Protein stability information

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. 

Analysis of the dependence of the structural thermodynamics of globular proteins on apolar surface area provides an estimation of the role of various contributions to protein stability. However, as mentioned above, proteins also show convergence temperatures that can yield similar information, given certain assumptions. 

The effects of single amino acid substitution on the conformational stability of the a subunit have been extensively investigated 43-46 58) These studies provide important information on factors that influence protein stability. They also help to characterize the effects of mutation on protein conformation. 

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. 

The combination of the energy distribution and the number of states that are allowed at each position constitute the alphabet. For model proteins, the complex dynamics of the side chains are often condensed into the energy term fij, whose purpose is usually to model protein stability. The simplest alphabet reduces the information contained in... 

ProTherm (16) is a large collection of thermodynamic data on protein stability, which has information on 1) protein sequence and stmcture (2) mutation details (wild-type and mutant amino acid hydrophobic to polar, charged to hydrophobic, aliphatic to aromatic, etc.), 3) thermodynamic data obtained from thermal and chemical denaturation experiments (free energy change, transition temperature, enthalpy change, heat capacity change, etc.), 4) experimental methods and conditions (pH, temperature, buffer and ions, measurement and method, etc.), 5) functionality (enzyme activity, binding constants, etc.), and 6) literature. 

In the above expressions, F2 (/) is the preferential binding parameter for site /, yj and 73 are the activity coefficients of water and cosolvent (in a molar fraction scale) in a protein-free mixed solvent, and is the equilibrium constant for the exchange equilibrium (eq 26) on site i. For an ideal mixed solvent, Ki = K-. In the Schellman model, it is necessary to know how the protein surface is subdivided into various kinds of sites. Such information is not available for real protein solutions. However, when some simplifications are made, the Schellman model can provide some information regarding the effect of various cosolvents on the protein stability. 

Mass spectrometry-based techniques have distinct advantages over existing methods in terms of sensitivity, protein stability, and extended mass range. Furthermore, the solubility and purity of a protein are of lesser concern. Other techniques are limited to provide information that represents an average of entire protein ensembles, whereas mass spectrometry can reveal structural details on transient or folding intermediates. 

Protein stability during encapsulation in biodegradable polymer microparticles has been reviewed in detail [6,23,27], In comparison, little information is available on lipid materials. However, conditions causing stability problems are not specific for polymer microparticle formulations. Lipids, being a hydrophobic material like many biodegradable polymers, may involve similar processing parameters [22,28],... 

It is possible to create simplified models that avoid atomic detail and instead rely on a more schematic residue-based view. A great deal is known from polymer theory about the behavior of these simplified models. Models can be constructed with a minimal amount of information and tested to see if they exhibit the behavior of actual proteins. If they do, then fundamental questions can be raised about protein stability. These include the relative importance of various constraints and intramolecular interactions. In this way, qualitative insights into protein conformation and folding can be gained. Because these models are simplified, many of the terms in the potential function do not correspond directly to actual energies, but instead are parameterized empirically to produce observed properties of interest. Skolnick and Kolinski have recently reviewed these topics. ... 

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