Conformations unfolding studies

The studies discussed above gives an idea of conformational stability of proteins in ILs as inferred from the t measurement and melting temperature determination followed by various spectroscopic methods. However, stability of a protein/enzyme has contributions from both thermodynamic and kinetic parameters [55]. While thermal unfolding studies help to extract important thermodynamic parameters, stability of the proteins could also arise from the kinetic barrier to the conformational changes in the protein scaffold [55]. However, except for monellin [48], the thermodynamic stability in other cases has not been examined. Thus, in context of stability of proteins/enzymes in ILs, it is important and imperative to study and probe thermal unfolding as well as the thermodynamics and kinetics of the conformational change of the proteins in ILs. 

From the drift-time distributions obtained in this setup, information about the number of isomers can be obtained. Some examples of IMS-mass spectrometry in conformation and folding-unfolding studies are for cytochrome c [51], apomyoglobin [51], lysozyme [52], and ubiquitin [53],... 

For any given protein, the number of possible conformations that it could adopt is astronomical. Yet each protein folds into a unique stmcture totally deterrnined by its sequence. The basic assumption is that the protein is at a free energy minimum however, calometric studies have shown that a native protein is more stable than its unfolded state by only 20—80 kj/mol (5—20 kcal/mol) (5). This small difference can be accounted for by the favorable... 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

When a protein reversibly unfolds in solution, it enters a large and diverse ensemble of conformations known as the denatured state. Studies of this reaction over the past 40 years have suggested it is a single,... 

A recent NMR study of the structure and dynamics of two amyloido-genic variants of human lysozyme (Chamberlain et al., 2001) showed that, although one variant destabilized the /6-domain much more than the other, it had no greater propensity to form amyloid fibrils. It was concluded that the increased ability of the variants to access substantially unfolded conformations of the protein is the origin of their amy-loidogenicity. This appears to reinforce the conclusions from ROA that a destabilized a-domain is involved in fibril formation. 

These studies suggest that the caseins, synucleins, and tau have natively unfolded structures in which the sequences are based largely on the PPII conformation and are held together in a loose noncooperative fashion. However, rather than describing them as random coil ,... 

Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. These rates are deduced from measurements of fluorescence fluctuations that arise as molecules with specific fluorescence properties enter or leave an open sample volume by diffusion, by undergoing a chemical reaction, or by other transport or reaction processes. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. 

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