Studies of Protein Denaturation

In this article we shall examine the main achievements of microcalorimetric studies of protein denaturation and of the dissolution of nonpolar substances in water. This analysis has led us to reconsider the popular point of view on the mechanism of hydrophobic interaction and its role in the stabilization of protein structures. 

Among the most important accomplishments of calorimetric studies of protein denaturation has been the establishment of the following general features. 

The native structure of large proteins is disrupted in several discrete stages in each of which discrete amounts of energy are absorbed. Each of these steps corresponds to the all-or-none breakdown of definite structural blocks of the protein molecule. Therefore, the large protein structure is not a monolith but appears to be composed of discrete, more or less independent, cooperative blocks, i.e., domains (Wetlaufer, 1973 Janin and Wodak, 1983 Privalov, 1982 see also Privalov et al., 1981 Privalov and Medved , 1982 Potekhin and Privalov, 1982 Novokhatny et al., 1984). 

It appears that the discreteness of a structure is a general principle of protein architecture, which not only reflects the evolution of the protein molecule but also has a deep physical ground (Privalov, 1985, 1986). It is just this unique thermodynamic property of the protein molecule that has made possible a quantitative definition of protein stability. 

Indeed, since the macroscopic states of a protein are discrete, they are described by discrete surfaces in the phase space of considered variables (Pfeil and Privalov, 1976c). The small globular proteins, or individual cooperative domains, which have only two stable macroscopic states, the native (N) and denatured (D), are described by two surfaces in the phase space, corresponding to their extensive thermodynamic functions. The transition between these states is determined by the differences of 

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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. 

Comparison of results on thermodynamic studies of protein denaturation and hydrocarbon dissolution in water shows a number of surprising similarities and differences between these two processes. The most surprising result is the close correspondence of the temperature of convergence of the enthalpy and entropy functions for the denaturation of proteins, Tx, and the temperature 7s for the dissolution of hydrocarbons in water. 

Denaturation was early observed to be a reversible process. Indeed, Anson (1 ) observed 35 years ago that hemoglobin could be heat denatured in a variety of ways and could be converted back to a state which had all the characteristics of its original native state, as determined by methods available at that time. Almost all studies of protein denaturation now revolve around not only the denaturation itself, but also its renaturation perhaps renaturation is a more interesting and provocative field for modern research. 

Chaotropic agents guanidine hydrochloride use for study of protein denaturation GTIC is considered to be more effective than GuCl GTIC used for nucleic acid extraction. 

The study of protein denaturation is relevant to food science and technology in different respects. 

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. 

There is yet another important origin of observed pressure changes in the NMR spectra of biomolecules in aqueous solutions. NMR studies of protein denaturation using temperature or chemical means are well established but pressure also leads to reversible denaturation as discussed in more detail in the section on applications. In the case of model membranes the use of pressure leads to very rich barotropic behaviour and high pressure NMR techniques can establish pressure-temperature phase diagrams for the membrane system studied. 

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