The spatial separation of basic amino acids is similar in RHAMM and hyaluronan binding peptide P15-1 despite different sequences and conformations.

Peptides that increase pro-reparative responses to injury and disease by modulating the functional organization of hyaluronan (HA) with its cell surface binding proteins (e.g., soluble receptor for hyaluronan-mediated motility [RHAMM] and integral membrane CD44) have potential therapeutic value. The binding of RHAMM to HA is an attractive target, since RHAMM is normally absent or expressed at low levels in homeostatic conditions, but its expression is significantly elevated in the extracellular matrix during tissue stress, response-to-injury, and in cancers and inflammation-based diseases. The HA-binding site in RHAMM contains two closely spaced sequences of clustered basic amino acids, in an alpha-helical conformation. In the present communication, we test whether an alpha-helical conformation is required for effective peptide binding to HA, and competitive disruption of HA-RHAMM interaction. The HA-binding RHAMM-competitive peptide P15-1, identified using the unbiased approach of phage display, was examined using circular dichroism spectroscopy and the conformation-predictive AI-based AlphaFold2 algorithm. Unlike the HA-binding site in RHAMM, peptide P15-1 was found to adopt irregular conformations in solution rather than alpha helices. Instead, our structural analysis suggests that the primary determinant of peptide-HA binding is associated with a specific clustering and spacing pattern of basic amino acids, allowing favorable electrostatic interaction with carboxylate groups on HA.

Hotspot Wizard-informed engineering of a hyperthermophilic ß-glucosidase for enhanced enzyme activity at low temperatures.

Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of ß-glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL-A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in ß-glucosidase activity at 25 and 50°C compared to the wild type, without compromising high-temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL-A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard-informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes.

Exploring the diversity of ß-glucosidase: Classification, catalytic mechanism, molecular characteristics, kinetic models, and applications.

High-value chemicals and energy-related products can be produced from biomass. Biorefinery technology offers a sustainable and cost-effective method for this high-value conversion. ß-glucosidase is one of the key enzymes in biorefinery processes, catalyzing the production of glucose from aryl-glycosides and cello-oligosaccharides via the hydrolysis of ß-glycosidic bonds. Although ß-glucosidase plays a critical catalytic role in the utilization of cellulosic biomass, its efficacy is often limited by substrate or product inhibitions, low thermostability, and/or insufficient catalytic activity. To provide a detailed overview of ß-glucosidases and their benefits in certain desired applications, we collected and summarized extensive information from literature and public databases, covering ß-glucosidases in different glycosidase hydrolase families and biological kingdoms. These ß-glucosidases show differences in amino acid sequence, which are translated into varying degrees of the molecular properties critical in enzymatic applications. This review describes studies on the diversity of ß-glucosidases related to the classification, catalytic mechanisms, key molecular characteristics, kinetics models, and applications, and highlights several ß-glucosidases displaying high stability, activity, and resistance to glucose inhibition suitable for desired biotechnological applications.

Oligomerization by co-assembly of ß-amyloid and α-synuclein.

Aberrant self-assembly of an intrinsically disordered protein is a pathological hallmark of protein misfolding diseases, such as Alzheimer's and Parkinson's diseases (AD and PD, respectively). In AD, the 40-42 amino acid-long extracellular peptide, ß-amyloid (Aß), self-assembles into oligomers, which eventually aggregate into fibrils. A similar self-association of the 140 amino acid-long intracellular protein, α-synuclein (αS), is responsible for the onset of PD pathology. While Aß and αS are primarily extracellular and intracellular polypeptides, respectively, there is evidence of their colocalization and pathological overlaps of AD and PD. This evidence has raised the likelihood of synergistic, toxic protein-protein interactions between Aß and αS. This mini review summarizes the findings of studies on Aß-αS interactions related to enhanced oligomerization via co-assembly, aiming to provide a better understanding of the complex biology behind AD and PD and common pathological mechanisms among the major neurodegenerative diseases.

α-synuclein-assisted oligomerization of ß-amyloid (1-42).

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative disorders, characterized by aggregation of amyloid polypeptides, ß-amyloid (Aß) and α-synuclein (αS), respectively. Aß and αS follow similar aggregation pathways, starting from monomers, to soluble toxic oligomeric assemblies, and to insoluble fibrils. Various studies have suggested overlaps in the pathologies of AD and PD, and have shown Aß-αS interactions. Unfortunately, whether these protein-protein interactions lead to self- and co-assembly of Aß and αS into oligomers - a potentially toxic synergistic mechanism - is poorly understood. Among the various Aß isoforms, interactions of Aß containing 42 amino acids (Aß (1-42), referred to as Aß42) with αS are of most direct relevance due to the high aggregation propensity and the strong toxic effect of this Aß isoform. In this study, we carefully determined molecular consequences of interactions between Aß42 and αS in their respective monomeric, oligomeric, and fibrillar forms using a comprehensive set of experimental tools. We show that the three αS conformers, namely, monomers, oligomers and fibrils interfered with fibrillization of Aß42. Specifically, αS monomers and oligomers promoted oligomerization and stabilization of soluble Aß42, possibly via direct binding or co-assembly, while αS fibrils hindered soluble Aß42 species from converting into insoluble aggregates by the formation of large oligomers. We also provide evidence that the interactions with αS were mediated by various parts of Aß42, depending on Aß42 and αS conformers. Furthermore, we compared similarities and dissimilarities between Aß42-αS and Aß40-αS interactions. Overall, the present study provides a comprehensive depiction of the molecular interplay between Aß42 and αS, providing insight into its synergistic toxic mechanism.

Engineering of a protein probe with multiple inputs and multiple outputs for evaluation of alpha synuclein aggregation states.

The aggregation of α-synuclein (αS) into oligomers and fibrils is implicated in the pathology of Parkinson's Disease (PD). While a molecular probe for rapid and comprehensive evaluation of αS aggregation states is critical for a better understanding of PD pathology, identification of therapeutic candidates, and the development of early diagnostic strategies, no such probe has yet to be developed. A structurally flexible αS variant, PG65, was previously developed as a target binding-driven, conformation-switching molecular probe for rapid αS oligomer detection. Though informative, detection using PG65 provides no comprehensive assessment of the αS aggregation states. In the present study, we report engineering of a molecular probe, PG65-MIMO (a PG65 variant with Multiple-Inputs and Multiple-Outputs), that rapidly (within 2 hr) produces comprehensive information on αS aggregation states. PG65-MIMO generates distinct fluorescence responses to the three major αS conformers (monomers, oligomers, and fibrils). PG65-MIMO also displays unique fluorescent signals for αS oligomers, depending on the tris(2-carboxyethyl)phosphine (TCEP) concentration. Our results suggest that the TCEP dependent signaling of PG65-MIMO may be associated with its conformational states. Overall, our study illustrates engineering of an αS variant to create a molecular probe for handling multiple inputs and multiple outputs, addressing the technological gap in αS detection.

Inhibition of alpha-synuclein aggregation by AM17, a synthetic resveratrol derivative.

Parkinson's disease (PD) is linked to the aberrant self-assembly of the amyloid protein, α-synuclein (αS), where αS monomers aggregate to form oligomers and fibrils. Out of the three conformers, αS oligomers are the major toxic agents in PD, while αS fibrils may work as a reservoir for toxic oligomeric conformers. Thus, compounds that inhibit aggregation of αS monomers and disaggregate αS oligomers and fibrils may serve as therapeutic agents against PD. In this regard, resveratrol and its synthetic derivatives (e.g., AM17, which contains a copper ion-selective ionophoric motif) have previously been examined for their inhibitory effects on aggregation of amyloid proteins, such as the ß-amyloid peptide implicated in Alzheimer's disease. In the current study, we employed an array of experimental tools, such as Thioflavin T fluorescence, transmission electron microscopy, immuno-dot blot assays, SDS- and native-PAGE, and circular dichroism, to determine the impact of AM17 and resveratrol on αS aggregation. To the best of our knowledge, we show for the first time that AM17 not only inhibits aggregation of αS monomers but also disaggregates αS oligomers and fibrils, independent of the copper ions. Similar αS aggregation inhibitory effects were observed with resveratrol only in the presence of the copper ion. The present study supports the high promise of applicability of AM17 as an effective amyloid aggregation inhibitor for various conformers and protein sequences.

MTADV 5-MER peptide suppresses chronic inflammations as well as autoimmune pathologies and unveils a new potential target-Serum Amyloid A.

Despite the existence of potent anti-inflammatory biological drugs e.g., anti-TNF and anti IL-6 receptor antibodies, for treating chronic inflammatory and autoimmune diseases, these are costly and not specific. Cheaper oral available drugs remain an unmet need. Expression of the acute phase protein Serum Amyloid A (SAA) is dependent on release of pro-inflammatory cytokines IL-1, IL-6 and TNF-α during inflammation. Conversely, SAA induces pro-inflammatory cytokine secretion, including Th17, leading to a pathogenic vicious cycle and chronic inflammation. 5- MER peptide (5-MP) MTADV (methionine-threonine-alanine-aspartic acid-valine), also called Amilo-5MER, was originally derived from a sequence of a pro-inflammatory CD44 variant isolated from synovial fluid of a Rheumatoid Arthritis (RA) patient. This human peptide displays an efficient anti-inflammatory effects to ameliorate pathology and clinical symptoms in mouse models of RA, Inflammatory Bowel Disease (IBD) and Multiple Sclerosis (MS). Bioinformatics and qRT-PCR revealed that 5-MP, administrated to encephalomyelytic mice, up-regulates genes contributing to chronic inflammation resistance. Mass spectrometry of proteins that were pulled down from an RA synovial cell extract with biotinylated 5-MP, showed that it binds SAA. 5-MP disrupted SAA assembly, which is correlated with its pro-inflammatory activity. The peptide MTADV (but not scrambled TMVAD) significantly inhibited the release of pro-inflammatory cytokines IL-6 and IL-1ß from SAA-activated human fibroblasts, THP-1 monocytes and peripheral blood mononuclear cells. 5-MP suppresses the pro-inflammatory IL-6 release from SAA-activated cells, but not from non-activated cells. 5-MP could not display therapeutic activity in rats, which are SAA deficient, but does inhibit inflammations in animal models of IBD and MS, both are SAA-dependent, as shown by others in SAA knockout mice. In conclusion, 5-MP suppresses chronic inflammation in animal models of RA, IBD and MS, which are SAA-dependent, but not in animal models, which are SAA-independent.

CO2 doping of organic interlayers for perovskite solar cells.

In perovskite solar cells, doped organic semiconductors are often used as charge-extraction interlayers situated between the photoactive layer and the electrodes. The π-conjugated small molecule 2,2',7,7'-tetrakis[N,N-di(4-methoxyphenyl)amino]-9,9-spirobifluorene (spiro-OMeTAD) is the most frequently used semiconductor in the hole-conducting layer1-6, and its electrical properties considerably affect the charge collection efficiencies of the solar cell7. To enhance the electrical conductivity of spiro-OMeTAD, lithium bis(trifluoromethane)sulfonimide (LiTFSI) is typically used in a doping process, which is conventionally initiated by exposing spiro-OMeTAD:LiTFSI blend films to air and light for several hours. This process, in which oxygen acts as the p-type dopant8-11, is time-intensive and largely depends on ambient conditions, and thus hinders the commercialization of perovskite solar cells. Here we report a fast and reproducible doping method that involves bubbling a spiro-OMeTAD:LiTFSI solution with CO2 under ultraviolet light. CO2 obtains electrons from photoexcited spiro-OMeTAD, rapidly promoting its p-type doping and resulting in the precipitation of carbonates. The CO2-treated interlayer exhibits approximately 100 times higher conductivity than a pristine film while realizing stable, high-efficiency perovskite solar cells without any post-treatments. We also show that this method can be used to dope π-conjugated polymers.

A KLVFFAE-Derived Peptide Probe for Detection of Alpha-Synuclein Fibrils.

Aggregation of an amyloid protein, α-synuclein (αS), is a critical step in the neurodegenerative pathway of Parkinson's diseases (PD). Specific detection of amyloid conformers (i.e., monomers, oligomers, and fibrils) produced during αS aggregation is critical in better understanding a molecular basis of PD and developing a diagnostic tool. While various molecular probes are available for detection of αS fibrils, which may serve as a reservoir of toxic αS aggregate forms, these probes suffer from limited conformer-specificity and operational flexibility. In the present study, we explored the potential of non-self-aggregating peptides derived from the highly aggregation-prone KLVFFAE region of an amyloid protein, ß-amyloid, as molecular probes for αS aggregates. We show that of the four peptides tested (KLVFWAK, ELVFWAE, and their C-terminal capping variants, all of which were attached with fluorescein isothiocyanate at their respective N-termini), KLVFWAK with C-terminal capping was selectively bound to αS fibrils over monomers and oligomers and readily used for monitoring αS fibrilization. Our analyses suggest that binding of the peptide to αS fibrils is mediated by both electrostatic and hydrophobic interactions. We anticipate that our peptide can readily be optimized for conformer-specificity and operational flexibility. Overall, this study presents the creation of a KLVFFAE-based molecular probe for αS fibrils and demonstrates fine-tuning of its conformer-specificity by terminal mutations and capping.

Interactions between Soluble Species of ß-Amyloid and α-Synuclein Promote Oligomerization while Inhibiting Fibrillization.

Aggregations of ß-amyloid (Aß) and α-synuclein (αS) into oligomeric and fibrillar assemblies are the pathological hallmarks of Alzheimer's and Parkinson's diseases, respectively. Although Aß and αS affect different regions of the brain and are separated at the cellular level, there is evidence of their eventual interaction in the pathology of both disorders. Characterization of interactions of Aß and αS at various stages of their aggregation pathways could reveal mechanisms and therapeutic targets for the prevention and cure of these neurodegenerative diseases. In this study, we comprehensively examined the interactions and their molecular manifestations using an array of characterization tools. We show for the first time that αS monomers and oligomers, but not αS fibrils, inhibit Aß fibrillization while promoting oligomerization of Aß monomers and stabilizing preformed Aß oligomers via coassembly, as judged by Thioflavin T fluorescence, transmission electron microscopy, and SDS- and native-PAGE with fluorescently labeled peptides/proteins. In contrast, soluble Aß species, such as monomers and oligomers, aggregate into fibrils, when incubated alone under the otherwise same condition. Our study provides evidence that the interactions with αS soluble species, responsible for the effects, are mediated primarily by the C-terminus of Aß, when judged by competitive immunoassays using antibodies recognizing various fragments of Aß. We also show that the C-terminus of Aß is a primary site for its interaction with αS fibrils. Collectively, these data demonstrate aggregation state-specific interactions between αS and Aß and offer insight into a molecular basis of synergistic biological effects between the two polypeptides.

Amyloid Aggregation of Bacillus circulans Xylanase under Native Conditions and its Modulation by ß-Amyloid-Derived Peptide Fragments.

The aggregation of intrinsically disordered proteins into fibrils is implicated in many neurodegenerative diseases. Amyloid aggregation is a generic property of proteins as evidenced by globular proteins that often form amyloid aggregates under partially denaturing conditions. Recently, multiple lines of evidence have suggested that the amyloid aggregation of globular proteins can also occur under native conditions. Unfortunately, amyloid aggregation under native conditions has been demonstrated in only a handful of cases. Engineering a globular protein's amyloid aggregation might benefit from its fusion to an amyloid-derived fragment with reduced aggregation propensity. Unfortunately, the impacts of such fragments on the amyloid aggregation under native conditions have yet to be examined. In this study, we show that a globular protein, Bacillus circulans xylanase (BCX), can aggregate to form amyloid fibrils under native conditions. When BCX was mixed with or fused to the non-self-aggregating fragments, KLVFWAK and ELVFWAE-which were derived from ß-amyloid (Aß)-they modulated the BCX amyloid aggregation to differing extents. This study also provides insight into a correlation between the kinetic stability and amyloid aggregation of BCX, and supports a view that Aß-derived fragments can be useful for the modulating amyloid aggregation of some, though not all, proteins.

Thermophilic Proteins as Versatile Scaffolds for Protein Engineering.

Literature from the past two decades has outlined the existence of a trade-off between protein stability and function. This trade-off creates a unique challenge for protein engineers who seek to introduce new functionality to proteins. These engineers must carefully balance the mutation-mediated creation and/or optimization of function with the destabilizing effect of those mutations. Subsequent research has shown that protein stability is positively correlated with "evolvability" or the ability to support mutations which bestow new functionality on the protein. Since the ultimate goal of protein engineering is to create and/or optimize a protein's function, highly stable proteins are preferred as potential scaffolds for protein engineering. This review focuses on the application potential for thermophilic proteins as scaffolds for protein engineering. The relatively high inherent thermostability of these proteins grants them a great deal of mutational robustness, making them promising scaffolds for various protein engineering applications. Comparative studies on the evolvability of thermophilic and mesophilic proteins have strongly supported the argument that thermophilic proteins are more evolvable than mesophilic proteins. These findings indicate that thermophilic proteins may represent the scaffold of choice for protein engineering in the future.

Hetero-assembly of a dual ß-amyloid variant peptide system.

Self-assembly of amyloid polypeptides (1) imparts biological effects depending on the size in over 20 amyloid diseases and (2) produces useful yet relatively untapped biomaterials. Unfortunately, our understanding of amyloid polypeptides, as related to biomedical implications and biomaterial applications, is limited by their self-assembling nature. In this study, we report the creation of a dual peptide system, where a pair of ß-amyloid (Aß) variants are not self-assembled but hetero-assembled in the presence of their assembly partners. We provide evidence that the resulting hetero-assemblies share molecular, structural and morphological similarities with typical amyloid self-assemblies formed by a single polypeptide (e.g., Aß). We anticipate that our dual peptide system may readily be adapted for precise control of amyloid assembly, for the study of size-dependent neurotoxicity and precise fabrication of amyloid biomaterials.

Laboratory Evolution of Bacillus circulans Xylanase Inserted into Pyrococcus furiosus Maltodextrin-Binding Protein for Increased Xylanase Activity and Thermal Stability Toward Alkaline pH.

High xylanase activity and stability toward alkaline pH is strongly desired for pulping and bleaching processes. We previously enhanced thermal stability of Bacillus circulans xylanase (BCX) by inserting into a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) (the resulting complex named as PfMBP-BCX165). In the present study, we aimed to evolve the inserted BCX domain within PfMBP-BCX165 for greater xylanase activity toward alkaline pH while maintaining enhanced thermal stability. No BCX sequence variation was required for the thermal stabilization, thus allowing us to explore the entire BCX sequence space for the evolution. Specifically, we randomized the BCX sequence within PfMBP-BCX165 and then screened the resulting libraries to identify a PfMBP-BCX165 variant, PfMBP-BCX165T50R. The T50R mutation enhanced xylanase activity of PfMBP-BCX165 toward alkaline pH without compromising thermal stability. When compared to PfMBP-BCX165T50R, the corresponding unfused BCX mutant, BCXT50R, exhibited similar pH dependence of xylanase activity, yet suffered from limited thermal stability. In summary, we showed that one can improve thermal stability and xylanase activity of BCX toward alkaline pH by inserting into PfMBP followed by sequence variation of the BCX domain. Our study also suggested that insertional fusion to PfMBP would be a useful stabilizing platform for evolving many proteins.

Stabilization of Bacillus circulans xylanase by combinatorial insertional fusion to a thermophilic host protein.

High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX). Specifically, we used an engineered transposon to construct a combinatorial library of randomly inserted BCX into PfMBP. The library was then subjected to functional screening to identify successful PfMBP-BCX insertion complexes, PfMBP-BCX161 and PfMBP-BCX165, displaying substantially improved kinetic stability at elevated temperatures compared to unfused BCX and other controls. Results from subsequent characterizations were consistent with the view that lowered aggregation of BCX and reduced conformational flexibility at the termini was responsible for increased thermal stability. Our stabilizing approach neither sacrificed xylanase activity nor required changes in the BCX amino acid sequence. Overall, the current study demonstrated the benefit of combinatorial insertional fusion to PfMBP as a systematic tool for the creation of enzymatically active and thermostable BCX variants.

Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris cutinase: Role of glycosylation.

Cutinase thermostability is important so that the enzymes can function above the glass transition of what are often rigid polymer substrates. A detailed thermal inactivation analysis was performed for two well-characterized cutinases, Aspergillus oryzae Cutinase (AoC) and Thiellavia terrestris Cutinase (TtC). Both AoC and TtC are prone to thermal aggregation upon unfolding at high temperature, which was found to be a major reason for irreversible loss of enzyme activity. Our study demonstrates that glycosylation stabilizes TtC expressed in Pichia pastoris by inhibiting its thermal aggregation. Based on the comparative thermal inactivation analyses of non-glycosylated AoC, glycosylated (TtC-G), and non-glycosylated TtC (TtC-NG), a unified model for thermal inactivation is proposed that accounts for thermal aggregation and may be applicable to other cutinase homologues. Inspired by glycosylated TtC, we successfully employed glycosylation site engineering to inhibit AoC thermal aggregation. Indeed, the inhibition of thermal aggregation by AoC glycosylation was greater than that achieved by conventional use of trehalose under a typical condition. Collectively, this study demonstrates the excellent potential of implementing glycosylation site engineering for thermal aggregation inhibition, which is one of the most common reasons for the irreversible thermal inactivation of cutinases and many proteins. Biotechnol. Bioeng. 2017;114: 63-73. © 2016 Wiley Periodicals, Inc.

Transposon for protein engineering.

Protein insertional fusion and circular permutation are 2 promising protein engineering techniques for creating integrated functionalities and sequence diversity of a protein, respectively. Finding insertion locations for protein insertional fusion and new termini for circular permutation through a rational approach is not always straightforward, especially, for proteins without detailed structural knowledge. On the contrary, a combinatorial approach facilitates a comprehensive search to evaluate all potential insertion sites and new termini locations. Conventional methods used to create random insertional fusion libraries generate sub-optimal inter-domain linker length and composition between fused proteins. There are also methods available for construction of random circular permutation libraries. However, these methods too, impose many drawbacks, such as significant sequence modification at the new termini of circular permutants and additionally, require re-design of transposons for tailored expression of circular permutants. Furthermore, these conventional methods employ relatively inefficient blunt-end ligation during library construction. In this commentary, we present a concise overview and key findings of engineered Mu transposons, which have recently been developed in our group as a facile and efficient tool to alleviate limitations realized from conventional methods and to construct high quality libraries for random insertional fusion and random circular permutation.

Molecular Determinants for Protein Stabilization by Insertional Fusion to a Thermophilic Host Protein.

A universal method that improves protein stability and evolution has thus far eluded discovery. Recently, however, studies have shown that insertional fusion to a protein chaperone stabilized various target proteins with minimal negative effects. The improved stability was derived from insertion into a hyperthermophilic protein, Pyrococcus furiosus maltodextrin-binding protein (PfMBP), rather than from changes to the target protein sequence. In this report, by evaluating the thermodynamic and kinetic stability of various inserted ß-lactamase (BLA) homologues, we were able to examine the molecular determinants of stability realized by insertional fusion to PfMBP. Results indicated that enhanced stability and suppressed aggregation of BLA stemmed from enthalpic and entropic mechanisms. This report also suggests that insertional fusion to a stable protein scaffold has the potential to be a useful method for improving protein stability, as well as functional protein evolution.