Denaturation unfolded proteins

The advantages of ESI-MS for studying conformational changes in proteins were recognized by Katta and Chait in 1991.31 ESI-MS is used not only to distinguish between native (folded) proteins and denatured (unfolded) proteins but also to follow the dynamics of the protein (un)folding process. In one case, an interesting conformational phenomenon that went unnoticed... 

Chymotrypsin specifically cleaves peptide bonds whose C-terminal amino acid is adjacent to non-polar aromatic amino acid residues or the bulky, hydrophobic methionine. Because these residues are often buried in the interior of proteins, including chymotrypsin, the self-hydrolysis of native, folded chymotrypsin is very inefficient. In fact, during digestion, chymotrypsin acts most effectively on partially degraded and denatured (unfolded) proteins. 

Fig. 7. Unfolding (a) isotherm, where the half-maximal unfolding for this protein occurs at 2.6 M denaturant and (b) free energy where in the absence of denaturant, the protein has an extrapolated stability,, of 17.6 kj/mol (4.2 kcal/mol) as shown. To convert to cal, divide by 4.184.

Denaturation is accompanied by changes in both physical and biological properties. Solubility is drastically decreased, as occurs when egg white is cooked and the albumins unfold and coagulate. Most enzymes also lose all catalytic activity when denatured, since a precisely defined tertiary structure is required for their action. Although most denaturation is irreversible, some cases are known where spontaneous renaturation of an unfolded protein to its stable tertiary structure occurs. Renaturation is accompanied by a full recovery of biological activity. 

Toward a Taxonomy of the Denatured State Small Angle Scattering Studies of Unfolded Proteins by Millett et al. assesses denatured states induced by heat, cold, and solvent for evidence of residual structure, while Insights into the Structure and Dynamics of Unfolded Proteins from NMR by Dyson and Wright describes their extensive investigations of residual structure in the unfolded state. 

The conformational plasticity supported by mobile regions within native proteins, partially denatured protein states such as molten globules, and natively unfolded proteins underlies many of the conformational (protein misfolding) diseases (Carrell and Lomas, 1997 Dobson et al., 2001). Many of these diseases involve amyloid fibril formation, as in amyloidosis from mutant human lysozymes, neurodegenerative diseases such as Parkinson s and Alzheimer s due to the hbrillogenic propensities of a -synuclein and tau, and the prion encephalopathies such as scrapie, BSE, and new variant Creutzfeldt-Jacob disease (CJD) where amyloid fibril formation is triggered by exposure to the amyloid form of the prion protein. In addition, aggregation of serine protease inhibitors such as a j-antitrypsin is responsible for diseases such as emphysema and cirrhosis. 

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. 

Privalov et al. (1989) also reported the temperature dependence of the ellipticity at 222 nm for the proteins studied at various pH values (Fig. 28). At the highest temperature studied (80°C), the 222 nm ellipticity value for the thermally unfolded, acid-unfolded, and Gdm-HCl-unfolded proteins appear to be converging, but show a range of 2000 deg cm2/dmol out of a total of 5000 deg cm2/dmol. (ApoMb is an exception in that, as noted before, the thermally denatured protein is apparently an associated /1-sheet. However, the acid- and Gdm HC1-unfolded forms of apoMb have similar [0] 222 values at 80°C.)... 

TOWARD A TAXONOMY OF THE DENATURED STATE SMALL ANGLE SCATTERING STUDIES OF UNFOLDED PROTEINS... 

Studies of thermally denatured proteins remain technically challenging owing to the propensity of thermally unfolded proteins to aggregate. Despite this potential difficulty, small-angle scattering techniques have been employed in the characterization of a number of thermally unfolded states. 

Because the molar volume of an unfolded protein is less than that of the native state, increasing pressure leads to denaturation (Gross and Jaenicke, 1994). Royer and co-workers have employed high-pressure SAXS to monitor the pressure-induced unfolding of Snase. They find that the unfolded ensemble achieves a pressure-independent Rg of... 

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. 

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