Proteins stability/engineering

The desymmetrization of l-alkylbicyclo[3.3.0]octane-2,8-diones can be achieved in a facile coenzyme-independent enzymatic reaction in buffer. Alkyl chains in the 1 -position of up to at least five carbon atoms are tolerated. The yields of the crude keto-acids are essentially quantitative, and the enantiotopic discrimination by the enzyme is usually excellent." Work remains to be done on the optimization of this biocatalyst with respect to protein stability and reaction engineering, but it remains a unique and intriguing possibility for the generation of interesting intermediates bearing multiple chiral centres. 

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. 

M. Niggemann and B. Steipe, Exploring local and non-local interactions for protein stability by structural motif engineering,... 

Because of the strict stereochemical requirements, it is not easy to find optimal sites for the introduction of disulfide bonds into proteins. Introduction of disulfide bonds into T4 lysozyme has been engineered by theoretical calculations and computer modeling.4 7 The results obtained from the mutant lysozymes illustrate several points relevant to the use of disulfide bonds for improving protein stability.6 (i) Introduction of the cysteine(s) should minimize the disruption or loss of interactions that stabilize the native structure, (ii) The size of the loop formed by the crosslink should be as large as possible, (iii) The strain energy introduced by the disulfide bond should be kept as low as possible. For this purpose, a location within the flexible part of the molecule is desirable. 

Under these conditions, it is possible to evaluate protein stability from denaturation experiments. This is not a subject of simply academic interest the increasing use of proteins in everyday use, for example as additives in washing powders, has made protein stability a matter of great practical importance, the more so since protein engineering has been used to improve stability properties. 

The combination of molecular modeling with genetic engineering to enhance protein stability has been successful in certain cases. For instance, introducing carefully sited novel disulfide bonds increased protein stability in T4 lysozyme (11-13) and in X-repressor (14). However, the results in other proteins, for instance, in subtilisin (15,16) and in dihydrofolate reductase (17) have been less predictable. 

Serrano L, Day AG, Fersht AR (1993) Step-wise mutation of bamase to binase. A procedure for engineering increased stability of proteins and an experimental analysis of the evolution of protein stability. J Mol Biol 233(2) 305-312... 

Dickinson, E. 1994. Protein-stabilized emulsions. Journal of Food Engineering 22 59-74. 

One of the most pretentious approaches for future biochemical engineering would consist of tailoring proteins to desired fvmctions by protein engineering. Pioneering work has for example been done in the area of biocatalysis, but it is commonplace that rational exploitation of protein engineering will require an enormous amount of additional knowledge on the primary - tertiary structure - function relationships. These again emphasize the importance of thermodynamics in the area of protein stability. 

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