Denaturation curves

Figure 12, Heat denaturation curves of hemoglobin from three members of a family with Hb-Leslie, a newly discovered unstable variant with a deletion of residue 131 of the p-chain, G.P.Sr. has Hb-Leslie p-thalassemia the %Hb-Leslie is 85% (DEAE-Sephadex chromatography) Gr.P. has Hb-LesUe- Hb-C %Hb--Leslie is 28% M.B. fm Hb LesUe trait %Hb Leslie is 28%,...

Fig. 23 Solvent denaturation curves of 45 measured by a the UV absorbance ratio A303M287 and b the circular dichroism (g bs at 316 nm) showing the need for higher vol% acetonitrile to bring about twist sense bias than that needed for folding...

The use of differential scanning microcalorimetry for measuring the thermal denaturation of proteins is described in Chapter 17, section Ale. Typically, 0.5-1 mg of protein in 1 mL of buffer, or 0.1-0.2 mg in 0.5 mL with the most sensitive apparatus, is required for an accurate determination of the enthalpy of denaturation. The thermodynamics of dissociation of a reversibly bound ligand may be calculated from its effects on the denaturation curve of a protein.14 The binding of ligands always raises the apparent Tm (temperature at 50% denaturation) of a protein because of the law of mass action the ligand does not bind to the denatured state of the protein, and so binding displaces the denaturation equilibrium toward the native state. 

Fig. 10. Predicted excess heat capacity function versus temperature for myoglobin. The curve simulates the experimental curve obtained at pH 3.83 by Privalov et al. (1986). Under those conditions both the cold and heat denaturation curves can be studied experimentally. The predicted values are Tm,cold = 4°C Tm>heat = 58°C A// = 59 kcal mol-1 ACp = 2.45 kcal K-1 mol-1. The experimental values are Tm>coid = 3°C Tm>heat = 57.5°C AH = 53 kcal mol-1 ACP = 2.5 kcal K-1 mol-1 (Privalov et al., 1986). [Reprinted from Freire and Murphy (1991).]...

Another common indication of protein stability is the concentration of either urea or GnHCl required to unfold half of the protein available. This concentration, given the symbol [D]i/2, is analogous to the Tm value from thermal denaturation curves. Increase or decrease in [D]i/2 is presumed to indicate a corresponding increase or decrease in protein stability, respectively. Analysis of these curves can also provide thermodynamic information [123-126]. As these experiments can be done at any temperature, they are more useful in that they can provide information regarding stability at or near room temperature. 

At the midpoint of the denaturation curve, the guanidine concentration is called Cm, and the free energy A Gy is zero. This yields... 

Figure 2 Thermal denaturation curves for wild-type MntR (8 xM) monitored by CD spectroscopy at 222 nm unfolding of apo MntR (open squares) and of MntR in the presence of 1.0mMMg +, Ca +, Mn +, Co +, Ni +, and Cd +. Buffer 20mM HEPES, pH 7.2 at 4 °C, 200 mM NaCl, 5% (v/v) glycerol. Pathlength 2 mm. (Reprinted with permission from Golynskiy, Davis, Hehnann and Cohen. 2005 American Chemical Society)...

Tubular membrane tension/mucosal DNA thermal melting point midpoint of thermal denaturation curve Transmembrane Trimethylamine oxide Transmembrane domain Trimethylsilyl thimersol Tobacco mosaic virus Treose nucleic acid 5-thio-2-nitrobenzoate... 

Figure 5-13. Protein unfolding by chemical denaturation. The unfolding and refolding of proteins caused by the addition of guanidinium chloride (GdnHCI) can be monitored from the CD spectrum, usually at 220 nm. The diagram on the left shows the denaturation curve as expressed by the concentration dependence of...

Unfolding can also be induced by temperature, and thermal denaturation curves (melting curves) are experimentally easier to obtain, since measurements are carried out only on a single protein solution. It is also easy to check the reversibility of the process by slowly cooling the denatured protein solution. The parameter A G°s can be obtained from melting curves provided either that the van t Hoff plot... 

Urea denaturation curves were determined by measuring the intrinsic fluorescence intensity (278 nm excitation and 320 nm emission) of solutions containing approximately 0.9 p.M protein in sodium acetate/acetic acid, pH 5.0 buffer and increasing concentrations of urea in a temperature regulated Peikin-Elmer MPF 44 B spectrophotometer. Solutions were incubated at 25 °C for 24 h before measurements were taken. The free energy of unfolding was calculated by the linear extrapolation method (17). The error in AG was 0.6 kcal/mol. 

Figure 2. Circular Dichroism of ZDD. A, Spectra of the fully zinc-coordinated form (native), a Zn2 species, and an apo-form of the fragment. B, Thermal denaturation curves of native ZDD as compared to a Zn2 species.

Equilibrium Denaturation. A variety of different techniques can be employed to monitor protein conformational changes in the presence of denaturants. Activity measurements reflect the extent of alterations of the active site environment. However, enzyme activity measurements may be affected the presence of denaturant in the assay mixture. The denaturation curves obtained by this method are difficult to inte ret and can only be taken as a first approximation of the unfolding transition. U.V. difference spectra indicate conformational changes by monitoring the degree of solvent exposure of aromatic amino-acid side chains. Finally, fluorescence intensity measurements can reveal the nature of the environment (polar, non-polar) of the four tryptophans of p-lactamase. 

Figure 5 Thermal denaturation curves (upper) and emulsifying properties (bottom) of polymannosyl and oligomannosyl lysozymes constructed by genetic engineering. , wild-type lysozyme O, polymannosyl lysozyme , oligomannosyl lysozyme.

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