Ribonuclease thermodynamic parameters

A good example of application is given by the protein structural changes of bovine ribonuclease A in the course of its denaturation by pressure. The UV spectrum of RNase is dominated by the absorbance of tyrosine - this RNase does not contain tryptophan. As shown in Figure 6, an increase of pressure from 1 to 500 MPa results in a blue-shift of the 4th derivative maximum from 285.7 0.05 to 283.5 0.05 nm. This shift of 2.2 nm corresponds to an increase of the mean dielectric constant from 25 to 59. It is characteristic of the exposure to the aqueous solvent of part of the 6 tyrosines, as it is expected for a partly denaturation. The transition is fully reversible with clear isosbestic points. The pressure effect can therefore be described by a simple two-state model between the native (e,. = 25) and the partially denatured (e,. = 59) state. A simulation on the basis of this model permitted us to determine the thermodynamic parameters of this transition AG° = 10.3 kJ/mol and AV = - 52 ml/mol. A comparison with results obtained by other methods indicates that the (e,. = 59) state corresponds to an intermediate in the defolding process which has molten globule like characteristics [12]. It thus appears that fourth derivative... 

There are now well over 200 publications in which microcalorimetry has specifically been used to study protein-hgand interactions of a variety of types. A fist of these studies is readily available by a MEDLINE search or from ITC equipment suppliers. Since the studies are too numerous to review here, perhaps a recent one might serve as a representative example of the technique and of its application. In this example [40] we determined the thermodynamic parameters associated with the binding of the reversible inhibitor 2 -CMP (2 -cytidine monophosphate) to RNAse-A (ribonuclease A). We were specifically interested in the binding under conditions that were relatively physiological, i.e., at body temperature and in a buffer that contained multiple ions at roughly cellular concentrations. 

A, B, C, D depend on the extracted protein and are functions of AGo, oc, e, A i, n, pi, Na, z. Their numerical value have been calculated from experimental data on solubilization of ribonuclease and concanavalin A in AOT/isooctane with a good correlation to the model equation. The great interest of this model is that all the assumptions necessary for its elaboration make it very simple, and at the same time, a promising tool of quantification of protein solubilization thermodynamics, even if some further refinements are still needed. It can be noted that there are more parameters than can be adjusted from experimental data. As a consequence, the model can provide no value for n, related to the micelle size, which could have permitted an interesting comparison with that predicted by Caselli et al. s model. 

The tertiary structure describes the complete three-dimensional stmcture of the whole polypeptide chain. It includes the relationship of different domains formed by the protein s secondary structure and the interactions of the amino acid substituent -R groups. An example of a protein chain with a-helices and /3-folding, the enzyme ribonuclease, is shown in Fig. 1.17. The specific folding of a protein is only thermodynamically stable within a restricted range of environmental parameters, i.e. the right temperature, pH and ionic strength. Outside of this range, the protein could unfold and lose its activity. 

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