Protein unfolding intermediates

Initial protein folding intermediates formed as a consequence of nonlocal interactions on the reaction path from unfolded to partially folded states. These loops are likely to determine the trajectory of later folding steps. The character of nonlocal interactions is likely to be much different than structures inferred from the results... 

Many proteins can be made to clump into fibrous amyloid deposits like those seen in Alzheimer s disease, Creutzfeldt-Jakob disease (the human counterpart of mad cow disease), and other serious ailments. To help prove this point, a natural enzyme to convert to amyloid fibrils—insoluble protein aggregates with a /3-pleated sheet structure—simply by maintaining protein for some time in the unfolded state. Until now, scientists have generally believed that only specific proteins such as amyloid /3-protein and prions are capable of being converted into amyloid fibrils.11 A variety of spectroscopic techniques have been used to confirm the gradual development of amyloid fibrils and to verify the fibrils predominant /3-pleated sheet structure. In the partially unfolded intermediates that form under denaturing conditions, hydrophobic amino acid residues and polypeptide backbone normally buried inside fully folded structures become exposed. Further work is needed to confirm and advance these findings. 

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. 

Permyakov et al. (1985) studied the binding of Na(I) and K(I), as well as of Ca(ll) and Mg(II), to bovine a-lactalbumin by intrinsic protein fluorescence. Urea- and alkali-induced unfolding transitions involve stable partially unfolded intermediates for the ion-bound forms of this protein (see also Section IX,E). 

The behaviors of apo- and Ca(Il)-bound forms of a-lactalbumin differ markedly upon denaturation with guanidine hydrochloride, as shown by Ikeguchi et al. (1986). Thus, at low Ca(ll) ion concentration a-lactalbumin unfolds to produce a stable intermediate, while at high Ca(ll) concentration the protein unfolds in a manner similar to that of lysozyme. 

If an intermediate forms that has a solubility less than that of N or D this can lead to aggregation and eventually to precipitation. For example, the addition of moderate amounts of denaturant to bovine growth hormone (bGH) can generate a partially unfolded intermediate of low solubility which aggregates. Similarly, y-interferon (IFN-y) is inactivated by acid treatment or by the addition of salt because the dimeric native state is converted into monomers, which are partially denamred. For both proteins the formation of intermediates leads to inactivation. 

Many of the unfolding processes are irreversible however, some are reversible with the return of full biological activity. For example, proteins dissolved in DMSO refold upon dilution in water (Chang et ah, 1991). Supercritical anti-solvent precipitation of insulin, lysozyme and trypsin from DMSO yielded partially unfolded intermediates, as characterized by spectroscopy (Yeo et al., 1994 Winters et ah, 1996). However, these structures were reversible upon reconstitution in aqueous media, with recovery of biological activity (Yeo et ah, 1994 Winters et ah, 1996). Chymotrypsin also completely unfolded in DMSO, but regained activity upon rehydration (Zaks and Klibanov, 1988a Yeo et ah, 1994). 

Homemann S, Glockshuber R (1998) A scrapie-like unfolding intermediate of the prion protein domain PrP(121-231) induced by acidic pH. Proc Natl Acad Sci USA 95 6010-6014... 

Fig. 3. Far-UV circular dichroism (CD) spectra of different conformational states of recombinant murine PrP(23-231) and PrP( 121-231) that can be populated in solution (1) oxidized PrP(121-231) at 4.0, (2) oxidized PrP(23-231) at pH 7.0, (3) acid-induced unfolding intermediate of oxidized PrP(121-231) in 3.5 M urea at pH. 4.0 (ionic strength 88 mM protein concentration 30 pM), (4) reduced PrP(23-231) at pH 4.0 and 10 mM ionic strength, and (5) reduced PrP(23-231) in 3 M urea at pH 7.4.

Fig. 4. Urea-induced equilibrium unfolding transitions of murine PrP (121-231) at 22°C. (A) Dependence of reversible, urea-induced unfolding on pH. Open symbols represent refolding experiments, and closed symbols represent unfolding experiments. (B) Dependence on ionic strength of the formation of the acid-induced unfolding intermediate of PrP(121-231) at pH 4.0 and a protein concentration of 29 pM. Unfolding experiments were performed at 22°C in 50 mM formic acid/NaOH, pH 4.0 (ionic strength 32 mM), containing 0 M (O), 50 mM ( ), 100 mM (A), or 150 mM ( ) sodium chloride.

The results of high-sensitivity DSC studies of protein denaturation have greatly helped to clarify the reversibility and the intermediate states issues, as indicated above. They have yielded a detailed analysis of the thermodynamical features of protein unfolding and led to a reassessment of the contributions of the different forces that determine protein stability. 

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