Consensus Protein Design without Phylogenetic Bias

Abstract

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 essentially free of phylogenetic bias. A small number of functional protein sequences selected from binary-patterned libraries suffices as input for 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. Keywordsprotein stabilization; multiple sequence alignments; consensus mutation; binary patterning; chorismate mutase Utilization of proteins outside of their normal biological context -for example in diagnostic, medical, or industrial applications or for the creation of novel receptors and catalysts1 ; 2 -often requires optimization of biophysical properties like stability.3 ; 4 For this purpose, engineering methods that exploit statistical amino acid frequencies from multiple sequence alignments (MSAs) are widely used.5 ; 6 Data-driven consensus design is based on the simple assumption that the frequency of a given residue in an MSA of homologous proteins correlates with that amino acid's contribution to protein stability.7 An artificial protein possessing the most frequent residue at each position should accordingly show maximum stability. Given the difficulty of predicting how individual residues contribute to overall stability,8 this approach to protein stabilization is often preferable to classical rational design, particularly as it does not depend on the availability of structural information.If an MSA were composed of fully independent protein sequences all selected for stable folding to the same structure, and if individual residues contributed additively to stability,

Protein stability: computation, sequence statistics, and new experimental methods

“…Effectively, consensus mutations are causing a compounding of stabilizing interactions that no one proteins needs to amass. A similar effect has been observed from mutations in directed evolution [55]. But it is also easy to think of reasons why this will not always work.…”

Stabilizing Proteins from Sequence Statistics: The Interplay of Conservation and Correlation in Triosephosphate Isomerase Stability

“…Interestingly, Hilvert recently reported that “consensus” mutations in libraries of chorismate mutase from directed evolution were also stabilizing, but the consensus variants ranged significantly in activity (from 2-fold higher to 30-fold lower). 49 …”

Phylogeny-Based Design of a B-Subunit of DNA Gyrase and Its ATPase Domain Using a Small Set of Homologous Amino Acid Sequences

“…55,56 However, the construction of the consGyrB-N sequence used only 16 GyrBs for MSA (hereafter referred to as bMSA-16Q); therefore, the sequence may be strongly biased towards the residues contained in the 16 sequences. To determine if sampling bias had occurred, we randomly chose 232 GyrB sequences and added them into the MSA-16 data set.…”