Protein unfolding temperature induced

Turning now to the chapters in this volume, a variety of complementary techniques and approaches have been used to characterize peptide and protein unfolding induced by temperature, pressure, and solvent. Our goal has been to assemble these complementary views within a single volume in order to develop a more complete picture of denatured peptides and proteins. The unifying observation in common to all chapters is the detection of preferred backbone conformations in experimentally accessible unfolded states. 

A major problem in unfolding studies of large proteins is irreversibility. In a study of elastase temperature-induced denaturation, second-derivative FTIR show a distinct loss of several sharp amide V features (dominant /3-sheet components and growth in broadened bands at 1645 and 1668 cm-1 (Byler et al., 2000). These features persisted on cooling, indicating lack of reversibility, a feature common to longer multidomain proteins. A graphic example of this is seen in the triosephosphate... 

That which we call a rose, by any other name would smell as sweet. The essence of the denatured state, in contrast, is rather more elusive. The difficulty stems, at least in part, from the wide variety of methods of inducing a protein to unfold. Some proteins appear to be natively unfolded that is, they remain unfolded in the cell under conditions in which they retain their biological activity (Plaxco and Gross, 1997 Wright and Dyson 1999 Dunker, 2002). Other proteins unfold only under the influence of changes in pH, high or low temperatures, or... 

Khechinashvili, N.N., Janin.J., and Rodier, F. (1995) Thermodynamics ofthe temperature induced unfolding of global proteins, Prot. Sci. 4, 1315-1324. 

The practical solution to the protein stability dilemma is to remove the water. Lyophilization (freeze-drying) is most commonly used to prepare dehydrated proteins, which, theoretically, should have the desired long-term stability at ambient temperatures. However, as will be described in this review, recent infrared spectroscopic studies have documented that the acute freezing and dehydration stresses of lyophilization can induce protein unfolding [8-11]. Unfolding not only can lead to irreversible protein denaturation, even if the sample is rehydrated immediately, but can also reduce storage stability in the dried solid [12,13]. 

Dong et al. [35] indicated that a successful lyophilized protein formulation is the preservation of the native conformation in the dried solid. They used FTIR as a tool to study lyophilized-induced unfolding and aggregation of proteins, namely, through the bands at 1620 cm to 1685 cm, common IR spectral features indicative of lyophilization- and temperature-induced protein aggregation, used to monitor and quantify aggregation even in the dried solid. 

The large positive activation energies observed for temperature induced unfolding of proteins is attributed to the unfolding step... 

The multiplicity and the partially unfolded character of the dena-turational states of proteins, especially upon temperature-induced denaturation, hamper any thermodynamical analysis of the data and their interpretation. In particular, evaluation of the conformational entropy terms is actually infeasible. 

The biophysical characterization of globular proteins will almost always include some type of study of the unfolding of protein to obtain thermodynamic parameters. The basic idea is that a transition between a native and unfolded state, induced by temperature, pH, or denaturant concentration, can serve as a standard reaction for obtaining a thermodynamic measure of the stability of the native state. For example, the free energy change for the unfolding reaction can be used to compare the stability of a set of mutant forms of a protein (1-4). 

The accepted relationship for the temperature induced unfolding of a protein is... 

The heat capacity change for unfolding of proteins has been foimd to be positive and to be related to the increase in solvent exposure of apolar side chains (1-3). In other words, a positive ACp is a result of the hydrophobic effect and a consequence is that the AG° (T) for unfolding of a protein will have a non-linear dependence on temperature, reaching a maximum at some temperature and showing both hi temperature and low temperature induced unfolding. 

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