Engineering/engineered protein folding

Engineering Protein Folding and Secretion Pathways to Enhance Productivity 701... 

Finkelslain A V 1997. Can Protein Unfolding Simulate Protein Folding Protein Engineering 10 843... 

Noncovalent Forces Stabilizing Protein Structure. Much of protein engineering concerns attempts to alter the stmcture or function of a protein in a predefined way. An understanding of the underlying physicochemical forces that participate in protein folding and stmctural stabilization is thus important. 

Through combined effects of noncovalent forces, proteins fold into secondary stmctures, and hence a tertiary stmcture that defines the native state or conformation of a protein. The native state is then that three-dimensional arrangement of the polypeptide chain and amino acid side chains that best facihtates the biological activity of a protein, at the same time providing stmctural stabiUty. Through protein engineering subde adjustments in the stmcture of the protein can be made that can dramatically alter its function or stabiUty. 

Fersht, A. R., Matouschek, A., and Serrano, L. (1992). The folding of an enzyme. 1. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771-782. 

In addition to identifying protein partners, yeast two-hybrid technology can be used to identify and study in detail the interaction domains between two proteins. Here, bait and/or fish truncation or deletion constructs of the parent proteins are engineered and characterized as described earlier (see 3.1 Selection and characterization of bait constructs). These are then investigated for association in a yeast two-hybrid interaction assay. Once the BD has been identified, it can be further refined by mutagenesis. The same caveat applies to these studies as for the identification of associating proteins, i.e., it is assumed that the respective fusion proteins fold and adopt the same or a similar three-dimensional conformation to the native protein. This is not always the case and results should be interpreted with caution and if possible, always validated by an alternative experimental approach. O Table 19-1 shows an example of mapping the... 

Because of its structural simplicity and instrumental role in protein function, the coiled coil is one of the most investigated protein folding motifs. Native coiled-coil sequences and their mutants have been synthesized and studied. Numerous model coiled-coil peptides have been designed de novo [22]. Although many theoretical questions remain unanswered, much has been learned about the sequence-structure relationship. It is even possible to design and engineer new coiled-coil sequences and structures that have never existed before [23]. 

Phillips, D C. Protein Engineering/ Review (Umv. of Wales), 46 (March 1987). Richards, F.M. The Protein Folding Problem, ScL Amer., 54 (January 1991). Radousky, H.B., G. Hammond, Z. Xu, et al. Gene Families Studies of DNA, RNA, Enzymes and Proteins, World Scientific Publishing Company. Inc., River Edge, NJ, 2001. 

Despite sharing only 25% sequence identity, structural analysis indicates that both proteins of E. coli NADP-IDH and T. thermophilus NAD-IMDH are homodimers which share a common protein fold that lacks the p p p motif characteristic of the nucleotide binding Rossmann fold [23], The strict and distinct specificities of these enzymes provide an attractive model system for engineering specificity, while the extensive knowledge of substrate and coenzyme binding and catalysis provide the sound foundation critical for rational design. 

Computational biochemistry and computer-assisted molecular modeling have rapidly become a vital component of biochemical research. Mechanisms of ligand-receptor and enzyme-substrate interactions, protein folding, protein-protein and protein-nucleic acid recognition, and de novo protein engineering are but a few examples of problems that may be addressed and facilitated by this technology. 

The correctness of hits corresponding to families with known structural information is further verified by employing the protein fold recognition method PHYRE (Protein Homology/analogY Recognition Engine) version 0.2, which assesses the compatibility of a sequence to a three-dimensional structure (29). 

The multidisciplinary approach described above has been used to study protein folding phenomena and protein SARs for protein engineering endeavors, as well as protein-based and pharmacophore drug design. The validity of the predicted structure obviously depends on many factors, including the accuracy of the structures... 

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