In silico screening of 393 mutants facilitates enzyme engineering of amidase activity in CalB.

Our previously presented method for high throughput computational screening of mutant activity (Hediger et al., 2012) is benchmarked against experimentally measured amidase activity for 22 mutants of Candida antarctica lipase B (CalB). Using an appropriate cutoff criterion for the computed barriers, the qualitative activity of 15 out of 22 mutants is correctly predicted. The method identifies four of the six most active mutants with ≥3-fold wild type activity and seven out of the eight least active mutants with ≤0.5-fold wild type activity. The method is further used to screen all sterically possible (386) double-, triple- and quadruple-mutants constructed from the most active single mutants. Based on the benchmark test at least 20 new promising mutants are identified.

Biocatalysts by evolution.

Proteins evolve by iterative cycles of mutation, selection and amplification. Analogous evolutionary strategies are being profitably exploited in the laboratory to generate and optimize biocatalysts for diverse biotechnological applications. In this review, we summarize recent efforts to improve this process by creating more effective protein libraries and more efficient screening/selection schemes. Targeted mutagenesis using simplified amino acid alphabets, statistical analyses of sequence-function-stability relationships, and neutral mutational drift have emerged as powerful tools for generating useful molecular diversity, while new techniques for controlling selection stringency and microfluidic methods for screening large populations of molecules promise to facilitate exploration of sequence space. Enzyme engineers interested in creating novel biocatalysts for abiological reactions are sure to profit from these advances.

Identification, characterization, and application of a recombinant antigen for the serological investigation of feline hemotropic Mycoplasma infections.

In felids, three hemotropic mycoplasma species (hemoplasmas) have been described: Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum," and "Candidatus Mycoplasma turicensis." In particular, M. haemofelis may cause severe, potentially life-threatening hemolytic anemia. No routine serological assays for feline hemoplasma infections are available. Thus, the goal of our project was to identify and characterize an M. haemofelis antigen (DnaK) that subsequently could be applied as a recombinant antigen in a serological assay. The gene sequence of this protein was determined using consensus primers and blood samples from two naturally M. haemofelis-infected Swiss pet cats, an experimentally M. haemofelis-infected specific-pathogen-free cat, and a naturally M. haemofelis-infected Iberian lynx (Lynx pardinus). The M. haemofelis DnaK gene sequence showed the highest identity to an analogous protein of a porcine hemoplasma (72%). M. haemofelis DnaK was expressed recombinantly in an Escherichia coli DnaK knockout strain and purified using Ni affinity, size-exclusion, and anion-exchange chromatography. It then was biochemically and functionally characterized and showed characteristics typical for DnaKs (secondary structure profile, thermal denaturation, ATPase activity, and DnaK complementation). Moreover, its immunogenicity was assessed using serum samples from experimentally hemoplasma-infected cats. In Western blotting or enzyme-linked immunosorbent assays, it was recognized by sera from cats infected with M. haemofelis, "Ca. Mycoplasma haemominutum," and "Ca. Mycoplasma turicensis," respectively, but not from uninfected cats. This is the first description of a full-length purified recombinant feline hemoplasma antigen that can readily be applied in future pathogenesis studies and may have potential for application in a diagnostic serological test.

Consensus protein design without phylogenetic bias.

Consensus design is an appealing strategy for the stabilization of proteins. It exploits amino acid conservation in sets of homologous proteins to identify likely beneficial mutations. Nevertheless, its success depends on the phylogenetic diversity of the sequence set available. Here, we show that randomization of a single protein represents a reliable alternative source of sequence diversity that is essentially free of phylogenetic bias. A small number of functional protein sequences selected from binary-patterned libraries suffice as input for the consensus design of active enzymes that are easier to produce and substantially more stable than individual members of the starting data set. Although catalytic activity correlates less consistently with sequence conservation in these extensively randomized proteins, less extreme mutagenesis strategies might be adopted in practice to augment stability while maintaining function.

Towards identifying preferred interaction partners of fluorinated amino acids within the hydrophobic environment of a dimeric coiled coil peptide.

Phage display technology has been applied to screen for preferred interaction partners of fluoroalkyl-substituted amino acids from the pool of the 20 canonical amino acids. A parallel, heterodimeric alpha-helical coiled coil was designed such that one peptide strand contained one of three different fluorinated amino acids within the hydrophobic core. The direct interaction partners within the second strand of the dimer were randomized and coiled coil pairing selectivity was used as a parameter to screen for the best binding partners within the peptide library. It was found that despite their different structures, polarities and fluorine contents, the three non-natural amino acids used in this study prefer the same interaction partners as the canonical, hydrophobic amino acids. The same technology can be used to study any kind of non-canonical amino acids. The emerging results will provide the basis not only for a profound understanding of the properties of these building blocks, but also for the de novo design of proteins with superior properties and new functions.

Selection of a buried salt bridge by phage display.

The alpha-helical coiled coil is a valuable folding motif for protein design and engineering. By means of phage display technology, we selected a capable binding partner for one strand of a coiled coil bearing a charged amino acid in a central hydrophobic core position. This procedure resulted in a novel coiled coil pair featuring an opposed Glu-Lys pair arranged staggered within the hydrophobic core of a coiled coil structure. Structural investigation of the selected coiled coil dimer by CD spectroscopy and MD simulations suggest that a buried salt bridge within the hydrophobic core enables the specific dimerization of two peptides.

Protein design by directed evolution.

While nature evolved polypeptides over billions of years, protein design by evolutionary mimicry is progressing at a far more rapid pace. The mutation, selection, and amplification steps of the evolutionary cycle may be imitated in the laboratory using existing proteins, or molecules created de novo from random sequence space, as starting templates. However, the astronomically large number of possible polypeptide sequences remains an obstacle to identifying and isolating functionally interesting variants. Intelligently designed libraries and improved search techniques are consequently important for future advances. In this regard, combining experimental and computational methods holds particular promise for the creation of tailored protein receptors and catalysts for tasks unimagined by nature.