Rational Protein Engineering Research Articles

Thermostability improvement of a Talaromyces leycettanus xylanase by rational protein engineering

Thermophilic xylanases with high catalytic efficiency are of great interest in the biofuel, food and feed industries. This study identified a GH11 xylanase gene, Tlxyn11B, in Talaromyces leycettanus JCM12802. Recombinant TlXyn11B produced in Pichia pastoris is distinguished by high specific activity (8259 ± 32 U/mg with beechwood xylan as substrate) and excellent pH stability (from 1.0 to 10.5). The beechwood xylan hydrolysates consisted mainly of xylobiose, xylotriose and xylotetraose, thus TlXyn11B could be used for the production of prebiotic xylooligosaccharide. By using the structure-based rational approach, the N-terminal sequence of TlXyn11B was modified for thermostability improvement. Mutants S3F and S3F/D35V/I/Q/M had elevated Tm values of 60.01 to 67.84 °C, with S3F/D35I the greatest. Homology modeling and molecular dynamics (MD) simulation analysis revealed that the substituted F3 and I35 formed a sandwich structure with S45 and T47, which may enhance the overall structure rigidity with lowered RMSD values. This study verifies the efficiency of rational approach in thermostability improvement and provides a xylanase candidate of GH11 with great commercialization potential.

Rational protein engineering approaches to further improve the keratinolytic activity and thermostability of engineered keratinase KerSMD

Keratinase has great potential to convert poultry waste into feed or fertilizers. In order to obtain excellent keratinase variants with improved thermostability and activity to degrade feather waste, we did rational protein engineering approaches. Using site-directed mutagenesis on keratinase FDD, two variants (Y94F and Y215F) showed higher extracellular keratinolytic activity and specific activity in Escherichia coli expression system. The C-terminus fusion on keratinase DDF created a new variant DDFD with the highest substrate specificity and keratinolytic activity of 6220±20U/mg. Besides, the performance of thermophilicity and theromostability of the variants was evaluated. The A218S and A218G showed more than 30% improvement of relative activity at 70°C while DDFD owned the most outstanding comprehensive performance between half-life (t1/2=146min) and keratinolytic activity. It is highly effective to obtain excellent keratinases by site-directed mutagenesis and C-terminus fusion based on model structure analysis, and it provides possibility of industrial application of new keratinases.

Rational Engineering of Photoconvertible Fluorescent Proteins for Dual‐Color Fluorescence Nanoscopy Enabled by a Triplet‐State Mechanism of Primed Conversion

AbstractGreen‐to‐red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super‐resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400‐nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far‐red/near‐infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate “primed” state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create “pr”‐ (for primed convertible) variants of most known green‐to‐red pcFPs.

Rational Engineering of Photoconvertible Fluorescent Proteins for Dual-Color Fluorescence Nanoscopy Enabled by a Triplet-State Mechanism of Primed Conversion.

Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create "pr"- (for primed convertible) variants of most known green-to-red pcFPs.

Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

Ancestral sequence reconstruction (ASR) represents a powerful approach for empirical testing structure-function relationships of diverse proteins. We employed ASR to predict sequences of five ancestral haloalkane dehalogenases (HLDs) from the HLD-II subfamily. Genes encoding the inferred ancestral sequences were synthesized and expressed in Escherichia coli, and the resurrected ancestral enzymes (AncHLD1-5) were experimentally characterized. Strikingly, the ancestral HLDs exhibited significantly enhanced thermodynamic stability compared to extant enzymes (ΔTm up to 24 °C), as well as higher specific activities with preference for short multi-substituted halogenated substrates. Moreover, multivariate statistical analysis revealed a shift in the substrate specificity profiles of AncHLD1 and AncHLD2. This is extremely difficult to achieve by rational protein engineering. The study highlights that ASR is an efficient approach for the development of novel biocatalysts and robust templates for directed evolution.

Rigidity theory for biomolecules: concepts, software, and applications

The mechanical heterogeneity of biomolecular structures is intimately linked to their diverse biological functions. Applying rigidity theory to biomolecules identifies this heterogeneous composition of flexible and rigid regions, which can aid in the understanding of biomolecular stability and long‐ranged information transfer through biomolecules, and yield valuable information for rational drug design and protein engineering. We review fundamental concepts in rigidity theory, ways to represent biomolecules as constraint networks, and methodological and algorithmic developments for analyzing such networks and linking the results to biomolecular function. Software packages for performing rigidity analyses on biomolecules in an efficient, automated way are described, as are rigidity analyses on biomolecules including the ribosome, viruses, or transmembrane proteins. The analyses address questions of allosteric mechanisms, mutation effects on (thermo‐)stability, protein (un‐)folding, and coarse‐graining of biomolecules. We advocate that the application of rigidity theory to biomolecules has matured in such a way that it could be broadly applied as a computational biophysical method to scrutinize biomolecular function from a structure‐based point of view and to complement approaches focused on biomolecular dynamics. We discuss possibilities to improve constraint network representations and to perform large‐scale and prospective studies. WIREs Comput Mol Sci 2017, 7:e1311. doi: 10.1002/wcms.1311This article is categorized under: Structure and Mechanism > Computational Biochemistry and Biophysics Computer and Information Science > Computer Algorithms and Programming Software > Molecular Modeling

Structure of NADP+-bound 7β-hydroxysteroid dehydrogenase reveals two cofactor-binding modes.

In mammals, bile acids/salts and their glycine and taurine conjugates are effectively recycled through enterohepatic circulation. 7β-Hydroxysteroid dehydrogenases (7β-HSDHs; EC 1.1.1.201), including that from the intestinal microbe Collinsella aerofaciens, catalyse the NADPH-dependent reversible oxidation of secondary bile-acid products to avoid potential toxicity. Here, the first structure of NADP+ bound to dimeric 7β-HSDH is presented. In one active site, NADP+ adopts a conventional binding mode similar to that displayed in related enzyme structures. However, in the other active site a unique binding mode is observed in which the orientation of the nicotinamide is different. Since 7β-HSDH has become an attractive target owing to the wide and important pharmaceutical use of its product ursodeoxycholic acid, this work provides a more detailed template to support rational protein engineering to improve the enzymatic activities of this useful biocatalyst, further improving the yield of ursodeoxycholic acid and its other applications.

Reweighting the Apo to the Holo Ensemble

Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site. Simulations can enable the discovery of allosteric binding sites and elucidate the mechanism of allostery, providing a foundation for applications including rational drug design and protein engineering. However, performing a separate simulation for every ligand can be computationally expensive. Here we demonstrate a proof of principle that conformations sampled from the apo state can be reweighted according to the binding potential of mean force (BPMF) to predict ensemble averages in the holo state. Using BPMFs for the binding of AMP and ADP to adenylate kinase, an enzyme that plays an important role in the process of energy homeostasis, we construct a hinge angle distribution consistent with alchemical binding free energy calculations.

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain.

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the β-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.

Active Hydrogen Bond Network (AHBN) and Applications for Improvement of Thermal Stability and pH-Sensitivity of Pullulanase from Bacillus naganoensis.

A method, so called “active hydrogen bond network” (AHBN), is proposed for site-directed mutations of hydrolytic enzymes. In an enzyme the AHBN consists of the active residues, functional residues, and conservative water molecules, which are connected by hydrogen bonds, forming a three dimensional network. In the catalysis hydrolytic reactions of hydrolytic enzymes AHBN is responsible for the transportation of protons and water molecules, and maintaining the active and dynamic structures of enzymes. The AHBN of pullulanase BNPulA324 from Bacillus naganoensis was constructed based on a homologous model structure using Swiss Model Protein-modeling Server according to the template structure of pullulanase BAPulA (2WAN). The pullulanase BNPulA324 are mutated at the mutation sites selected by means of the AHBN method. Both thermal stability and pH-sensitivity of pullulanase BNPulA324 were successfully improved. The mutations at the residues located at the out edge of AHBN may yield positive effects. On the other hand the mutations at the residues inside the AHBN may deprive the bioactivity of enzymes. The AHBN method, proposed in this study, may provide an assistant and alternate tool for protein rational design and protein engineering.

Development of an Optical Zn2+ Probe Based on a Single Fluorescent Protein

Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn2+. Here, we present Green Zinc Probe (GZnP), a novel genetically encoded Zn2+ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering, and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1, which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn2+ in HeLa cells (Fmax/Fmin = 2.6, Kd = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn2+ (Fmax/Fmin = 2.2). This sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn2+ function.

Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations.

Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest.

Capitalizing on Directed Evolution and Rational Protein Engineering to Expand the Neuroscientist's Imaging Toolbox.

Breakthroughs in neuroscience research are accelerated by technological innovations. For example, patch-clamp electrophysiology significantly transformed the brain research landscape in the late 20th century. Neuroscience has now entered into an “age of light” ([Scanziani and Hausser, 2009][1

Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site

The aim of this study was to improve a useful molecular tool-TaqII restriction endonuclease-methyltransferase-by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue-TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.

A Comprehensive Study of Molecular Evolution at the Self-Incompatibility Locus of Rosaceae.

The family Rosaceae includes a range of important fruit trees, most of which have the S-RNase-based self-incompatibility (SI). Several models have been developed to explain how pollen (SLF) and pistil (S-RNase) components of the S-locus interact. It was discovered in 2010 that additional SLF proteins are involved in pollen specificity, and a Collaborative Non-Self Recognition model has been proposed for SI in Solanaceae; however, the validity of such model remains to be elucidated for other species. The results of this study support the divergent evolution of the S-locus genes from two Rosaceae subfamilies, Prunoideae/Amygdaloideae and Maloideae, The difference identified in the selective pressures between the two lineages provides evidence for positive selection at specific sites in both the S-RNase and the SLF proteins. The evolutionary findings of this study support the role of multiple SLF proteins leading to a Collaborative Non-Self Recognition model for SI in the Maloideae. Furthermore, the identification of the sites responsible for SI specificity determination and the mapping of these sites onto the modelled tertiary structure of ancestor proteins provide useful information for rational functional redesign and protein engineering for the future engineering of new functional alleles providing increased diversity in the SI system in the Maloideae.

Mechanism-based suicide inactivation of white Spanish broom (Cytisus multiflorus) peroxidase by excess hydrogen peroxide

Suicide inactivation is a common mechanism observed for haem peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by intermediate highly oxidizing enzyme forms during the catalytic cycle. The time-dependence and the inactivation mechanism of Cytisus multiflorus peroxidase (CMP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferulic acid, guaiacol and o-dianisidine). Catalytic activity decreased following the sequence ABTS>guaiacol>ferulic acid>o-dianisidine. Once the intermediate complex (compound III-H2O2) had been formed, competition was established between the catalytic pathway and the suicide inactivation pathway. One mole of CMP afforded around 3790 turnovers of H2O2 for ABTS before its complete inactivation. These results suggest that CMP follows a suicide mechanism, the enzyme not being protected in this case. The mechanism of suicide inactivation is discussed with a view to establishing a broad knowledge base for future rational protein engineering.

Rational Protein Engineering Guided by Deep Mutational Scanning.

Sequence–function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and three-dimensional structures is rapidly widening. A recently developed method termed deep mutational scanning explores the functional phenotype of thousands of mutants via massive sequencing. Coupled with a highly efficient screening system, this approach assesses the phenotypic changes made by the substitution of each amino acid sequence that constitutes a protein. Such an informational resource provides the functional role of each amino acid sequence, thereby providing sufficient rationale for selecting target residues for protein engineering. Here, we discuss the current applications of deep mutational scanning and consider experimental design.

Structural analysis of enzymes used for bioindustry and bioremediation.

Microbial enzymes have been widely applied in the large-scale, bioindustrial manufacture of food products and pharmaceuticals due to their high substrate specificity and stereoselectivity, and their effectiveness under mild conditions with low environmental burden. At the same time, bioremedial techniques using microbial enzymes have been developed to solve the problem of industrial waste, particularly with respect to persistent chemicals and toxic substances. And finally, structural studies of these enzymes have revealed the mechanistic basis of enzymatic reactions, including the stereoselectivity and binding specificity of substrates and cofactors. The obtained structural insights are useful not only to deepen our understanding of enzymes with potential bioindustrial and/or bioremedial application, but also for the functional improvement of enzymes through rational protein engineering. This review shows the structural bases for various types of enzymatic reactions, including the substrate specificity accompanying cofactor-controlled and kinetic mechanisms.

Creating a completely "cell-free" system for protein synthesis.

Cell-free protein synthesis is a promising tool to take biotechnology outside of the cell. A cell-free approach provides distinct advantages over in vivo systems including open access to the reaction environment and direct control over all chemical components for facile optimization and synthetic biology integration. Promising applications of cell-free systems include portable diagnostics, biotherapeutics expression, rational protein engineering, and biocatalyst production. The highest yielding and most economical cell-free systems use an extract composed of the soluble component of lysed Escherichia coli. Although E. coli lysis can be highly efficient (>99.999%), one persistent challenge is that the extract remains contaminated with up to millions of cells per mL. In this work, we examine the potential of multiple decontamination strategies to further reduce or eliminate bacteria in cell-free systems. Two strategies, sterile filtration and lyophilization, effectively eliminate contaminating cells while maintaining the systems' protein synthesis capabilities. Lyophilization provides the additional benefit of long-term stability at storage above freezing. Technologies for personalized, portable medicine and diagnostics can be expanded based on these foundational sterilized and completely "cell-free" systems.

Improving the catalytic performance of a GH11 xylanase by rational protein engineering.

XynCDBFV from Neocallimastix patriciarum is among the most effective xylanases and holds great potentials in a wide variety of industrial applications. In the present study, several active site residues were modified referring to the instrumental information of the complex structure of XynCDBFV and xylooligosaccharides. Among the 12 single active site mutants, W125F and F163W show increased activity comparing to the wild-type protein. The double mutant W125F/F163W was then generated which displayed nearly 20 % increase in the enzyme activity. Although W125F/F163W showed 5 °C reduction in the optimal temperature, it still preserves similar thermostability and is more active than the wild-type enzyme at temperatures lower than 60 °C. These properties make the double mutant a suitable candidate for commercial applications that involve lower operating temperatures. Furthermore, we investigated the effect of N-glycosylation on the thermostability of XynCDBFV when expressed in the yeast strain Pichia pastoris for industrial use. Two potential glycosylation sites (Asn-37 and Asn-88) were examined, and their roles in enzyme performance were validated. We found that the N-glycosylations of XynCDBFV are related to both catalytic activity and heat stability, with Asn-37 motif playing a dominant role. Collectively, the enzymatic properties of XynCDBFV were improved by molecular engineering, and the influences of N-glycosylations on the enzyme have been clearly elucidated herein.