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  • Title: Protein design through systematic catalytic loop exchange in the (beta/alpha)8 fold.
    Author: Ochoa-Leyva A, Soberón X, Sánchez F, Argüello M, Montero-Morán G, Saab-Rincón G.
    Journal: J Mol Biol; 2009 Apr 10; 387(4):949-64. PubMed ID: 19233201.
    Abstract:
    Protein engineering by directed evolution has proven effective in achieving various functional modifications, but the well-established protocols for the introduction of variability, typically limited to random point mutations, seriously restrict the scope of the approach. In an attempt to overcome this limitation, we sought to explore variant libraries with richer diversity at regions recognized as functionally important through an exchange of natural components, thus combining design with combinatorial diversity. With this approach, we expected to maintain interactions important for protein stability while directing the introduction of variability to areas important for catalysis. Our strategy consisted in loop exchange over a (beta/alpha)(8) fold. Phosphoribosylanthranilate isomerase was chosen as scaffold, and we investigated its tolerance to loop exchange by fusing variant libraries to the chloramphenicol acetyl transferase coding gene as an in vivo folding reporter. We replaced loops 2, 4, and 6 of phosphoribosylanthranilate isomerase with loops of varied types and sizes from enzymes sharing the same fold. To allow for a better structural fit, saturation mutagenesis was adopted at two amino acid positions preceding the exchanged loop. Our results showed that 30% to 90% of the generated mutants in the different libraries were folded. Some variants were selected for further characterization after removal of chloramphenicol acetyl transferase gene, and their stability was studied by circular dichroism and fluorescence spectroscopy. The sequences of 545 clones show that the introduction of variability at "hinges" connecting the loops with the scaffold exhibited a noticeable effect on the appearance of folded proteins. Also, we observed that each position accepted foreign loops of different sizes and sequences. We believe our work provides the basis of a general method of exchanging variably sized loops within the (beta/alpha)(8) fold, affording a novel starting point for the screening of novel activities as well as modest diversions from an original activity.
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