Kinetics and thermodynamics of amyloid formation from direct measurements of fluctuations in fibril mass

Abstract

Aggregation of proteins and peptides is a widespread and muchstudied problem, with serious implications in contexts ranging from biotechnology to human disease. An understanding of the proliferation of such aggregates under specific conditions requires a quantitative knowledge of the kinetics and thermodynamics of their formation; measurements that to date have remained elusive. Here, we show that precise determination of the growth rates of ordered protein aggregates such as amyloid fibrils can be achieved through real-time monitoring, using a quartz crystal oscillator, of the changes in the numbers of molecules in the fibrils from variations in their masses. We show further that this approach allows the effect of other molecular species on fibril growth to be characterized quantitatively. This method is widely applicable, and we illustrate its power by exploring the free-energy landscape associated with the conversion of the protein insulin to its amyloid form and elucidate the role of a chemical chaperone and a small heat shock protein in inhibiting the aggregation reaction.protein aggregation ͉ quartz crystal microbalance ͉ biosensors

Quantitative approaches to defining normal and aberrant protein homeostasis

“…[35][36][37][38] (2) New ideas about protein aggregation, 10,28 including the finding that the ability to assemble into stable and highly organised structures (e.g. amyloid fibrils) is not an unusual feature exhibited by a small group of peptides and proteins with special sequence or structural properties, but rather a property shared by most, if not all, proteins; (3) The discovery that specific aspects of protein behaviour, including their aggregation propensities 21,23,39,40 and the cellular toxicity associated with the aggregation process, 24,41 can be predicted with a remarkable degree of accuracy from the knowledge of their amino acid sequences; (4) The realisation that a wide variety of techniques originally devised for applications in nanotechnology can be used to probe the nature of protein aggregation and assembly and of the structures that emerge; 30,[42][43][44] and (5) The development of powerful approaches using model organisms for probing the origins and progression of misfolding diseases by linking concepts and principles emerging from in vitro studies to in vivo phenomena such as neurodegeneration. 24 An analysis of these results, which span across a wide range of subjects from neuroscience to nanoscience, reveals that the ability to keep proteins in their soluble form is absolutely central for the maintenance of cell homeostasis.…”

“…Powerful techniques are being developed to complement more established methods to overcome the challenges posed by the task of providing such a description. 30,[42][43][44]46 Our own approach is based primarily on methods that directly combine experimental and computational techniques. 5,6,35 These procedures involve the use of experimental data, largely derived from NMR spectroscopy, as restraints in computer simulations.…”

“…[70][71][72] Together with the strategy mentioned above, nanoscience techniques are also emerging as powerful tools that can provide insight into the factors that stabilise amyloid fibrils. 30,42,44 In an initial study, by using atomic force microscopy (AFM) imaging we described the changes in the distribution of inter-versus intra-molecular bonding interactions associated with the transition of proteins from their native globular structures into ordered supramolecular assemblies. This work reveals that hydrogen bonded arrays of polypeptide chains exhibit material properties intermediate between those of hard materials such as steel and carbon nanotubes and softer biological fibres such as tubulin and actin (Fig.…”

Kinetic theory of amyloid fibril templating

“…The key timescales in the growth process can be summarized k −1 grow t wait , t diff k −1 + , with an impressive 8 to 9 orders of magnitude separating the macroscopic growth rate from the microscopic timescale of H-bond formation. 26,27 In addition, Eq. (11) shows that the timescale governing growth in the reaction limited regime, t wait t diff , is not the annealing of newly bound molecules, but the clearance of off-pathway intermediates.…”

“…A rough estimate of t diff can be obtained from the reaction rate for particles approaching an absorbing sphere, 4π acD p , where a is the radius of the absorbing surface, c is the concentration far from the surface, and D p is the diffusion constant of the particles. To describe the growth rates of fibrils bound to a surface 26,27 this must be reduced by half to account for the 2π solid angle restriction…”