A protein sequence encodes its energy landscape-all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs-questioning the differences between them and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the consensus protein derived from our full Ribonuclease H sequence alignment is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted set of sequences is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order sequence correlations using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

Acrylamide is present in thermally processed foods, and it possesses toxic and carcinogenic properties. L-asparaginases could effectively regulate the formation of acrylamide at the source. However, current L-asparaginases have drawbacks such as poor thermal stability, low catalytic activity, and poor substrate specificity, thereby restricting their utility in the food industry. To address this issue, this study employed consensus design to predict the crucial residues influencing the thermal stability of Corynebacterium glutamicum L-asparaginase (CgASNase). Subsequently, a combination of site-point saturating mutation and combinatorial mutation techniques was applied to generate the double-mutant enzyme L42T/S213N. Remarkably, L42T/S213N displayed significantly enhanced thermal stability without a substantial impact on its enzymatic activity. Notably, its half-life at 40 °C reached an impressive 13.29 ± 0.91 min, surpassing that of CgASNase (3.24 ± 0.23 min). Moreover, the enhanced thermal stability of L42T/S213N can be attributed to an increased positive surface charge and a more symmetrical positive potential, as revealed by three-dimensional structural simulations and structure comparison analyses. To assess the impact of L42T/S213N on acrylamide removal in biscuits, the optimal treatment conditions for acrylamide removal were determined through a combination of one-way and orthogonal tests, with an enzyme dosage of 300 IU/kg flour, an enzyme reaction temperature of 40 °C, and an enzyme reaction time of 30 min. Under these conditions, compared to the control (464.74 ± 6.68 µg/kg), the acrylamide reduction in double-mutant-enzyme-treated biscuits was 85.31%, while the reduction in wild-type-treated biscuits was 68.78%. These results suggest that L42T/S213N is a promising candidate for industrial applications of L-asparaginase.

A protein sequence encodes its energy landscape - all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs - questioning the differences and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the overall consensus protein is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted region is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order couplings using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

Plant legumains are Asn/Asp-specific endopeptidases that have diverse functions in plants. Peptide asparaginyl ligases (PALs) are a special legumain subtype that primarily catalyze peptide bond formation rather than hydrolysis. PALs are versatile protein engineering tools but are rarely found in nature. To overcome this limitation, here we describe a two-step method to design and engineer a high-yield and efficient recombinant PAL based on commonly found asparaginyl endopeptidases. We first constructed a consensus sequence derived from 1500 plant legumains to design the evolutionarily stable legumain conLEG that could be produced in E. coli with 20-fold higher yield relative to that for natural legumains. We then applied the ligase-activity determinant hypothesis to exploit conserved residues in PAL substrate-binding pockets and convert conLEG into conPAL1-3. Functional studies showed that conLEG is primarily a hydrolase, whereas conPALs are ligases. Importantly, conPAL3 is a superefficient and broadly active PAL for protein cyclization and ligation.

Protein stability, dynamics and function are intricately linked. Accordingly, protein designers leverage dynamics in their designs and gain insight to their successes and failures by analyzing their proteins\' dynamics. Molecular dynamics (MD) simulations are a powerful computational tool for quantifying both local and global protein dynamics. This review highlights studies where MD simulations were applied to characterize the stability and dynamics of designed proteins and where dynamics were incorporated into computational protein design. First, we discuss the structural basis underlying the extreme stability and thermostability frequently observed in computationally designed proteins. Next, we discuss examples of designed proteins, where dynamics were not explicitly accounted for in the design process, whose coordinated motions or active site dynamics, as observed by MD simulation, enhanced or detracted from their function. Many protein functions depend on sizeable or subtle conformational changes, so we finally discuss the computational design of proteins to perform a specific function that requires consideration of motion by multi-state design.

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.

Staphylococcus aureus sortase A (SaSrtA) is an enzyme that anchors proteins to the cell surface of Gram-positive bacteria. During the transpeptidation reaction performed by SaSrtA, proteins containing an N-terminal glycine can be covalently linked to another protein with a C-terminal LPXTG motif (X being any amino acid). Since the sortase reaction can be performed in vitro as well, it has found many applications in biotechnology. Although sortase-mediated ligation has many advantages, SaSrtA is limited by its low enzymatic activity and dependence on Ca2+. In our study, we evaluated the thermodynamic stability of the SaSrtA wild type and found the enzyme to be stable. We applied consensus analysis to further improve the enzyme\'s stability while at the same time enhancing the enzyme\'s activity. As a result, we found thermodynamically improved, more active and Ca2+-independent mutants. We envision that these new variants can be applied in conjugation reactions in low Ca2+ environments.

Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion.

Consensus-based protein engineering strategy has been applied to various proteins and it can lead to the design of proteins with enhanced biological performance. Histone-like HUs comprise a protein family with sequence variety within a highly conserved 3D-fold. HU function includes compacting and regulating bacterial DNA in a wide range of biological conditions in bacteria. To explore the possible impact of consensus-based design in the thermodynamic stability of HU proteins, the approach was applied using a dataset of sequences derived from a group of 40 mesostable, thermostable, and hyperthermostable HUs. The consensus-derived HU protein was named HUBest, since it is expected to perform best. The synthetic HU gene was overexpressed in E. coli and the recombinant protein was purified. Subsequently, HUBest was characterized concerning its correct folding and thermodynamic stability, as well as its ability to interact with plasmid DNA. A substantial increase in HUBest stability at high temperatures is observed. HUBest has significantly improved biological performance at ambience temperature, presenting very low Kd values for binding plasmid DNA as indicated from the Gibbs energy profile of HUBest. This Kd may be associated to conformational changes leading to decreased thermodynamic stability and, therefore, higher flexibility at ambient temperature.