Protein unfolding calorimetry

B. Chowdhry, S. Leharne. Simulation andAnalysis of Differential Scanning Calorimetry Output Protein Unfolding Studies 1. J. Chem. Educ. 1997, 74, 236-241. 

Simulation and Analysis of Differential Scanning Calorimetry Output Protein Unfolding Studies 1 87... 

Fig. 4. Profile of a differential scanning calorimetry experiment done on a synthetic lysozyme. The heat capacity (kilocalories per degree per mole) of the unfolding process was monitored as a function of temperature on a Micro-Cal MC2 instrument. The transition midpoint of protein unfolding corresponds to the temperature at the peak of the curve, and the thermodynamic parameters A H and A Cp are evaluated by the procedure of Privalov.33...

One of the principal goals of calorimetry of proteins is to define the protein stability curve (Fig. 1), i.e., to define T , AH°(T ), and ACp for protein unfolding. Because ACp is positive for protein unfolding, " the curve is concave downwards, and AG° is zero at two temperatures (assuming more than marginal stability). These are the heat denaturation iT ) and cold denaturation midpoint temperatures. AH° controls the steepness of the curve at any temperature, and... 

Therefore a plot of InK vs 1/T gives a line whose slope is the van t Hoff enthalpy AHvh divided by J . An estimate of K at any temperature can be obtained by the partial area ratio, as shown in Figure 9.23(a). The comparison of A Hvh with A H can be instructive. A broader transition due, for example, to the unfolding transition not being two state leads to A fJvH being less than the calorimetric value. Similarly if the protein unfolds cooperatively as a dimer, the transition will be sharper and A Hvh will be greater than that measured by calorimetry. Therefore this method can determine the so-called cooperativity or the cooperative unit of the system. 

The thermal unfolding of proteins is best measured by differential scanning calorimetry, which measures the heat absorbed by a protein as it is slowly heated through its melting transition (Figure 17.1). A solution of about 1 mg of protein in 1 mL of buffer and a separate reference sample of buffer alone are heated electrically.6 The additional current required to heat the protein solution is recorded. As the protein denatures, there is a large uptake of heat because the process is highly endothermic. The temperature at the maximum of the peak is... 

A study of two of the most prominent and widespread osmolytes, betaine and beta-hydroxyectoine, by differential scanning calorimetry (DSC) on bovine ribonu-clease A (RNase A) revealed an increase in the melting temperature Tm of RNase A of more than 12 K and of protein stability AG of 10.6 kj mol-1 at room temperature at a 3 M concentration of beta-hydroxyectoine. The heat capacity difference ACp between the folded and unfolded state was significantly increased. In contrast, betaine stabilized RNase A only at concentrations less than 3 M. When enzymes are applied in the presence of denaturants or at high temperature, beta-hydroxyectoine should be an efficient stabilizer. 

D. T. Haynie and E. Freire, Estimation of the folding/unfolding energetics of marginally stable proteins using differential scanning calorimetry, Anal. Biochem. 1994, 216, 33-41. 

Equations (l)-(4) provide the basic statistical thermodynamic framework necessary to deal with the protein folding problem. Several years ago, Freire and Biltonen (1978a) showed that scanning calorimetry data could be used to evaluate the protein folding/unfolding partition function experimentally by a double integration procedure ... 

Abstract. Walter Kauzmann stated in a review of protein thermodynamics that volume and enthalpy changes are equally fundamental properties of the unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behaviour (Nature 325 763-764, 1987). While the thermodynamic basis for pressure effects has been known for some time, the molecular mechanisms have remained rather mysterious. We, and others in the rather small field of pressure effects on protein structure and stability, have attempted since that time to clarify the molecular and physical basis for the changes in volume that accompany protein conformational transitions, and hence to explain pressure effects on proteins. The combination of many years of work on a model system, staphylococcal nuclease and its large numbers of site-specific mutants, and the rather new pressure perturbation calorimetry approach has provided for the first time a fundamental qualitative understanding of AV of unfolding, the quantitative basis of which remains the goal of current work. 

Protein stability is the free energy difference (AG) between the folded and unfolded states at physiological conditions, and it is in the range of 5-25 kcal/mol. Site-directed mutagenesis experiments provided a wealth of data for understanding the importance of chemical interactions for the stability of proteins during amino acid substitutions. Protein stability is experimentally measured with differential scanning calorimetry, circular dichroism, fluorescence spectroscopy, and so forth. The availability of such data in an electronically accessible database would be a valuable resource for the analysis and prediction of protein mutant stability. 

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