Rational protein design

There have been many attempts to improve protein stability and protein properties, utilizing methods such as random mutagenesis, directed evolution, and rational protein design approaches. In general, these methods are far from straightforward and can be time-consuming. In addition, the stabilization of proteins without loss of function is not a trivial problem. 

Hellinga, H. W. (1997). Rational protein design Combining theory and experiment. Proc. Natl. Acad. Sci. USA 94, 10015-10017. 

U. T. Bornscheuer and M. Pohl, Improved biocatalysts by directed evolution and rational protein design, Cun. Opin. Chem. Biol. 2001, 5, 137-143. 

Schomburg D (1994) Rational protein design of proteins with new properties. In Wrede P, Schneider G (eds) Concepts in protein engineering and design. De Gruyter, Berlin, p 169... 

Major advances in the knowledge of biochemical pathways and the establishment of computer-based predictions of three-dimensional structures of proteins led to the development of new microbiological methods, such as rational protein design or directed evolution, giving scientists the possibility to provide tailor-made biocatalysts [15-19]. These methodological works on the disclosure of new efficient biocatalysts are not explicitly mentioned in this review unless they were applied in natural product synthesis. Biotransformations do not always compete with known chemical syntheses, but rather complement the portfolio of catalytic methods in organic chemistry. 

The lipase-catalysed access to enantiomerically pure compounds remains a versatile method for the separation of enantiomers. The selected examples shown in this survey demonstrate the broad applicability of lipases in terms of substrate structures and enantioselectivity. More recently, modem molecular biology methods such as rational protein design and especially directed evolution103 will further boost the development of tailor-made lipases for future applications in the synthesis of optically pure compounds. It has been already shown that a virtually non-enantioselective lipase (E=l.l in the resolution of 2-methyldecanoate) could be evolved to become an effective biocatalyst (E>50). Furthermore, variants were identified which showed opposite enantiopreference. 

Fig. 13.3. Correlation of required mechanistic information, required structural information, importance of screening and number of possible enzyme variants in protein engineering by rational protein design and directed evolution.

The examples provided in this Chapter demonstrate that directed evolution resembles a very useful tool to create enzyme activities hardly accessible by means of rational protein design (Table 14.1). Even if the desired substrate specificity is known from other biocatalysts - e.g. phospholipase A1 activity - the advantage of the directed evolution approach resides in the already achieved functional expression of a particular protein. Thus bottlenecks arising from the identification of enzymes by traditional screening and cultivation methods can be circumvented. In addition, directed evolution can dramatically reduce the time required for the provision of a suitable tailor-made enzyme, also because cloning and functional expression of the biocatalyst has already been achieved. 

Hellinga, H.W. 1997. Rational protein design Combining theory and experiments. Proceedings of the National Academy of Science USA, 94 10015-7. 

During the past decade, many enzymes have been improved by directed evolution using random mutagenesis, rational protein design, or via a combination of both methods [12, 13]. Early examples were committed to the enhancement of enzyme stability under unusual conditions such as in the presence of organic solvents or towards increased activity. More recently, directed evolution has been used for tlie creation of enantioselective biocatalysts or, even more, for the reversal of enantioselectivity of an enzyme-catalyzed reaction [72, 73]. 

An inverse task is given when there is demand for a macromolecule that specifically binds a small ligand. This question has only recently been addressed by peptide chemistry. For example, antiparallel bundles of four a-helices, which were assembled on a cyclic peptide structure as template, have been used to create hydro-phobic cavities for heme as a low-molecular-weight compound [3]. The specific complexation of Fe " protoporphyrin IX was facilitated by the proper positioning of liganding His residues. While this approach could be interesting from the perspective of rational protein design, it may be limited to special applications, and detailed structural information about the complex is not yet available. 

Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den Heuvel, J., Gruber, A.D., Heinz, D.W., Lengeling, A., Schubert, W.D. 2007. Extending the host range of Listeria monocytogenes by rational protein design. Cell 129 891-902. 

The above-mentioned incremental nature of protein stability has been established by detailed studies on model proteins such as phage T4 lysozyme, and by rational protein design. As mentioned, stabilization may involve all levels of the hierarchy of protein structure, local packing of the polypeptide chain, secondary and supersecondary structural elements, domains, and subunits. Approaches to assign specific structural alterations to changes in stability are summarized in Table I. 

Rational protein design based on structural knowledge of the protein of interest and computational modeling and... 

Rational Protein Design Process of making desired changes to a protein based on the knowledge of the structure and function of that protein. 

Ability to improve lipase properties (e.g. by genetic engineering and/or directed evolution and/or rational protein design and/or mutagenesis) Immobilized lipases can be used many times... 

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