Stability of the protein

In Figure 7b, the data are plotted as AG yielding a linear function. Extrapolation to 2ero denaturant provides a quantitative estimate of the intrinsic stability of the protein, AG, which in principle is the free energy of unfolding for the protein in the absence of denaturant. Comparison of the AG values between mutant and wild-type proteins provides a quantitative means of assessing the effects of point mutations on the stability of a protein. 

If unidentified peaks are detected the stability of the protein under the chromatographic conditions should be checked. In all analytical investigations of proteins on SEC columns it is desirable to be able to monitor the eluted peaks at a very high sensitivity of the ultraviolet detector. Therefore, very pure (analytical grade) salts and buffers should be used. 

Fig. 3.2.7 Left panel Effects of temperature on the luminescence intensity and stability of the protein P from Meganyctiphanes. The initial light intensity was measured with F plus P in 5 ml of 20 mM Tris-HCl/0.15 M NaCl, pH 7.5, at various temperatures. In the stability test, P was kept at the indicated temperature for 10 min, then mixed with 5 ml of 25 mM Tris-HCl/1 M NaCl, pH 7.59, containing F, to measure initial light intensity. Right panel Effect of the concentration of salts on the light intensity of the luminescence of F plus P, in 25 mM Tris-HCl, pH 7.6, at near 0°C. In the case of NaCl, the light intensity decreased to about a half after 10 min. From Shi-momura and Johnson, 1967, with permission from the American Chemical Society.

Using a random mutagenesis approach, respiratory-deficient (34) and temperature-sensitive (46, 47) mutants of the Rieske protein of the yeast bc complex have been selected. A large fraction of the point mutants had changes of residues in the bottom of the cluster binding subdomain (the loop /S7-/38) and in the Pro loop comprising residues 174-180 of the ISF (Fig. 9 see Section III,B,3) this indicates the importance of the Pro loop for the stability of the protein. Amino... 

In summary, it appears that the protein has to adopt the correct fold before the Rieske cluster can be inserted. The correct folding will depend on the stability of the protein the Rieske protein from the thermoacidophilic archaebacterium Sulfolobus seems to be more stable than Rieske proteins from other bacteria so that the Rieske cluster can be inserted into the soluble form of the protein during expression with the help of the chaperonins. If the protein cannot adopt the correct fold, the result will be either no cluster or a distorted iron sulfur cluster, perhaps using the two cysteines that form the disulfide bridge in correctly assembled Rieske proteins. 

The storage modulus (G ) was recorded at a frequency of IHz under 0.015 strain amplitude until stabilization of the protein network. In order to reduce stress in the sample, G recording started just before the gelation time which corresponds to the time at which G deviated from the baseline. Data were collected and rheological parameters were calculated using Carri-Med 50 software. For each system, the experiments were performed in triplicate. 

An examination of mutant PKA proteins was undertaken. Phosphorylation of Thr-197 is required to activate PKA and phosphorylation of Ser-338 enhances stability of the protein. Replacement of Thr-197 and/or Ser-338 by Ala was examined to determine any conformational changes in the protein. Both single substitution mutants were expressed in Escherichia coli in similar levels to wild-type protein. However, both mutants were found to be less stable, as had been previously described. The double mutant... 

In addition to the additives used in a formulation to help stabilize the protein to freezing, the residual moisture content of the lyophilized powder needs to be considered. Not only is moisture capable of affecting the physicochemical stability of the protein itself, equally important is the ability of moisture to affect the Tg of the formulation. Water acts as a plasticizer and depresses the Tg of amorphous solids [124,137,138]. During primary drying, as water is gradually removed from the product, the Tg increases accordingly. The duration and temperature of the secondary drying step of the lyophilization process determines how much moisture remains bound to the powder. Usually lower residual moisture in the finished biopharmaceutical product leads to enhanced stability. Typically, moisture content in lyophilized formulations should not exceed 2% [139]. The optimal moisture level for maximum stability of a particular product must be demonstrated on a case-by-case basis. 

Environmental factors such as temperature (increases), humidity (violent change), pH and exposure to UV radiation can also influence the stability of the proteins, causing changes in their structure and lowering the resistance to biodeterioration. This decay is also influenced by the presence of other components such as lipids, carbohydrates, mineral... 

Disulfide exchange reactions occur over a broad range of conditions—from acid to basic pH—and in a wide variety of buffer constituents. Most crosslinking reactions involving disulfide exchange are done under physiological conditions or those most appropriate to maintain stability of the protein or other molecule being modified. 

Calculation of the internal cell potential is a very complicated matter because the electrochemistry of all of the species within the protocell would have to be balanced subject to their composition quotient Q, after which the standard free energy would have to be established from tabulations. The transport of Na+ would also change this balance, along with the ionic strength of the solution and the stability of the proteins or prebiotic molecules within the protocell. Such non-equilibrium thermodynamics forms the basis of the protocell metabolism. The construction... 

Temperature-sensitive mutations usually arise from a single mutation s effect on the stability of the protein. Temperature-sensitive mutations make the protein just unstable enough to unfold when the normal temperature is raised a few degrees. At normal temperatures (usually 37°C), the protein folds and is stable and active. However, at a slightly higher temperature (usually 40 to 50°C) the protein denatures (melts) and becomes inactive. The reason proteins unfold over such a narrow temperature range is that the folding process is very cooperative—each interaction depends on other interactions that depend on other interactions. 

The required high concentration of ligands limits the number of compounds that can be tested simultaneously, since the total amount of ligands is normally limited for reasons of solubility and stability of the protein. 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

The structure and function of enzymes is determined by both the amino acid sequence and the surrounding solvent. The overall stability of proteins is characterized by a subtle balance of into- and inter-molecular interactions. The basic principle of the structure (and of the stability) of the proteins is related to the nature of its normal enviromnent for (water) soluble globular proteins this is the minimization of the hydrophobic surface area, whereas the contrary is the case for membrane proteins (Jaenicke, 1991). 

Corn Corn has been used extensively for the expression of biopharmaceuticals. A great advantage is the stability of the protein in the seed, which is in turn easy to store and transport. 

---