Protein stability, measurement

Site directed mutagenesis combined with protein stability measurements, as well as calorimetric studies and ensuing analyses have shown that protein stability results from a delicate balance between different physical contributions. Theoretical studies, aimed at rationalizing the experimental measures in terms of structural parameters and dynamic properties of the protein, " support this view. 

Aucamp, J.P., Cosme, A.M., Lye, G.J. andDalby, P.A. (2005) High-throughput measurement of protein stability in microtiter plates. Biotechnology and Bioengineering, 89, 599-607. 

As described in the following chapter, there are many biopharmaceutical applications of protein assays. Assigning the protein concentration for the drug substance, drug product, or in-process sample is often the first task for subsequent analytical procedures because assays for purity, potency, or identity require that the protein concentration be known. Hence it is typical for several different methods to be employed under the umbrella of protein concentration measurement, depending on the requirements of speed, selectivity, or throughput. The protein concentration is valuable as a stand-alone measurement for QC and stability of a protein. However, protein concentration methods provide no valuable... 

Powell, K.D. Fitzgerald, M.C. Measurements of protein stability by H/D exchange and matrix-assisted laser desorption/ionization mass spectrometry using picomoles of material. Ancd. Chem. 2001, 73, 3300-3304. 

Activity and stability measurements of the enzyme entrapped inside POPC vesicles against externally added Bz-Tyr-pNA. No activity against the larger substrate Suc-Ala-Ala-Pro-Phe-pNA or casein. Inhibition of externally present enzyme by an inhibitor protein. 

The important contribution of protein engineering to the study of protein stability is that defined structural changes can be made in a protein and the change in stability measured. In this way, the relationship between structure and stability can be established experimentally. Fortunately, it is easier to measure changes in stability accurately than it is absolute values of 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). 

Surface diffusion measurements in protein-stabilized films... 

C. N. Pace, Measuring and increasing protein stability, TIBTECH 1990, 8, 93-98. 

The term thermal stability (also thermostability) refers to the resistance of a protein to adverse intrinsic and extrinsic environmental influences, i.e., the thermal characteristic of the protein to remain steady against the dena-turation of its molecular integrity and inactivation of its biologic activity on facing high temperatures or other deleterious agents (6). One of the most important indices to measure protein stability is the decimal reduction time, or D-value, the time required to reduce 90% of the initial protein concentration exposed to the reference temperature. The D-value was used... 

Rief M, Gautel M, Schemmel A, Gaub HE (1998b) The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy. Biophys. J. 75 3008-3014... 

One global property suitable for evaluation of evolutionary intermediates is protein stability. The unique biological and physical properties of an enzyme are highly dependent on the integrity of its native conformation. The structural stability of a protein can be measured by its resistance to heat, acid, and various chaotropic agents. Measurements of the thermostability of lysozymes provide an easy and an accurate way to assess the protein stability. 

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