Rational Mutation Research Articles

Immobilized high-performance heparin lyase III for efficient preparation of low molecular weight heparin

Heparin lyase III has garnered widespread attention due to its high specificity and minimal loss of anticoagulant activity during the preparation of low molecular weight heparin (LMWH), a crucial anticoagulant drug in clinical practice. However, low expression levels and complex preparation processes limit its practical application. To address these challenges, high-performance Bacteroides thetaiotaomicron heparin lyase III (Bhep III) variants were engineered and immobilized for LMWH preparation. First, we enhanced enzyme expression by adding a solubility-enhancing tag and optimizing the N-terminal coding sequence, which resulted in a Bhep III activity level of 2.9 × 103 U/L with 8-fold increase. After evolution guided the design of rational mutations, the variant Bhep III K85A/Q95F/S471T generated higher activity (5.4 × 104 U/L in 5-L fermenter), which is, to our knowledge, the highest reported to date in the literature, being 1.7-fold that of the wild type and demonstrating 2-fold increase of the thermal stability. By screening and optimizing the C-terminal self-assembling tag, we successfully immobilized Bhep III, further increasing its thermal stability by 12-fold, and allowing for the multi-batch preparation of LMWH with simple centrifugation. The immobilized heparin lyase III demonstrated sufficient reusability in enzymatic reactions, facilitating efficient industrial-scale production of LMWH.

Molecular basis of one-step methyl anthranilate biosynthesis in grapes, sweet orange, and maize.

Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate (MeAA) is an anti-herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one-step anthranilate methyltransferase (AAMT), grapes have been thought to use a two-step pathway for MeAA biosynthesis. By mining available transcriptomics data, we identified two AAMTs in Vitis vinifera (wine grape), as well as one ortholog in "Concord" grape. Many angiosperms methylate the plant hormone salicylic acid (SA) to produce methyl salicylate, which acts as a plant-to-plant communication molecule. Because the Citrus sinensis (sweet orange) SA methyltransferase can methylate both anthranilate (AA) and SA, we used this enzyme to examine the molecular basis of AA activity by introducing rational mutations, which identified several active site residues that increase activity with AA. Reversing this approach, we introduced mutations that imparted activity with SA in the maize AAMT, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of the Vitis AAMTs shares an ancestor with jasmonic acid methyltransferases, similar to the AAMT from strawberry (Frageria sp.). Collectively, these data demonstrate the molecular mechanisms underpinning AA activity across methyltransferases and identify one-step enzymes by which grapes synthesize MeAA.

Tailored expression of ICCM cutinase in engineered Escherichia coli for efficient polyethylene terephthalate hydrolysis

Enzymatic depolymerization of PET waste emerges as a crucial and sustainable solution for combating environmental pollution. Over the past decade, PET hydrolytic enzymes, such as PETase from Ideonella sakaiensis (IsPETases), leaf compost cutinases (LCC), and lipases, have been subjected to rational mutation to enhance their enzymatic properties. ICCM, one of the best LCC mutants, was selected for overexpression in Escherichia coli BL21(DE3) for in vitro PET degradation. However, overexpressing ICCM presents challenges due to its low productivity. A new stress-inducible T7RNA polymerase-regulating E. coli strain, ASIAhsp, which significantly enhances ICCM production by 72.8 % and achieves higher enzyme solubility than other strains. The optimal cultural condition at 30 °C with high agitation, corresponding to high dissolved oxygen levels, has brought the maximum productivity of ICCM and high PET-hydrolytic activity. The most effective PET biodegradation using crude or pure ICCM occurred at pH 10 and 60 °C. Moreover, ICCM exhibited remarkable thermostability, retaining 60 % activity after a 5-day reaction at 60 °C. Notably, crude ICCM eliminates the need for purification and efficiently degrades PET films.

Evolutionarily rational mutations in structured populations

This paper considers an evolutionary dynamic to study games played in large populations that consist of many identical, potentially small subpopulations, where individuals interact with others in their subpopulation, rarely moving between them.Since long-run behavior depends on the used mutation strategies, a criterion loosely inspired by Maynard Smith's (1972) notion of an evolutionary stable strategy is imposed on mutation strategies – we ask which mutation strategies are robust to “invasions” by others. Non-degenerate mutation strategies lead to behavioral diversity. Populations that use different interaction strategies in different subpopulations can be robust to “invasions,” even if each interaction strategy would not be robust if used by the entire population.Mutation strategies satisfying the proposed criterion always exist. A simple characterization is possible for the case of weak selection. The developed theory yields a rich class of predictions that can be inconsistent with standard concepts like Nash equilibrium. For instance, for finitely repeated games, the model always predicts relatively high payoffs if the stage game is repeated sufficiently often.

Modeling and affinity maturation of an anti-CD20 nanobody: a comprehensive in-silico investigation

B-cell Non-Hodgkin lymphomas are the malignancies of lymphocytes. CD20 is a membrane protein, which is highly expressed on the cell surface of the B-cells in NHL. Treatments using monoclonal antibodies (mAbs) have resulted in failure in some cases. Nanobodies (NBs), single-domain antibodies with low molecular weights and a high specificity in antigen recognition, could be practical alternatives for traditional mAbs with superior characteristics. To design an optimized NB as a candidate CD20 inhibitor with raised binding affinity to CD20, the structure of anti-CD20 NB was optimized to selectively target CD20. The 3D structure of the NB was constructed based on the optimal templates (6C5W and 5JQH), and the key residues were determined by applying a molecular docking study. After identifying the key residues, some mutations were introduced using a rational protocol to improve the binding affinity of the NB to CD20. The rational mutations were conducted using the experimental design (Taguchi method). Six residues (Ser27, Thr28, Phe29, Ile31, Asp99, and Asn100) were selected as the key residues, and five residues were targeted for rational mutation (Trp, Phe, His, Asp, and Tyr). Based on the mutations suggested by the experimental design, two optimized NB structures were constructed. NB2 showed a remarkable binding affinity to CD20 in docking studies with a binding energy of − 853 kcal/mol. The optimized NB was further evaluated using molecular dynamics simulation. The results revealed that CDR1 (complementarity determining regions1) and CDR3 are essential loops for recognizing the antigen. NB2 could be considered as a potential inhibitor of CD20, though experimental evaluations are needed to confirm it.

Expanding the Substrate Scope of a Bacterial Nucleotidyltransferase via Allosteric Mutations.

Bacterial glycoconjugates, such as cell surface polysaccharides and glycoproteins, play important roles in cellular interactions and survival. Enzymes called nucleotidyltransferases use sugar-1-phosphates and nucleoside triphosphates (NTPs) to produce nucleoside diphosphate sugars (NDP-sugars), which serve as building blocks for most glycoconjugates. Research spanning several decades has shown that some bacterial nucleotidyltransferases have broad substrate tolerance and can be exploited to produce a variety of NDP-sugars in vitro. While these enzymes are known to be allosterically regulated by NDP-sugars and their fragments, much work has focused on the effect of active site mutations alone. Here, we show that rational mutations in the allosteric site of the nucleotidyltransferase RmlA lead to expanded substrate tolerance and improvements in catalytic activity that can be explained by subtle changes in quaternary structure and interactions with ligands. These observations will help inform future studies on the directed biosynthesis of diverse bacterial NDP-sugars and downstream glycoconjugates.

In silico and in vitro analysis of a rational mutation in gIII signal peptide and its effects on periplasmic expression of rhGH in E. coli

The secretion efficiency of a heterologous protein in E. coli is mainly dictated by the N-terminal signal peptide fused to the desired protein. In this study, we aimed to select and introduce mutations into the – 1, – 2 and – 3 positions of the gIII signal peptide (originated from filamentous phage fd Gene III) fused to the N-terminus of the human growth hormone (hGH), and study its effect on the secretion efficiency of the recombinant hGH into the periplasmic space of E. coli Top10. Bioinformatics software such as SignalP-5.0 and PrediSi were employed to predict the effects of the mutations on the secretion efficiency of the recombinant hGH. Site-directed mutagenesis was applied to introduce the desired mutations into the C-terminus of the gIII signal peptide. The periplasmic expression and the secretion efficiency of the recombinant hGH using the native and mutant gIII signal peptides were compared in E. coli Top10 under the control of araBAD promoter. Our results from bioinformatics analysis indicated that the mutant gIII signal peptide was more potent than the native one for secretion of the recombinant hGH in E. coli. While our experimental results revealed that the mutation had no effect on hGH secretion. This result points to the importance of experimental validation of bioinformatics predictions.

Single-chain fragment variable produced by phage display technology: Construction, selection, mutation, expression, and recent applications in food safety.

Immunoassays are reliable, efficient, and accurate methods for the analysis of small-molecule harmful substances (such as pesticides, veterinary drugs, and biological toxins) that may be present in food. However, traditional polyclonal and monoclonal antibodies are limited by animal hosts and hinder further development of immunoassays. With the gradual application of phage display technology as an efficient in vitro selection technology, the single-chain fragment variable (scFv) now provides an exciting alternative to traditional antibodies. Efficiently constructed scFv source libraries and specifically designed biopanning schemes can now yield scFvs possessing specific recognition capabilities. A rational mutation strategy further enhances the affinity of scFv, and allows it to reach a level that cannot be achieved by immunization. Finally, appropriate prokaryotic expression measures ensure stable and efficient production of scFv. Therefore, when developing excellent scFvs, it is necessary to focus on three key aspects of this process that include screening, mutation, and expression. In this review, we analyze in detail the preparation and affinity improvement process for scFv and provide insights into the research progress and development trend of scFv-based immunoassay methods for monitoring small-molecule harmful substances.

An Engineered Arginine Residue of Unusual pH-Sensitive Reactivity Facilitates Site-Selective Antibody Conjugation.

Monoclonal antibody h38C2 is a humanized catalytic antibody that has been used to generate various immunoconjugate species such as chemically programmed antibodies, antibody-drug conjugates, and antibody-siRNA conjugates. Highly efficient and specific conjugation of h38C2 occurs at its uniquely reactive lysine (Lys) residue buried inside the antibody's catalytic pocket. We recently reported the rational mutation of this Lys residue at position 99 in the heavy chain variable domain to an arginine (Arg) residue. The Lys99Arg mutation can be site-selectively conjugated with molecules containing a hapten-like triazolyl-phenylglyoxal (TPG) unit. Here we show that this conjugation is facilitated by the unusual pH-sensitive reactivity of the Arg99 residue, consistent with an indirectly measured pKa of 5.2. The Arg99/TPG conjugation holds promise to further expand the versatility of the h38C2 conjugation platform, such as for the generation of antibody conjugates with dual payloads.

Advancing Rational Control of Peptide-Surface Complexes.

Understanding peptide-surface interactions is crucial for programming self-assembly of peptides at surfaces and in realizing their applications, such as biosensors and biomimetic materials. In this study, we developed insights into the dependence of a residue's interaction with a surface on its neighboring residue in a tripeptide using molecular dynamics simulations. This knowledge is integral for designing rational mutations to control peptide-surface complexes. Using graphene as our model surface, we estimated the free energy of adsorption (ΔAads) and extracted predominant conformations of 26 tripeptides with the motif LNR-CR-Gly, where LNR and CR are variable left-neighboring and central residues, respectively. We considered a combination of strongly adsorbing (Phe, Trp, and Arg) and weakly adsorbing (Ala, Val, Leu, Ser, and Thr) amino acids on graphene identified in a prior study to form the tripeptides. Our results indicate that ΔAads of a tripeptide cannot be estimated as the sum of ΔAads of each residue indicating that the residues in a tripeptide do not behave as independent entities. We observed that the contributions from the strongly adsorbing amino acids were dominant, which suggests that such residues could be used for strengthening peptide-graphene interactions irrespective of their neighboring residues. In contrast, the adsorption of weakly adsorbing central residues is dependent on their neighboring residues. Our structural analysis revealed that the dihedral angles of LNR are more correlated with that of CR in the adsorbed state than in bulk state. Together with ΔAads trends, this implies that different backbone structures of a given CR can be accessed for a similar ΔAads by varying the LNR. Therefore, incorporation of context effects in designing mutations can lead to desired peptide structure at surfaces. Our results also emphasize that these cooperative effects in ΔAads and structure are not easily predicted a priori. The collective results have applications in guiding rational mutagenesis techniques to control orientation of peptides at surfaces and in developing peptide structure prediction algorithms in adsorbed state from its sequence.

An Evolutionary Based Strategy for Predicting Rational Mutations in G Protein-Coupled Receptors

Capturing conserved patterns in genes and proteins is important for inferring phenotype prediction and evolutionary analysis. The study is focused on the conserved patterns of the G protein-coupled receptors, an important superfamily of receptors. Olfactory receptors represent more than 2% of our genome and constitute the largest family of G protein-coupled receptors, a key class of drug targets. As no crystallographic structures are available, mechanistic studies rely on the use of molecular dynamic modelling combined with site-directed mutagenesis data. In this paper, we hypothesized that human-mouse orthologs coding for G protein-coupled receptors maintain, at speciation events, shared compositional structures independent, to some extent, of their percent identity as reveals a method based in the categorization of nucleotide triplets by their gross composition. The data support the consistency of the hypothesis, showing in ortholog G protein-coupled receptors the presence of emergent shared compositional structures preserved at speciation events. An extreme bias in synonymous codon usage is observed in both the conserved and non-conserved regions of many of these receptors that could be potentially relevant in codon optimization studies. The analysis of their compositional structures would help to design new evolutionary based strategies for rational mutations that would aid to understand the characteristics of individual G protein-coupled receptors, their subfamilies and the role in many physiological and pathological processes, supplying new possible drug targets.

Analysis of binding residues in monoclonal antibody with high affinity for the head domain of the rat P2X4 receptor.

P2X4 receptor is known to be involved in neuropathic pain. In order to detect the expression of P2X4 receptor on microglia at the time of onset of neuropathic pain, one approach consists on the preparation of the monoclonal antibodies with both selective binding and high affinity. We have recently established a monoclonal antibody (named 12-10H) which had high affinity to rat P2X4 receptor expressed in 1321N1 cells. The dissociation constants of the complex between the monoclonal antibodies obtained so far and the head domain (HD) in the rat P2X4 receptor were in the nanomolar range. To improve the affinity by rational mutations, we need to know the precious location of the binding site in these monoclonal antibodies. Here, we have analysed and identified the binding residues in the monoclonal antibody (12-10H) with high affinity for the HD of the rat P2X4 receptor by site-directed mutagenesis.

Influenza Hemagglutinin Head Domain Mimicry by Rational Design.

Despite diligent vaccination efforts, influenza virus infection remains a major cause for respiratory-related illness across the globe. The less-than-optimal immunity conferred by the currently prescribed seasonal vaccines and protracted production times warrant the development of novel vaccines. Induction of an epitope-focused antibody response targeting known neutralization epitopes is a viable strategy to enhance the breadth of protection against rapidly evolving infectious viruses. We report the development of a design framework to mimic the hemagglutinin (HA) head fragment of H1-subtype viruses by delineating the interaction network of invariant residues lining the receptor binding site (RBS); a site targeted by cross-reactive neutralizing antibodies. The incorporation of multiple sequence alignment information in our algorithm to fix the construct termini and engineer rational mutations facilitates the facile extension of the design to heterologous (subtype-specific) influenza strains. We evaluated our design protocol by generating head fragments from divergent influenza A H1N1 A/Puerto Rico/8/34 and pH1N1 A/California/07/2009 strains that share a sequence identity of only 74.4% within the HA1 subunit. The designed immunogens exhibited characteristics of a well-ordered protein, and bound conformation-specific RBS targeting antibodies with high affinity, a desirable feature for putative vaccine candidates. Additionally, the bacterial expression of these immunogens provides a low-cost, rapidly scalable alternative.

The Phospholipid:Diacylglycerol Acyltransferase-Mediated Acyl-Coenzyme A-Independent Pathway Efficiently Diverts Fatty Acid Flux from Phospholipid into Triacylglycerol in Escherichia coli.

Researchers have long endeavored to accumulate triacylglycerols (TAGs) or their derivatives in easily managed microbes. The attempted production of TAGs in Escherichia coli has revealed barriers to the broad applications of this technology, including low TAG productivity and slow cell growth. We have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in E. coli mediated by Chlamydomonas reinhardtii phospholipid:diacylglycerol acyltransferase (CrPDAT) without interfering with membrane functions. We then showed the synergistic effect on TAG accumulation via the acyl-CoA-independent pathway mediated by PDAT and the acyl-CoA-dependent pathway mediated by wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT). Furthermore, CrPDAT led to synchronous TAG accumulation during cell growth, and this could be enhanced by supplementation of arbutin. We also showed that rationally mutated CrPDAT was capable of decreasing TAG lipase activity without impairing PDAT activity. Finally, ScPDAT from Saccharomyces cerevisiae exhibited similar activities as CrPDAT in E. coli Our results suggest that the improvement in accumulation of TAGs and their derivatives can be achieved by fine-tuning of phospholipid metabolism in E. coli Understanding the roles of PDAT in the conversion of phospholipids into TAGs during the logarithmic growth phase may enable a novel strategy for the production of microbial oils.IMPORTANCE Although phospholipid:diacylglycerol acyltransferase (PDAT) activity is presumed to exist in prokaryotic oleaginous bacteria, the corresponding gene has not been identified yet. In this article, we have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in Escherichia coli mediated by exogenous CrPDAT from Chlamydomonas reinhardtii without interfering with membrane functions. In addition, the acyl-CoA-independent pathway and the acyl-CoA-dependent pathway had the synergistic effect on TAG accumulation. Overexpression of CrPDAT led to synchronous TAG accumulation during cell growth. In particular, CrPDAT possessed multiple catalytic activities, and the rational mutation of CrPDAT led to the decrease of TAG lipase activity without impairing acyltransferase activity. The present findings suggested that applying PDAT in E. coli or other prokaryotic microbes may be a promising strategy for accumulation of TAGs and their derivatives.

Common activation mechanism of class A GPCRs.

Class A G-protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology. Understanding receptor activation mechanism is critical for discovering novel therapeutics since about one-third of all marketed drugs target members of this family. GPCR activation is an allosteric process that couples agonist binding to G-protein recruitment, with the hallmark outward movement of transmembrane helix 6 (TM6). However, what leads to TM6 movement and the key residue level changes of this movement remain less well understood. Here, we report a framework to quantify conformational changes. By analyzing the conformational changes in 234 structures from 45 class A GPCRs, we discovered a common GPCR activation pathway comprising of 34 residue pairs and 35 residues. The pathway unifies previous findings into a common activation mechanism and strings together the scattered key motifs such as CWxP, DRY, Na+ pocket, NPxxY and PIF, thereby directly linking the bottom of ligand-binding pocket with G-protein coupling region. Site-directed mutagenesis experiments support this proposition and reveal that rational mutations of residues in this pathway can be used to obtain receptors that are constitutively active or inactive. The common activation pathway provides the mechanistic interpretation of constitutively activating, inactivating and disease mutations. As a module responsible for activation, the common pathway allows for decoupling of the evolution of the ligand binding site and G-protein-binding region. Such an architecture might have facilitated GPCRs to emerge as a highly successful family of proteins for signal transduction in nature.

PH and Charged Mutations Modulate Cold Shock Protein Folding and Stability: A Constant pH Monte Carlo Study.

The folding and stability of proteins is a fundamental problem in several research fields. In the present paper, we have used different computational approaches to study the effects caused by changes in pH and for charged mutations in cold shock proteins from Bacillus subtilis (Bs-CspB). First, we have investigated the contribution of each ionizable residue for these proteins to their thermal stability using the TKSA-MC, a Web server for rational mutation via optimizing the protein charge interactions. Based on these results, we have proposed a new mutation in an already optimized Bs-CspB variant. We have evaluated the effects of this new mutation in the folding energy landscape using structure-based models in Monte Carlo simulation at constant pH, SBM-CpHMC. Our results using this approach have indicated that the charge rearrangements already in the unfolded state are critical to the thermal stability of Bs-CspB. Furthermore, the conjunction of these simplified methods was able not only to predict stabilizing mutations in different pHs but also to provide essential information about their effects in each stage of protein folding.

Homology Modeling-Based in Silico Affinity Maturation Improves the Affinity of a Nanobody.

Affinity maturation and rational design have a raised importance in the application of nanobody (VHH), and its unique structure guaranteed these processes quickly done in vitro. An anti-CD47 nanobody, Nb02, was screened via a synthetic phage display library with 278 nM of KD value. In this study, a new strategy based on homology modeling and Rational Mutation Hotspots Design Protocol (RMHDP) was presented for building a fast and efficient platform for nanobody affinity maturation. A three-dimensional analytical structural model of Nb02 was constructed and then docked with the antigen, the CD47 extracellular domain (CD47ext). Mutants with high binding affinity are predicted by the scoring of nanobody-antigen complexes based on molecular dynamics trajectories and simulation. Ultimately, an improved mutant with an 87.4-fold affinity (3.2 nM) and 7.36 °C higher thermal stability was obtained. These findings might contribute to computational affinity maturation of nanobodies via homology modeling using the recent advancements in computational power. The add-in of aromatic residues which formed aromatic-aromatic interaction plays a pivotal role in affinity and thermostability improvement. In a word, the methods used in this study might provide a reference for rapid and efficient in vitro affinity maturation of nanobodies.

A role for antibiotic biosynthesis monooxygenase domain proteins in fidelity control during aromatic polyketide biosynthesis

The formicamycin biosynthetic gene cluster encodes two groups of type 2 polyketide antibiotics: the formicamycins and their biosynthetic precursors the fasamycins, both of which have activity against methicillin-resistant Staphylococcus aureus. Here, we report the formicapyridines which are encoded by the same gene cluster and are structurally and biosynthetically related to the fasamycins and formicamycins but comprise a rare pyridine moiety. These compounds are trace-level metabolites formed by derailment of the major biosynthetic pathway. Inspired by evolutionary logic we show that rational mutation of a single gene in the biosynthetic gene cluster encoding an antibiotic biosynthesis monooxygenase (ABM) superfamily protein leads to a significant increase both in total formicapyridine production and their enrichment relative to the fasamycins/formicamycins. Our observations broaden the polyketide biosynthetic landscape and identify a non-catalytic role for ABM superfamily proteins in type II polyketide synthase assemblages for maintaining biosynthetic pathway fidelity.

From general base to general acid catalysis in a sodium-specific DNAzyme by a guanine-to-adenine mutation.

Recently, a few Na+-specific RNA-cleaving DNAzymes were reported, where nucleobases are likely to play critical roles in catalysis. The NaA43 and NaH1 DNAzymes share the same 16-nt Na+-binding motif, but differ in one or two nucleotides in a small catalytic loop. Nevertheless, they display an opposite pH-dependency, implicating distinct catalytic mechanisms. In this work, rational mutation studies locate a catalytic adenine residue, A22, in NaH1, while previous studies found a guanine (G23) to be important for the catalysis of NaA43. Mutation with pKa-perturbed analogs, such as 2-aminopurine (∼3.8), 2,6-diaminopurine (∼5.6) and hypoxanthine (∼8.7) affected the overall reaction rate. Therefore, we propose that the N1 position of G23 (pKa ∼6.6) in NaA43 functions as a general base, while that of A22 (pKa ∼6.3) in NaH1 as a general acid. Further experiments with base analogs and a phosphorothioate-modified substrate suggest that the exocyclic amine in A22 and both of the non-bridging oxygens at the scissile phosphate are important for catalysis for NaH1. This is an interesting example where single point mutations can change the mechanism of cleavage from general base to general acid, and it can also explain this Na+-dependent DNAzyme scaffold being sensitive to a broad range of metal ions and molecules.

Stabilizing and Understanding a Miniprotein by Rational Redesign.

Miniproteins reduce the complexity of the protein-folding problem allowing systematic studies of contributions to protein folding and stabilization. Here, we describe the rational redesign of a miniprotein, PPα, comprising a polyproline II helix, a loop, and an α helix. The redesign provides a de novo framework for interrogating noncovalent interactions. Optimized PPα has significantly improved thermal stability with a midpoint unfolding temperature (TM) of 51 °C. Its nuclear magnetic resonance structure indicates a density of stabilizing noncovalent interactions that is higher than that of the parent peptide, specifically an increased number of CH-π interactions. In part, we attribute this to improved long-range electrostatic interactions between the two helical elements. We probe further sequence-stability relationships in the miniprotein through a series of rational mutations.