Chemical denaturation, unfolded proteins

CD spectroscopy has also provided valuable insight into the chemical stability and chemical denaturation of proteins. A recent study by Rumfeldt etal. examines the guanidinium-chloride induced denaturation of mutant copper-zinc superoxide dismutases (SODs). These mutant forms of the Cu, Zn-SOD enzyme are associated with toxic protein aggregation responsible for the pathology of amyotrophic lateral sclerosis. In this study, CD spectroscopy was used in conjunction with tryptophan fluorescence, enzyme activity, and sedimentation experiments to study the mechanism by which the mutated enzyme undergoes chemical denaturation. The authors found that the mutations in the enzyme structure increased the susceptibihty of the enzyme to form partially unfolded destabilized monomers, rather than the stable metaUated monomer intermediate or native metallated dimer. 

Proteins unfolded by GdmHCl or urea will have a dominant conformation, Pn- At low temperatures we find about one-third of the residues in chemically denatured proteins in the Pn-helix conformation, with two-thirds in the form of the high-temperature ensemble. Since at least one-third of the residues in this ensemble are isolated Pn residues or in Pn helices of two or three residues, the total Pn content will be 50% or greater. The Pn content of cold- and acid-denatured proteins will be substantial, probably >40%, but not as large as in chemically denatured proteins. 

Chemical denaturants, excessive heat, or low temperatures are typically not the reasons proteins unfold in the cell. Several groups have... 

An excluded-volume random-coil conformation will be achieved when the solvent quality exceeds the theta point, the temperature or denatu-rant concentration at which the solvent-monomer interactions exactly balance the monomer—monomer interactions that cause the polymer to collapse into a globule under more benign solvent conditions. A number of lines of small-angle scattering—based evidence are consistent with the suggestion that typical chemical or thermal denaturation conditions are good solvents (i.e., are beyond the theta point) and thus that chemically or thermally unfolded proteins adopt a near random-coil conformation. 

Any chemical or enzymatic process that modifies a therapeutic protein in the body is known as protein metabolism. Unfolded proteins are, in general, more susceptible to metabolism via proteolysis because of increased access to critical peptide sequences. Chemical modification of a protein can mark it for further degradation. This is thought to occur by altering the folding equilibrium in favor of the denatured conformation [3]. 

Several studies since then have supported this suggestion, and now it is widely accepted that conformational change/structural perturbation is a prerequisite for amyloid formation. Structural perturbation involves destabilization of the native state, thus forming nonnative states or partially unfolded intermediates (kinetic or thermodynamic intermediates), which are prone to aggregation. Mild to harsh conditions such as low pH, exposure to elevated temperatures, exposure to hydrophobic surfaces and partial denaturation using urea and guanidinium chloride are used to achieve nonnative states. Stabilizers of intermediate states such as trimethylamine N-oxide (TMAO) are also used for amyloidogenesis. However, natively unfolded proteins, such as a-synuclein, tau protein and yeast prion, require some structural stabilization for the formation of partially folded intermediates that are competent for fibril formation. Conditions for partial structural consolidation include low pH, presence of sodium dodecyl sulfate (SDS), temperature or chemical chaperones. 

Temperatures above (and sometimes below) the normal range will cause thermally unstable proteins to unfold or denature . High concentrations of solutes, extremes of pH, mechanical forces and the presence of chemical denaturants can do the same. A fuUy denatured protein lacks both tertiary and secondary structure, and exists as a random coil . In most cases denaturation of proteins is irreversible. 

Figure 5-13. Protein unfolding by chemical denaturation. The unfolding and refolding of proteins caused by the addition of guanidinium chloride (GdnHCI) can be monitored from the CD spectrum, usually at 220 nm. The diagram on the left shows the denaturation curve as expressed by the concentration dependence of...

As stated earlier, proteins can be denatured by heat or by chemical denaturants such as urea or guanidium chloride. For many proteins, a comparison of the degree of unfolding as the concentration of denaturant increases has revealed a relatively sharp transition from the folded, or native, form to the unfolded, or denatured, form, suggesting that only these two conformational states are present to any significant extent (Figure 3.56). A similar sharp transition is observed if one starts with unfolded proteins and removes the denaturants, allowing the proteins to fold. 

On the other hand, the observation that unfolded proteins undergo irreversible chemical destruction at these extremely high temperatures would mean that a real equilibrium between native and unfolded state cannot exist in these hyperthermophiles. Once unfolded, the proteins become irreversibly denatured. One might speculate that the strategy of protein stabilization at these temperatures aims mainly at an increase of the activation energy of unfolding, i.e., at a retardation of the unfolding process. 

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