Guanidinium chloride, unfolded proteins

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. 

The Up Ug reactions in unfolded proteins have properties that are characteristic of prolyl peptide bond isomerizations in small peptides. The equilibrium is independent of temperature (Schmid, 1982) and independent of the concentration of additives, such as guanidinium chloride (GdmCl) (Schmid and Baldwin, 1979), that strongly decrease protein stability but do not affect prolyl peptide bond isomerization. The reaction is catalyzed by strong acid and it shows an activation energy of 88 kj/ mol, as expected for prolyl isomerization (Schmid and Baldwin, 1978). 

Fig. 7. Oxidative refolding of reduced RNase Tl. Reoxidation conditions were 0.1 M Tris-HCl, pH 7.8, 0.2 Af guanidinium chloride, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 0.2 mM EDTA, and 2.5 nM RNase Tl at 25°C. The kinetics of oxidative refolding were followed by the increase in tryptophan fluorescence intensity at 320 nm ( ), by an unfolding assay (Kiefhaber el ai, 1990b) that measures the formation of native protein molecules (A), and by the increase in the intensity of the band for native RNase Tl in native polyacrylamide gel electrophoresis ( ). Fluorescence emission in the presence of 10 mM reduced dithioerythritol to block disulfide bond formation (O). The small decrease in signal after several hours is caused by slight aggregation of the reduced and unfolded protein. (From Schonbrunner and Schmid (1992).

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. 

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 denatu-rants such as urea or guanidinium chloride. For many proteins, a comparison of the degree of unfolding as the concentration of denaturant increases reveals 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 2.63). A similar sharp transition is observed if denaturants are removed from unfolded proteins, allowing the proteins to fold. 

FIGURE 7.4 Transition of proteins from the native to the unfolded state or vice versa, (a) Ribonuclease at pH 3.15, as a function of temperature, (b) Lysozyme as a function of guanidinium chloride concentration, (c) Nuclease A as a function of pH. 

FlGURE 7.7 Combined effects of two variables on conformational stability of globular proteins, (a) Denaturation (unfolding) temperature as a function of pH for papain (P), lysozyme (L), cytochrome C (C), parvalbumin (A), and myoglobin (M). (b) Effect of concentration of guanidinium chloride concentration and temperature on conformation of lysozyme at pH 1.7. (c) Effect of pressure (1 kbar= 10s Pa) and temperature on conformation of chymotrypsinogen. (d) Effect of pressure and pH on conformation of myoglobin (20°C). 

Fig. 1. Unfolding isothenn of the trimeric adenylate kinase from Sulfolobus acidocaldarius using CD spectroscopy. The squares represent the measured CD values at 222 nm versus the corresponding guanidinium chloride concentration at 40°. A linear dependence outside the transition region is assumed. The linear extrapolations of the pre- and posttransitional parts of the isotherms (solid lines) and the corresponding expressions are shown. The dotted line represents a fit using a function from Backmann et of for a trimeric protein.

FIGURE 13.10 Patterns for cooperative unfolding of proteins induced by changing environmental conditions, that is, (a) pH, (b) temperature, and (c) concentration of guanidinium chloride. 

FIGURE 13.12 Gibbs energy of the unfolding of 3-phosphoglycerate kinase in guanidinium chloride solutions. (Adapted from Tanford, C., Adv. Protein Chem., 24,1, 1970.)... 

Investigation of the equilibrium between native and unfolded conformations formed on solvent denaturation, temperature, and pressure have now been performed on a wide range of proteins. For example, James and Sawan have studied the effect of increasing guanidinium chloride concentration on the mobility of individual histidine residues in ribonuclease (pancreatic) C n.m.r. spectroscopy using spin-lattice relaxation in an off-resonance rotating frame. They found that L-histidines-12, -105, and -119 increase in mobility up to a denaturant concentra-... 

Guanidinium chloride and urea deserve special attention in that they are among the most effective and most widely used denaturants. Their outstanding capacity to form hydrogen bonds modifles the water structure, thereby altering the protein-water interactions as a result, the protein unfolds. 

Compounds such as urea and guanidinium chloride (GnCl) cause unfolding of the proteins they also cause unwinding of double helical DNA. In both cases the effect is caused by a decrease in the hydrophobic effect. This effect is a principal force that organizes protein structure, by packing the hydrocarbon sidechains of proteins into the interior, and it also contributes to the stability of DNA since the hydrophobic purines and pyrimidines can stack on each other away from water. The way in which such denaturants as urea act to overcome the hydrophobic effect has only recently been clarified, by studies we conducted aimed at distinguishing between the two likely mechanisms [8]. 

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