Urea denaturation, unfolded proteins

The denaturation of proteins generally involves at least partial unfolding, with the loss of secondary and tertiary structure. In the present context, we are interested in the end point of this process — proteins that are unfolded to the maximal extent by various agents heat, cold, acid, urea, Gdm-HCl.1 Three major questions concerning unfolded proteins are of interest in the present chapter. Do different unfolding agents... 

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

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 very high resolution of the chemical shifts gives information on a per-residue basis, and it also leads to spectral crowding in simple experiments. Figure 3 shows three very different ID proton spectra of the 14-kDa protein called a-lactalbumin, in three different states native folded, molten globule, and unfolded (urea denatured). 

Urea denaturation curves were determined by measuring the intrinsic fluorescence intensity (278 nm excitation and 320 nm emission) of solutions containing approximately 0.9 p.M protein in sodium acetate/acetic acid, pH 5.0 buffer and increasing concentrations of urea in a temperature regulated Peikin-Elmer MPF 44 B spectrophotometer. Solutions were incubated at 25 °C for 24 h before measurements were taken. The free energy of unfolding was calculated by the linear extrapolation method (17). The error in AG was 0.6 kcal/mol. 

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. 

Expression of proteins in E. coli often results in the formation of insoluble aggregates called inclusion bodies, probably comprising fully or partially unfolded proteins. Inclusion bodies are brought to their monomeric form by extraction with a denaturant (e.g., 8 M urea) under reducing conditions (e.g., 0.1 M cysteine). 

As noted previously and indicated in Figure 16-26, ATP hydrolysis by Hsc70 chaperone proteins in both the c rt osol and the mitochondrial matrix is required for Import of mitochondrial proteins. Cytosolic Hsc70 expends energy to maintain bound precursor proteins in an unfolded state that is competent for translocation into the matrix. The Importance of ATP to this function was demonstrated in studies in which a mitochondrial precursor protein was purified and then denatured (unfolded) by urea. When tested in the cell-free mitochondrial translocation system, the denatured protein was incorporated into the matrix in the absence of ATP. In contrast, import of the native, undenatured precursor required ATP for the normal unfolding function of c3rt osollc chaperones. 

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