Cold denaturation

Cp X) T) function have been obtained from equations 65 and 66 - see section 5.5. 

If A Cp is zero there is no cold denaturation, because only one temperature exists at which A G vanishes. For a reliable extrapolation of protein stability over a long temperature interval it is therefore of utmost importance to know accurately the heat capacities of the native and denatured state of the protein in the same temperature region. 

Often ACp has been determined by performing measurements at differ- 

The slope of this curve is then assumed to correspond to ACp according to equation 6. But this procedure is problematic, since the slope of the plot is not only determined by the intrinsic change in heat capacity of the polypeptide chain, but also by ligation effects which include piotonatioii changes [9,22], Furthermore ACp generally cannot be expected to be temperature independent, due to the curvature in the Cp D function and the linearity of Cp,N [23]. 

To derive such a stability relation for a two-state transition N nD of an oligomeric protein, N,j, consisting of n non-covalently linked monomers (subunits), we proceed in the following manner. Aj G (T) is written as a Taylor series 

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Korhonen, H., Pihlanto-Leppala, A., Rantamaki, P., and Tupasela, T. (1998). Impact of processing on bioactive proteins and peptides. Trends Food Sci. Technol. 9,307-319. Kunugi, S. and Tanaka, N. (2002). Cold denaturation of proteins under high pressure. Biochim. Biophys. Acta 1595, 329-344. 

Near-UV CD of denatured proteins also provides evidence for some order in the side chains, especially in urea- and cold-denatured proteins. Nolting et al (1997) found a broad positive band with possible vibrational... 

Table V shows the results of this analysis for the Pn-helix fraction of several proteins denatured by heat, cold, acid, and Gdm HCl/urea. There is rather good consistency among the estimated Pn-helix contents for proteins denatured by a given agent, except for acid-denatured proteins, which show more variability. The chemically denatured proteins have 30 5% Pn-helix content near 0°C. At the other extreme, heat-denatured proteins have Pn-helix contents near 0%, with lysozyme having the highest value (8%). Although there are only two examples of cold-denatured proteins in Table V,2 they both have Pn-helix contents of about 20%. Acid-denatured proteins have Pn-helix contents ranging from 0 to 16%.

The cold-denatured state clearly has residual structure in that it is collapsed and exhibits a fairly globular Kratky scattering profile. Truncated, equilibrium unfolded states appear similarly compact (and have been shown by other spectroscopic means to have structure). But what of the highly expanded, urea, GuHCl, or thermally denatured states ... 

D. 4a(S),8a(R)-2-Benzoyloctahydro-6(2H)-isoquinolinone (4). Palladium (Pd), 10% on carbon, 4.0 g, (Note 21) is placed in a 500-mL Parr bottle under N2 and carefully wetted with 50 mL of cold denatured ethanol (EtOH). A slurry of 34.7 g of enone 3 (0.14 mol) in denatured EtOH (250 mL) is added and the Parr shaker apparatus assembled. After the system is purged with nitrogen-hydrogen (N2/H2), the reaction is shaken at 50 psi H2 and 50°C until H2 uptake is complete (1 hr, Note 22). The catalyst is filtered over a Celite pad (Note 23) and rinsed with warm chloroform (CHCI3) (4 x 75 mL). The filtrate is concentrated under reduced pressure, dissolved in 90 mL of CH2CI2 and crystallized with 200 mL of hexanes. The crystalline solid is filtered, rinsed with hexanes and dried to afford 34.3 g (98%, Note 24) of the ketone 4, representing a 51% yield over four steps. 

Figure 13.9 Temperature response trace (at optimal Tm solution conditions) of AGu, for pepsinogen at pH 6, with and without 20% ethanol (EtOH). Labels shown are identified as Ted = cold denaturation temperature, Tms = temperature of maximum stability, and Tra...

This phosphotransferase [EC 2.7.2.1] catalyzes the thermodynamically favored phosphorylation of ADP to form ATP Aeq = [ATP][acetate]/ [acetyl phosphate] [ADP] = 3000). GDP is also an effective phosphoryl group acceptor. This enzyme is easily cold-denatured, and one must use glycerol to maintain full catalytic activity. Initial kinetic evidence, as well as borohydride reduction experiments, suggested the formation of an enzyme-bound acyl-phosphate intermediate, but later kinetic and stereochemicaT data indicate that the kinetic mechanism is sequential and that there is direct in-line phosphoryl transfer. Incidental generation of a metaphosphate anion during catalysis may explain the formation of an enzyme-bound acyl-phosphate. Acetate kinase is ideally suited for the regeneration of ATP or GTP from ADP or GDP, respectively. 

Selected entries from Methods in Enzymology [vol, page(s)] Acetate assay with, 3, 269 activation, 44, 889 activity assay, 44, 893, 894 alternative substrates, 87, 11 bridge-to-nonbridge transfer, 87, 19-20, 226, 232 chiral phosphoryl-ATP, 87, 211, 258, 300 cold denaturation, 63, 9 cysteine residues, 44, 887-889 equilibrium constant, 63, 5 exchange properties, 64, 9, 39, 87,... 

This formalism may also be valuable in addressing protein unfolding, DNA unwinding, cold denaturation, and effects of site-direct mutagenesis on lysozyme unfolding. ... 

C and Tm = -19°C and +57°C, i.e., the protein is denatured by either cooling below 18°C or heating above 57°C, a behavior that is common for many proteins. Cold denaturation is observed whenever the unfolded state has a higher heat capacity than does the folded state.647... 

The early stages of folding of barstar have been measured on the microsecond time scale by temperature jumping its cold-denatured state from 2 to 10°C.65,66 There is the fast formation of a folding intermediate (tm 200 fxs) with the peptidy 1-proline 48 bond trans, followed by the formation with ty2 60 ms of a second intermediate that is highly native-like because it binds to and inhibits barnase. The native-like intermediate then undergoes trans cis peptidyl-proline isomerization on the time scale of minutes to give the final native structure (equation 19.2). 

Ts is the temperature of maximum stability, at which the AG(T) versus T curve passes through its maximum, i.e., AG(TS) > 0. At Tm, the maximum temperature of stability, AG (T) = 0. By necessity, Ts < Tm. There is another special point in the temperature stability (AG(T)) curve, however as the temperature stability curve resembles a inverted parabola, there must be a second intersection point with the line of minimum stability, AG(T) = 0. This intersection point is the minimum of the range of temperature stability and is called cold denaturation temperature, Tc. The cold denaturation temperature is practically never taken into consideration in the discussion of temperature stability of biocatalysts. One reason lies in its frequent inaccessibility often, Tc is below 0 °C and thus cannot be measured in water or any other mostly aqueous medium. 

Mikulecky, P. J., and Feig, A. L. (2004). Heat capacity changes in RNA folding Application of perturbation theory to hammerhead ribozyme cold denaturation. Nucleic Adds Res. 32, 3967-3976. 

Figure 5 also illustrates other general features of globular protein stability. The positive overall ACp results in the existence of two temperatures at which AG° is zero. The low temperature point defines the so-called cold denaturation of the protein and the high temperature point defines the heat denaturation. Additionally, the curvature in AG° implies the existence of a temperature of maximal stability. This temperature occurs at the point where AS0 is equal to zero. The... 

Fig. 8. Experimental ( — ) and theoretical heat capacity functions for the thermal folding/unfolding transition of phosphoglycerate kinase at pH 6.5 in the presence of 0.7 M GuHCl. The heat denaturation transition is characterized by a single peak, whereas the cold denaturation displays two peaks corresponding to the independent unfolding of the N and C domains. The experimental curve has been published before (Griko et al., 1989). As discussed in the text, the theoretical curve does not represent the best fit to the experimental data, but rather the calculated curve using structural information in conjunction with thermodynamic information for elementary interactions. [Reprinted from Freire et al. (1991).]...

Fig. 9. Calculated overall free energy of stabilization (AGtota ) for yeast phos-phoglycerate kinase at pH 6.5 and 0.7 M GuHCl. This curve displays two zeros, corresponding to the temperatures of cold and heat denaturation. Also shown in the curve are the cooperative Gibbs free energies (AG ) associated with the uncompensated exposure of apolar surfaces on unfolding of each of the domains. For both domains, AG is positive for the heat denaturation and close to zero for the cold denaturation. This behavior results in a cooperative heat denaturation and a non-cooperative cold denaturation. [Reprinted from Freire el al. (1991).]...

The thermal stability of metmyoglobin and apomyoglobin has been extensively studied under different solvent conditions (Privalov et al., 1986). In particular, it was shown that at low pH values both the heat and cold denaturation peaks are clearly visible in the calorimetric scans. Figure 10 shows the excess heat capacity function for apomyoglobin predicted by the hierarchical partition function and the thermodynamic parameters described above. In order to simulate the experimental curve obtained at pH 3.83 (Privalov et al., 1986), the protonation of five specific histidine residues on unfolding was... 

As seen in Fig. 10, the model accurately predicts the presence, location, and area of the cold and heat denaturation peaks. Under these conditions, the hierarchical partition function predicts a heat denaturation peak centered at 58°C and a cold denaturation peak centered at 4°C. The enthalpy change for the heat denaturation peak is 59 kcal mol-1 and the ACp is equal to 2.45 kcal K-1 mol-1. The experimental values reported by Privalov et al. (1986) are 57.5 and 3°C for the heat and cold denaturation transition temperatures, 53 kcal mol-1 for the enthalpy change, and 2.5 kcal K-1 mol-1 for ACp. Analysis of the theoretical curve indicates that it corresponds to a two-state transition, in agreement with the experimental data. The population of partially folded intermediates is never greater than 10-5 during the heat denaturation transition. 

One expects a significant amount of both the native and denatured protein structure in the vicinity of these two temperatures. The disruption of the native state on heating is usually called heat denaturation, since it proceeds with heat absorption and, consequently, with an increase in the molecular enthalpy and entropy. The disruption of the native structure on cooling, which we can call by analogy cold denaturation, should then proceed with a release of heat and, hence, with a decrease in enthalpy and entropy, because both of these functions have reversed their signs before reaching temperature 7 en. ... 

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