Solubility is a key requirement for the functioning of a protein within the complex network of cellular components. [1][2][3][4] A class of highly debilitating disorders, including Alzheimers and Parkinsons diseases, is related to the loss of solubility of peptides and proteins that is accompanied by their aggregation into ordered amyloid fibrils. [5] It has been found that, under at least some physiological conditions, these aggregates are thermodynamically more stable than the native forms of biological polypeptides. [6] This finding raises questions as to the factors governing the crucial ability of native proteins to remain soluble even under conditions where they do not necessarily correspond to global minima on free energy landscapes. In order to address this question, we have studied in detail the kinetics of elongation of amyloid fibrils formed by a wide range of polypeptides. The formation of amyloid fibrils from soluble protein molecules involves at least a primary nucleation step, an elongation step and, in general, a secondary nucleation process such as fibril fragmentation. [7] In addition, multiple interconverting oligomeric intermediates can be involved. [8] Measurements of amyloid growth in bulk solution often reflect all of these processes, and it can therefore be extremely challenging to determine accurately the concentrations of the different species and the rate constants for the individual elementary steps.In order to overcome these difficulties, surface-based sensing techniques, notably those based on quartz crystal microbalance (QCM) measurements, have been developed in recent years, by which the growth of a constant, surfacebound ensemble of fibrils can be monitored. [9][10][11] These methods make use of the fact that in the presence of preexisting fibrils, aggregation can be highly accelerated through seeding. [12] This seeding process corresponds to the elongation of existing fibrils and can be well described as diffusional motion over a single free energy barrier, [13] involving no intermediate species between monomeric and fibrillar peptide. The elongation of the fibrils is monitored through the increase in hydrodynamic mass bound to the quartz crystal, as the rate of change of the resonant frequency is proportional to the average elongation rate of the fibrils. [9] The opportunity to image the sensor surface enables an estimation of the surface density of fibrils, an important factor in the determination of the rate constants in this bimolecular reaction, the overall rate of which depends on both the concentration of soluble protein and the number of available fibril ends. In addition, the lengths of the fibrils before and after an experiment can be compared and therefore an independent measurement of the length increase can be made and used to calibrate the frequency response of the microbalance. The covalent irreversible attachment of the preformed fibrils to the sensor surface [14] and the subsequent passivation of the remaining surface, as well as the short duration of individual ...
We describe the changes in structure and dynamics that occur in the second PDZ domain of human tyrosine phosphatase 1E upon binding the small peptide RA-GEF2 by an analysis of NMR data based on their use as ensemble-averaged restraints in molecular dynamics simulations. This approach reveals the presence of two interconnected networks of residues, the first exhibiting structural changes and the second dynamical changes upon binding, and it provides a detailed mapping of the regions of increased and decreased mobility upon binding. Analysis of the dynamical properties of the residues in these networks reveals that conformational changes are transmitted through pathways of coupled side-chain reorientations. These results illustrate how the strategy we described, in which NMR data are used in combination with molecular dynamics simulations, can be used to characterize in detail the complex organization of the changes in structure and dynamics that take place in proteins upon binding.
Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.
The partial unfolding of human lysozyme underlies its conversion from the soluble state into amyloid fibrils observed in a fatal hereditary form of systemic amyloidosis. To understand the molecular origins of the disease, it is critical to characterize the structural and physicochemical properties of the amyloidogenic states of the protein. Here we provide a high-resolution view of the unfolding process at low pH for three different lysozyme variants, the wild-type protein and the mutants I56T and I59T, which show variable stabilities and propensities to aggregate in vitro. Using a range of biophysical techniques that includes differential scanning calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that thermal unfolding under amyloidogenic solution conditions involves a cooperative loss of native tertiary structure, followed by progressive unfolding of a compact, molten globule-like denatured state ensemble as the temperature is increased. The width of the temperature window over which the denatured ensemble progressively unfolds correlates with the relative amyloidogenicity and stability of these variants, and the region of lysozyme that unfolds first maps to that which forms the core of the amyloid fibrils formed under similar conditions. Together, these results present a coherent picture at atomic resolution of the initial events underlying amyloid formation by a globular protein.
Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release.
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