Proteins, stability denaturation

Cell Disruption Intracellular protein products are present as either soluble, folded proteins or inclusion bodies. Release of folded proteins must be carefully considered. Active proteins are subject to deactivation and denaturation, and thus require the use of gentle conditions. In addition, due consideration must be given to the suspending medium lysis buffers are often optimized to promote protein stability and protect the protein from proteolysis and deactivation. Inclusion bodies, in contrast, are protected by virtue of the protein agglomeration. More stressful conditions are typically employed for their release, which includes going to higher temperatures if necessaiy. For native proteins, gentler methods and temperature control are required. 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

The following protocol for passive adsorption is based on methods reported for use with hydrophobic polymeric particles, such as polystyrene latex beads or copolymers of the same. Other polymer particle types also may be used in this process, provided they have the necessary hydrophobic character to promote adsorption. For particular proteins, conditions may need to be optimized to take into consideration maximal protein stability and activity after adsorption. Some proteins may undergo extensive denaturation after immobilization onto hydrophobic surfaces therefore, covalent methods of coupling onto more hydrophilic particle surfaces may be a better choice for maintaining native protein structure and long-term stability. 

Protein stability is just the difference in free energy between the correctly folded structure of a protein and the unfolded, denatured form. In the denatured form, the protein is unfolded, side chains and the peptide backbone are exposed to water, and the protein is conformationally mobile (moving around between a lot of different, random structures). The more stable the protein, the larger the free energy difference between the unfolded form and the native structure. 

Interfacial interaction between silicone and protein/starch microparticle, 3 and the use of polysiloxanes having hydrophilic groups for the stabilization of proteins against denaturation, 4 were studied. 

The study of reaction rates or kinetics of a particular denaturation process of a protein therapeutic can provide valuable information about the mechanism, i.e., the sequence of steps that occur in the transformation of the protein to chemically or conformationally denatured products. The kinetics tell something about the manner in which the rate is influenced by such factors as concentration, temperature, excipients, and the nature of the solvent as it pertains to properties of protein stability. The principal application of this information in the biopharmaceutical setting is to predict how long a given biologic will remain adequately stable. 

Shortle, D. 1996. The denatured state (the other half of the folding equation) and its role in protein stability. Faseb J 10 27-34. 

Commercial wines are commonly tested for protein stability. Wine proteins, upon denaturation by heat or cold, may cause cloudiness and unsightly deposits after bottling. In addition, proteins may combine with iron and copper salts to form flocculate material in bottled wines. The reaction and absorption of proteins on bentonite is an effective means of removing protein from wines (109, 110, 111). Therefore, fining wines... 

Helix stability and protein stability. We can predict the stability of helixes more reliably than we can any other element of protein structure. This provides a means for increasing the stability of proteins, because naturally occurring helixes are not always optimized for stability. If we make a mutation in the face of a helix that is exposed to solvent, and the mutation does not affect interactions elsewhere in the protein, then the overall free energy of folding of the protein generally changes by the same amount as the stability of the helix.47 This rule breaks down if we overstabilize the helix if the helix becomes so stable that it is still present as a helix in the denatured state, then increasing its stability further does not increase the stability of the protein, because both the native and denatured states are increased equally in stability. 

In Chapter 17, we discuss the effects of mutation of side chains on protein stability. A protein that has a native structure N and a denatured state D is converted in lo N and D by substitution of one of its amino acid residues. D and N differ only in their noncovalent interactions because none of the covalent bonds are altered on denaturation, as are D and N. We can measure the free energies of de-naturation directly (AGD N and AGD N>) and draw a cycle (scheme 4). 

Why denaturants such as urea and GdmCl cause proteins to denature may be considered empirically. Those denaturants solubilize all the constituent parts of a protein, from its polypeptide backbone to its hydrophobic side chains. To a first approximation, the free energy of transfer of the side chains and polypeptide backbone from water to solutions of denaturant is linearly proportional to the concentration of denaturant.7,8 Because the denatured state is more exposed to solvent than the native state, the denatured state is preferentially stabilized by denaturant. Thus, the free energy of denaturation at any particular concentration of denaturant is given by... 

CD spectra, particularly in the near-UV (see Support Protocol I) reflect the dynamics of the chromophore and may therefore show dependence on temperature. It is important to stabilize the temperature reproducibly. Accurate temperature control is particularly important in denaturation experiments to determine protein stability. 

One of the major problems that a biochemical engineer will encounter is that of the stability of protein materials. The biological function of the molecule is determined by its secondary and tertiary structures and if these are upset irreversibly then the protein becomes denatured. Denaturation can occur under relatively mild conditions as proteins are usually stable over only very narrow ranges of pH (e.g. 5-8) and of temperature (e.g. 10-40°C). The boiling of an egg illustrates this point. In its uncooked form the white is a slimy clear protein solution, but under acidic conditions, or when put in boiling water, the solution gels to white solid. 

Unlike most uncatalysed reactions as the temperature is raised the rate of an enzyme-catalysed reaction rises to a maximum and then decreases as the protein is denatured by the heat. Temperature affects not only activity but also the stability of the enzyme since the tertiary structure is particularly susceptible to thermal damage. 

Experimental studies of protein stabilities are numerous and there are still points of serious disagreement concerning the conclusions to be drawn from these studies. Areas of discord include the extent to which thermal denaturation corresponds to denaturation by chemical agents and the extent to which hydrophobic, van der Waals, and/or hydrogen bonds stabilize the native state. In this discussion, we will focus on the work of Peter L. Privalov.k Privalov has developed much of the microcalorimetric instrumentation that has made the calorimetric studies of proteins feasible. He has also published numerous review articles that summarize experimental data and formulate general observations concerning protein denaturation. His 1995 paper in Advances in Protein Chemistry9 presents a recent, comprehensive, review of the experimental results... 

A study of two of the most prominent and widespread osmolytes, betaine and beta-hydroxyectoine, by differential scanning calorimetry (DSC) on bovine ribonu-clease A (RNase A) revealed an increase in the melting temperature Tm of RNase A of more than 12 K and of protein stability AG of 10.6 kj mol-1 at room temperature at a 3 M concentration of beta-hydroxyectoine. The heat capacity difference ACp between the folded and unfolded state was significantly increased. In contrast, betaine stabilized RNase A only at concentrations less than 3 M. When enzymes are applied in the presence of denaturants or at high temperature, beta-hydroxyectoine should be an efficient stabilizer. 

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... 

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