Clarkbattle5262

The quest for an enzyme with desired property is high for biocatalyic production of valuable products in industrial biotechnology. Synthetic biology and metabolic engineering also increasingly require an enzyme with unusual property in terms of substrate spectrum and catalytic activity for the construction of novel circuits and pathways. β-Aminopropionitrile purchase Structure-guided enzyme engineering has demonstrated a prominent utility and potential in generating such an enzyme, even though some limitations still remain. In this chapter, we present some issues regarding the implementation of the structural information to enzyme engineering, and exemplify the structure-guided rational approach to the design of an enzyme with desired functionality such as substrate specificity and catalytic efficiency.The functional properties of proteins are decided not only by their relatively rigid overall structures, but even more importantly, by their dynamic properties. In a protein, some regions of structure exhibit highly correlated or anti-correlated motions with others, some are highly dynamic but uncorrelated, while other regions are relatively static. The residues with correlated or anti-correlated motions can form a so-called dynamic cross-correlation network, through which information can be transmitted. Such networks have been shown to be critical to allosteric transitions, and ligand binding, and have also been shown to be able to mediate epistatic interactions between mutations. As a result, they are likely to play a significant role in the development of new enzyme engineering strategies. In this chapter, protocols are provided for the assessment of dynamic cross-correlation networks, and for their application in protein engineering. Transketolase from E. coli is used as a model and the software GROMACS is applied for carrying out MD simulations to generate trajectories containing structural ensembles. The trajectory is then used for a dynamic cross correlation analysis using the R package, Bio3D. A matrix of all atom-wise cross-correlation coefficients is finally obtained, which can be displayed in a graphical representation termed a dynamical cross-correlation matrix.The goal of protein design is to create proteins that are stable, soluble, and active. Here we focus on one approach to protein design in which sequence information is used to create a "consensus" sequence. Such consensus sequences comprise the most common residue at each position in a multiple sequence alignment (MSA). After describing some general ideas that relate MSA and consensus sequences and presenting a statistical thermodynamic framework that relates consensus and non-consensus sequences to stability, we detail the process of designing a consensus sequence and survey reports of consensus design and characterization from the literature. Many of these consensus proteins retain native biological activities including ligand binding and enzyme activity. Remarkably, in most cases the consensus protein shows significantly higher stability than extant versions of the protein, as measured by thermal or chemical denaturation, consistent with the statistical thermodynamic model. To understand this stability increase, we compare various features of consensus sequences with the extant MSA sequences from which they were derived. Consensus sequences show enrichment in charged residues (most notably glutamate and lysine) and depletion of uncharged polar residues (glutamine, serine, and asparagine). Surprisingly, a survey of stability changes resulting from point substitutions show little correlation with residue frequencies at the corresponding positions within the MSA, suggesting that the high stability of consensus proteins may result from interactions among residue pairs or higher-order clusters. Whatever the source, the large number of reported successes demonstrates that consensus design is a viable route to generating active and in many cases highly stabilized proteins.The consensus sequence approach to predicting stabilizing substitutions in proteins rests on the notion that conserved amino acids are more likely to contribute to the stability of a protein fold than non-conserved amino acids. To implement a prediction for a target protein sequence, one finds homologous sequences and aligns them in a multiple sequence alignment. The sequence of the most frequently occurring amino acid at each position is the consensus sequence. Replacement of a rarely occurring amino acid in the target with a frequently occurring amino acid from the consensus sequence is predicted to be stabilizing. Consensus Finder is an open-source web tool that automates this prediction. This chapter reviews the rationale for the consensus sequence approach and explains the options for fine-tuning this approach using Staphylococcus nuclease A as an example.The remolding active site loops via residue insertion/deletion as well as substitution is thought to play a key role in enzyme divergent evolution. However, enzyme engineering by residue insertion in active site loops often severely perturbs the protein structural integrity and causes protein misfolding and activity loss. We have designed a stepwise loop insertion strategy (StLois), in which a pair of randomized residues is introduced in a stepwise manner, efficiently collating mutational fitness effects. The strategy of StLois constitutes three key steps. First, the target regions should be identified through structural and functional analysis on the counterpart enzymes. Second, pair residues can be introduced in loop regions through insertion with NNK codon degeneracy. Third, the best hit used as a template for the next round mutagenesis. The residue insertion process can repeat as many times as necessary. By using the StLois method, we have evolved the substrate preference of a lactonase to phosphotriesterase. In this chapter, we describe the detailed StLois technique, which efficiently expands the residue in the loop region and remolds the architecture of enzyme active site for novel catalytic properties.Employing the homologous DNA recombination apparatus of Saccharomyces cerevisiae as a dynamic engineering tool allows mutant libraries to be constructed in a rapid and efficient manner. Among the plethora of methods based on the yeast's splicing apparatus, site-directed recombination (SDR) is often useful to gather information from mutations discovered in directed evolution experiments. When using SDR, the target gene is divided in segments carrying the selected mutation positions so that the resulting PCR fragments show 50% mutated and 50% wild type residues at the codons of interest. The PCR products are then assembled and cloned into yeast through one-pot transformations with the help of homologous overlapping flanking regions. By screening SDR libraries, the effect of the mutations/reversions at the different positions can be rapidly sorted out in a combinatorial manner. As such, SDR can serve as the `final polishing step´ in a laboratory evolution campaign, revealing beneficial synergies among mutations and/or overriding deleterious mutations.