Protein folding half-concentration

To check for the presence of an intermediate in a protein folding process, the temperatures at which the secondary structure (Tsec) and the tertiary structure (Ttert) of the folded conformation are half-formed can be compared. If both coincide, the protein loses the tertiary and the secondary structures simultaneously, and only a native conformation with secondary and tertiary structures ordered or an unfolded conformation with both structural levels unordered describe the process. If significant differences are observed in the crossing temperatures of concentration profiles, a new, intermediate third species with the secondary structure ordered and the tertiary unordered may be needed to explain the shift in the appearance of the tertiary and secondary structures. The difference of almost 20°C found between Tsec and Ttert in the above two experiments seems to guarantee the presence of an intermediate conformation in the folding of a-apolactalbumin, but only the multivariate resolution analysis of the suitable measurements (far-UV and near-UV CD spectra) together can confirm this hypothesis and model the appearance of the intermediate conformation. 

A partially purified cyclic GMP-activated protein kinase from lobster muscle was later found to have a for cyclic GMP of about 0.08 /x.M and for cyclic AMP of about 4 /aM. A cyclic AMP-activated protein kinase isolated from the same tissue had an apparent Ka for cyclic AMP of about 0.02 /J.M and for cyclic GMP of about 1.2 ptM [76]. Cyclic GMP-dependent phosphorylation of endogenous protein has been demonstrated in membranes of mammalian smooth muscle [78]. In the presence of 10 mM Mn, a half-maximal increase in the phosphorylation of these proteins occurred with 20-30 nM cyclic GMP, but ten-fold higher concentrations of cyclic AMP were required to produce the same increase in phosphorylation. 

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... 

Mixed systems of sodium caseinate + phosphatidylcholine have been recently investigated (Semenova el al., 2008). It was found that lecithin oxidation was much reduced (or even absent) when the phospholipid was complexed with the protein (see Figure 2.4). In addition, the half-life of foams stabilized by complexes of sodium caseinate (1 wt%) with phosphatidylcholine (10 6— 10 3 mol/dm3) showed a four-fold increase, as compared to the protein alone over the range of experimental conditions studied (pH 5.5-7.0 ionic strength 0.001-0.01 M). It was also noted that a solution of pure phosphatidylcholine did not give a fine stable foam for these concentrations and under these experimental conditions. 

The specific effects of purified dnaj and grpE proteins on the dnaK protein have been characterized (Liberek et al., 1991a). The dnaj and grpE proteins, acting in concert, can stimulate the basal ATPase activity of dnaK 50-fold dnaj appears to enhance the rate of nucleotide hydrolysis by dnaK, but does not substantially affect nucleotide release. The grpE protein appears to enhance the rate of release of nucleotide bound to dnaK. Consequently, neither protein alone exerts a dramatic effect on the overall rate of ATP turnover by dnaK however, the effect of the two proteins combined is to accelerate both the rate of ATP hydrolysis and the rate of nucleotide release, resulting in enhanced ATP turnover. The concentrations of dnaj and grpE required for half-maximal effect are both 0.1-0.2 fiM. 

For complete alkylation of half-cystine residues, after the DTT reduction procedure described in 3.8.2.1, (2-bromoethyl) trimethyl-ammonium bromide is added in a 3-fold molar excess over the DTT concentration. After reaction for 24 to 48 hr under nitrogen, at room temperature, the protein derivative is desalted either by gel filtration, or exhaustive dialysis, and lyophilized. 

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