecular level processes involved in the overall conversion reaction. A master equation approach is discussed\footnote{Cohen et al, J Chem Phys 2011, 135, 065106} \footnote{Knowles et al, Science, 2009, 326, 1533-1537} and used in combination with kinetic measurements to yield mechanistic insights into the amyloid growth phenomenon.   

Scaling exponents report on changes in the mechanism of filamentous growth

We analyse the scaling behaviour in the proliferation and growth of protein nanostructures. We show that changes in scaling exponents that govern the lag time of the reaction within a given system can be identified with mechanistic changes that affect a molecular step in the assembly pathway. In this study, we focused on fibril growth from a representative protein, insulin, and an unstructured peptide, A$\beta$42. Our results reveal that the scaling exponent contains contributions not only from the dominant secondary nucleation mechanism in the systems studied, but also from the primary nucleation step even in cases where the generation of nuclei from the primary pathway is significantly smaller than from the secondary pathway. These results shed light on the origin of the scaling behaviour of filamentous growth.   

Control the kinetics and pathway of insulin fibril formation

Protein fibrils have been proposed as possible toxic agents for many amyloid related diseases, such as Alzheimer's disease, however the reaction pathway toward the amyloid fibrillation remain inadequately understood. In this work, we examine the conformational transition of human insulin as the model amyloid protein by single-molecule fluorescence spectroscopy and imaging. By controlling the pH cycling, insulin monomer and oligomers are indentified at given pH variation condition. Furthermore, low frequency ac-electric fields are employed to control the insulin aggregation from its monomers in a microchannel. It is observed that lag time to induce insulin fibrillation can be significantly shortened, in compassion to the commonly used cooling and seeding methods, and exhibits a strong dependence on applied ac-field strength. Additionally, the structure of insulin aggregates under ac-electric fields is observed to be drastically different from that under the temperature control.   

Time-Dependent and Low Temperature Studies of Amyloidogenic and Non-amyloidogenic Proteins by Dielectric Relaxation Spectroscopy

We present dielectric relaxation spectroscopy measurements of amyloidogenic Abeta (1-42) and non-amyloidogenic Abeta (42-1) proteins over a frequency range of 10 mHz to 10 MHz. Measurements were performed as a function of time from 0 to 24 h and temperature range of 193K-283K. Two relaxation peaks, alpha and beta, were observed at temperatures above 193 K. These peaks are attributed to the bulk and bound water. As a function of time, the dielectric signal of the Abeta (1-42) shifts towards the dielectric signal of the solvent while for the Abeta (42-1) the dielectric signal does not change. The activation energies of Abeta (1-42) and Abeta (42-1) were calculated and significant differences were found. We attribute these variations to structural changes that affect the hydration map of Abeta (1-42) aggregates. Our results are in agreement with theoretical predictions.   

Applying microfluidic techniques in quantitative studies of protein aggregation

Protein aggregation and fibrillation is involved in a number of devastating diseases, of which we have a limited understanding at present. Microfluidic techniques can be used in developing quantitative assays to study individual aspects of protein aggregation. Under certain conditions bovine insulin aggregates to give spherulites; spherical structures with fibrils growing and branching out from a central core. Drawing a parallel to actin polymerisation of the cell's cytoskeleton, fibril growth generates force. The force generated by polymerisation at fibril ends during spherulite growth can be measured in a microfluidic environment (TPJ Knowles et al, PNAS, 2009). By measuring the bending of four polydimethylsiloxane walls by a growing spherulite positioned in the centre, the force generated by polymerisation at fibril termini can be calculated. By growing the spherulites with a constant flow of monomer, the maximum force able to be generated by fibril growth, the stall force, can be calculated. This gives insight into the energy landscape of protein aggregation.   

Anomalous Formation of Multilayer Protein Aggregates on the Surface of Nanotubular $TiO_{2}$

Significant evidence links protein aggregation to the pathology and progression of most protein misfolding diseases. Protein aggregation also poses a significant problem for the safe and cost-effective production of therapeutic proteins. A comprehensive understanding of these problems requires both a detailed understanding native protein-protein interactions as well as an understanding of how protein-material interactions may alter protein aggregation phenomenon. Here we report on the anomalous formation of multilayered protein aggregates of globular proteins on the surface of $TiO_{2}$ nanotubes. Our findings suggest that minor alterations of the surface hydration properties of the nanotubes may drastically alter protein aggregation phenomenon. We further highlight the role of electrostatic and Van der Waals forces in this aggregation process.   

Effect of Lipid Bilayer on Human Islet Amyloid Polypeptide Self Assembly

Aggregates of human islet amyloid polypeptides (hIAPP, also known as human amylin) are commonly found in the pancreatic $\beta$-cells of type II diabetes patients. Experimental studies have shown that small aggregates of hIAPP, that arise during the assembly process, lead to membrane leakage and are highly cytotoxic. Due to the fast assembly kinetics, it is difficult to study the early aggregation of hIAPP experimentally. In this work, we use molecular simulation with a coarse grained (CG) model to investigate the oligomerization of hIAPP with and without the presence of lipid bilayers. We develop a CG protein model that reproduces the three thremodynamically stable structures of hIAPP, namely $\alpha$-helix, $\beta$-hairpin, and unstructured coil, and the corresponding free energy differences calculated by atomistic molecular simulations. The aggregated structure of hIAPP also agrees with that proposed by NMR experiments. We further investigate the assembly of hIAPP in the presence of a lipid bilayer and its effect on the membrane leakage. Comparing our results with the mechanism proposed based on experimental data provides a better understanding of the origins of hIAPP self assembly and its toxicity.