Protein folding determination from temperature

Even extremophilic organisms and their proteins contain the same 20 amino acids with bonds similar to those in mesophiles. As the difference in free enthalpy between folded and unfolded states of globular proteins AG N >G is only about 45 15 kj mol-1 the sequence and structure of extremophilic proteins should differ from those of ordinary species. However, the main question, namely which properties cause the increase in denaturation temperature of thermostable proteins, is still debated (Rehaber, 1992). Theoretical and experimental analyses have shown that thermal stability is largely achieved by small but relevant changes at different locations in the structure involving electrostatic interactions and hydro-phobic effects (Karshikoff, 2001). There is no evidence for a common determinant or for just one effect causing thermostability. 

Folded proteins can be caused to spontaneously unfold upon being exposed to chaotropic agents, such as urea or guanidine hydrochloride (Gdn), or to elevated temperature (thermal denaturation). As solution conditions are changed by addition of denaturant, the mole fraction of denatured protein increases from a minimum of zero to a maximum of 1.0 in a characteristic unfolding isotherm (Fig. 7a). From a plot such as Figure 7a one can determine the concentration of denaturant, or the temperature in the case of thermal denaturation, required to achieve half maximal unfolding, ie, where... 

Since the active ester end of the molecule is subject to hydrolysis (half-life of about 20 minutes in phosphate buffer at room temperature conditions), it should be coupled to an amine-containing protein or other molecule before the photolysis reaction is done. During the initial coupling procedure, the solutions should be protected from light to avoid decomposition of the phenyl azide group. The degree of derivatization should be limited to no more than a 5- to 20-fold molar excess of sulfo-SBED over the quantity of protein present to prevent possible precipitation of the modified molecules. For a particular protein, studies may have to be done to determine the optimal level of modification. 

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