Analysis Of Protein Aggregation Research Articles

Modelling and analysis of protein aggregation - Competing pathways in prion (PrP) polymerisation

Protein aggregation leading to the formation of amyloid fibrils is involved in several neurodegenerative diseases such as prion diseases. To clarify how these fibrils are able to incorporate additional units, prion fibril aggregation and disaggregation kinetics were experimentally studied using Static Light Scattering (SLS). Values that are functions of the second order moment of c_i, with c_i being the concentration of fibrils of size i, were then measured as a function of time. An initial model, adapted from the Becker-Doring system that considers all fibrils to react similarly is not able to reproduce the observed in vitro behaviour. Our second model involves an additional compartment of fibrils unable to incorporate more prion units. This model leads to kinetic coefficien ts which are biologically plausible and correctly simulates the first experimental steps for prion aggregation.

Biophysical Underpinnings of the Repeat Length Dependence of Polyglutamine Amyloid Formation

There are now 10 expanded CAG repeat diseases in which both disease risk and age of onset are strongly dependent on the repeat length of the polyglutamine (polyQ) sequence in the disease protein. Large, polyQ-rich inclusions in patient brains and in cell and animal models are consistent with the involvement of polyQ aggregation in the disease mechanism. This possibility is reinforced by studies showing strong repeat length dependence to the aggregation process, qualitatively mirroring the repeat length dependence of disease risk. Our understanding of the underlying biophysical principles that mediate the repeat length dependence of aggregation, however, is far from complete. A previous study of simple polyQ peptides showed that N*, the size of the critical nucleus that controls onset of aggregation, decreases from unfavorable tetramer to favorable monomer over the range Q23 to Q26. These data, however, do not explain why, for all peptides exhibiting N* ∼ 1, spontaneous aggregation rates continue to increase with increasing repeat length. Here we describe a novel kinetics analyses that maps out the nonlinear dependence with repeat length of a nucleation efficiency term that is likely related to aspects of nucleus structure. This trend accounts for why nucleus size increases to tetrameric at repeat lengths of Q23 or below. Intriguingly, both aggregation and age of onset trend with repeat length in similar ways, exhibiting large changes per added Gln at low repeat lengths and small changes per added Gln at relatively long repeat lengths. Fibril stability also increases with repeat length in a nonlinear fashion.

Analysis of Protein Aggregation in Neurodegenerative Disease

Pathological protein and peptide aggregation are key events in a number of chronic and devastating neurodegenerative conditions including dementias such as Alzheimer's and Creutzfeldt-Jakob's disease and other central nervous system diseases such as Parkinson's and Huntington's disease and amyotrophic lateral sclerosis. Analytical methods for studying protein aggregation in these diseases are important for mapping pathophysiological events and ultimately for the development of new therapies and better diagnostic tools.

Simultaneous Detection and Analysis of Protein Aggregation and Protein Unfolding by size Exclusion Chromatography with Post Column Addition of the Fluorescent Dye BisANS

For development and optimization of protein formulation sensitive analytical tools are required to follow both aggregation and changes in protein structure. The latter can be seen as the beginning of physical instability leading to aggregation. The focus of this work laid on the development of a novel analysis simultaneously detecting changes in protein conformation and the formation of oligomers. By adding the extrinsic fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (BisANS) after size exclusion chromatography (SEC) and UV detection, it was possible to separate protein monomer and oligomers by size, analyze the amount of formed oligomers quantitatively using UV detection, and observe changes in protein structure of different protein species by fluorescence detection. This enabled us to distinguish between native-like and denatured oligomers and monomers formed under different stress conditions. Correspondingly, increased fluorescence reflecting partial unfolding was assigned specifically to monomer, oligomer, or both. The unfolding of monomer is not traceable by commonly used detection methods, but its monitoring may provide important information about activity and long-term stability. By adding the dye after SEC and UV detection, interferences with prior detectors are precluded, excipients are separated avoiding interferences with the protein-dye interaction and, in addition, the dye-protein interaction cannot impact the aggregation formation, as added after the separation of monomer and aggregates.

Analysis of Protein Aggregation in Sub-Femtoliter Volume by Two-Photon-Based Fluorescence Correlation Spectroscopy

Analysis of Protein Aggregation in Sub-Femtoliter Volume by Two-Photon-Based Fluorescence Correlation Spectroscopy

Detection and Analysis of Protein Aggregation with Confocal Single Molecule Fluorescence Spectroscopy

The misfolding and aggregation of proteins is a common phenomenon both in the cell, in in vitro protein refolding, and the corresponding biotechnological applications. Most importantly, it is involved in a wide range of diseases, including some of the most prevalent neurodegenerative disorders. However, the range of methods available to analyze this highly heterogeneous process and the resulting aggregate structures has been very limited. Here we present an approach that uses confocal single molecule detection of FRET-labeled samples employing four detection channels to obtain information about diffusivity, anisotropy, fluorescence lifetimes and Förster transfer efficiencies from a single measurement. By combining these observables, this method allows the separation of subpopulations of folded and misfolded proteins in solution with high sensitivity and a differentiation of aggregates generated under different conditions. We demonstrate the versatility of the method with experiments on rhodanese, an aggregation-prone two-domain protein.

Methods for Measuring Protein Aggregation

Misfolded proteins can aggregate into complexes of varying size and structure, generally with a concomitant loss of protein function. These aggregates frequently impede expression, purification, analysis, and storage of wild-type and mutant proteins to be used for research or as pharmaceuticals. Human health can be severely affected by protein misfolding and aggregation resulting from genetic mutations, medical treatment, or prion diseases (tauopathies). Analysis of protein aggregation is therefore an important facet of many areas of protein chemistry and related fields. Three categories of aggregation experiments are reviewed. First, characteristic data anomalies from common protein methods indicate the presence of aggregates. Second, once aggregation has been identified, several techniques are available to screen for solvent conditions that promote protein solubility. Finally, methods that characterize aggregate structure allow investigation of the process of protein coagulation. These methods vary in the required amount and purity of sample, the cost and difficulty of implementation, the type of aggregate detected, and the ability to be applied for quantification and kinetics. Approaches to improve protein solubility through the addition of co-solvents or the alteration of over-expression strategies are also reviewed. Keywords: Aggregate, Aggresome, Inclusion body, Amyloid fibril, Chaperone, Proteolysis

2P065 単純化配列と系統的変異体解析を用いた蛋白質凝集現象の熱力学解析(蛋白質 C) 物性(安定性、折れたたみなど)))

2P065 単純化配列と系統的変異体解析を用いた蛋白質凝集現象の熱力学解析(蛋白質 C) 物性(安定性、折れたたみなど)))