Protein Engineering Applications Research Articles

Probing Biomolecular Interactions with Paramagnetic Nuclear Magnetic Resonance Spectroscopy.

Recent advances in computational methods like AlphaFold have transformed structural biology, enabling accurate modeling of protein complexes and driving applications in drug discovery and protein engineering. However, predicting the structure of systems involving weak, transient, or dynamic interactions, or of complexes with disordered regions, remains challenging. Nuclear Magnetic Resonance (NMR) spectroscopy offers atomic-level insights into biomolecular complexes, even in weakly interacting and dynamic systems. Paramagnetic NMR, in particular, provides long-range structural restraints, easily exceeding distances over 25 Å, making it ideal for studying large protein complexes. Advances in chemical tools for introducing paramagnetic tags into proteins, combined with progress in electron paramagnetic resonance (EPR) spectroscopy, have enhanced the method's utility. This perspective article discusses paramagnetic NMR approaches for analyzing biomolecular complexes in solution and in the solid state, emphasizing quantities like pseudocontact shifts, residual dipolar couplings, and paramagnetic relaxation enhancements. Additionally, dynamic nuclear polarization offers a promising method to amplify NMR signals of large complexes, even in complex environments. The integration of AlphaFold protein structure prediction with paramagnetic NMR holds great potential for advancing our understanding of biomolecular interactions.

Prions: structure, function, evolution, and disease.

Prions are proteinaceous infectious particles implicated in fatal neurodegenerative disorders known as prion diseases. Herein, we provide an overview of prion biology, emphasizing the structural, functional, and evolutionary aspects of prions, along with their potential applications in protein engineering. Understanding the structure-function relationships of both healthy and disease-associated prion proteins enables a deeper understanding of the mechanisms of prion-induced neurotoxicity. Furthermore, we describe how insights into prion evolution have begun to shed light on their ancient origins and evolutionary resilience, offering deeper insights into the potential roles of prions in primordial chemical processes.

Thermodynamic and Conformational Analysis of GTRNase and Lysozyme Proteins Under Thermal Variations using Molecular Dynamics Simulations

This study examines the thermodynamic and conformational dynamics of GTRNase and lysozyme proteins under varying temperature conditions using molecular dynamics (MD) simulations. The objective is to evaluate their structural stability, folding behavior, and thermodynamic properties to understand their responses to thermal fluctuations. Protein structures were retrieved from the Protein Data Bank (PDB), refined to remove extraneous molecules, and simulated using GROMACS 2022.2 under NVT and NPT ensembles, spanning temperatures from 270 K to 380 K. The results revealed distinct behaviors for the two proteins. GTRNase exhibited a slight escalation in radius of gyration (Rg) from 1.434 nm at 270 K to 1.445 nm at 380 K, suggesting a marginal conformational expansion. In contrast, lysozyme maintained a consistent Rg of 1.38 nm over the same temperature range, indicating structural compactness. Root mean square deviation (RMSD) data demonstrated increased flexibility in both proteins, with GTRNase escalating from 0.115 nm at 270 K to 0.179 nm at 380 K and lysozyme rising from 0.102 nm to 0.142 nm across the temperature range. Solvent-accessible surface area (SASA) for GTRNase fluctuated between 69 nm² and 73 nm², with the lowest value observed at 300 K and the highest at 370 K. These findings highlight that GTRNase is more susceptible to thermal perturbations than lysozyme, showing greater conformational flexibility and expansion. This research underscores the utility of MD simulations in exploring protein behavior and provides valuable insights for applications in protein engineering and drug design.

Amino acid variability at W194 of Staphylococcus aureus sortase A alters nucleophile specificity.

Bacterial sortases are a family of cysteine transpeptidases in Gram-positive bacteria of which sortase A (SrtA) enzymes are responsible for ligating proteins to the peptidoglycan layer of the cell surface. Engineered versions of sortases are also used in sortase-mediated ligation (SML) strategies for a variety of protein engineering applications. Although a versatile tool, substrate recognition by Staphylococcus aureus SrtA (saSrtA), the most commonly utilized enzyme in SML, is stringent and relies on an LPXTG pentapeptide motif. Previous structural studies revealed that the requirement of a glycine in the binding motif may be due to potential steric hindrance of amino acids possessing a β-carbon by W194, a tryptophan located in the β7-β8 loop of the enzyme. Here, we measured the effect of seven single point mutants of W194 (A, D, F, G, N, S, Y) saSrtA using a FRET-based activity assay. We found that while the LPXTG motif remains a requirement for initial proteolytic cleavage, the nucleophile specificity of our variants is altered. In particular, W194A and W194S saSrtA recognize a D-Ala nucleophile and are able to perform ligation reactions. Notably, an LPXT(D-Ala) peptide was not cleaved by either mutant enzyme. We hypothesize that these variants may potentially be utilized to develop an irreversible sortase-mediated reaction. Taken together, this experiment reveals new insight into sortase specificity and possible future SML strategies.

Advancements in the Engineering Modification of Sucrose Phosphorylase

Sucrose phosphorylase (SPase) is a member of the glycoside hydrolase family 13, catalyzing the reversible phosphorolysis of sucrose to produce α–glucose–1–phosphate and exhibiting transglycosylation activity toward multiple substrates. Its wide substrate specificity enables the synthesis of various glycosides, which are broadly applied in food, cosmetics, and pharmaceuticals. However, the industrial application of SPase is constrained by its poor thermostability and limited transglycosylation activity. Therefore, current research focuses on enhancing the thermostability and transglycosylation activity of SPase through efficient engineering strategies based on its crystal structure and catalytic mechanism. This paper systematically reviews the crystal structure and catalytic mechanism of SPase, outlines the application of protein engineering and immobilization strategies in improving the thermostability of SPase, and analyzes how modifications at key amino acid sites affect the synthesis of typical glycosylation products. It also summarizes the limitations of SPase engineering modification strategies and explores the potential of diversified approaches for SPase modification, highlighting its broad application prospects in industrial production and laying a solid foundation for further advancements in SPase engineering modification and its industrial application.

Tuning the Stability and Kinetics of Dioxazaborocanes.

We investigated the equilibrium reaction of boronic acid (BA), diethanolamines (DEA), and 1,3,6,2-dioxazaborocanes (DOAB) in aqueous solutions, both theoretically and experimentally. Our findings show that the association constant can be adjusted by substituting BA and DEA derivatives, ranging from 100 to 103 M-1, exhibiting a bell-shaped pH dependency. The highest stability was achieved when the pKa values of DEA and BA were closely matched. This approach enabled the preparation of a highly stable DOAB under physiological conditions. Furthermore, the hydrolysis kinetics of DOABs were controllable over a range of five orders of magnitude based on the substituent's steric effect. In the slowest case, this resulted in quasi-static stability with only 1 % cleavage in the first hour, followed by a week-long cleavage period to reach equilibrium. These insights could establish a unique chemistry platform for designing scheduled cleavability on a day-to-week timescale, relevant to protein engineering, immunotherapy, and other smart drug delivery applications.

Visible-Light-Driven Synthesis of N-Alkyl α-Amino Acid Derivatives from Unactivated Alkyl Bromides and In Situ Generated Imines.

One-pot, multicomponent reactions are known for their green and efficient nature. We report a novel three-component reaction of alkyl amines, alkyl glyoxylates, and unactivated alkyl bromides under visible-light-induced palladium catalysis, yielding N-alkyl unnatural α-amino acid derivatives. This method offers mild conditions, broad substrate scope, and excellent functional group tolerance without requiring stoichiometric organometallic reagents. The approach has promising applications in protein engineering and drug discovery.

Kinetic Resolution of Epimeric Proteins Enables Stereoselective Chemical Mutagenesis.

Chemical mutagenesis via dehydroalanine (Dha) is a powerful method to tailor protein structure and function, allowing the site-specific installation of post-translational modifications and non-natural functional groups. Despite the impressive versatility of this method, applications have been limited, as products are formed as epimeric mixtures, whereby the modified amino acid is present as both the desired l-configuration and a roughly equal amount of the undesired d-isomer. Here, we describe a simple remedy for this issue: removal of the d-isomer via proteolysis using a d-stereoselective peptidase, alkaline d-peptidase (AD-P). We demonstrate that AD-P can selectively cleave the d-isomer of epimeric residues within histone H3, GFP, Ddx4, and SGTA, allowing the installation of non-natural amino acids with stereochemical control. Given the breadth of modifications that can be introduced via Dha and the simplicity of our method, we believe that stereoselective chemoenzymatic mutagenesis will find broad utility in protein engineering and chemical biology applications.

Rapid protein evolution by few-shot learning with a protein language model.

Directed evolution of proteins is critical for applications in basic biological research, therapeutics, diagnostics, and sustainability. However, directed evolution methods are labor intensive, cannot efficiently optimize over multiple protein properties, and are often trapped by local maxima. In silico-directed evolution methods incorporating protein language models (PLMs) have the potential to accelerate this engineering process, but current approaches fail to generalize across diverse protein families. We introduce EVOLVEpro, a few-shot active learning framework to rapidly improve protein activity using a combination of PLMs and protein activity predictors, achieving improved activity with as few as four rounds of evolution. EVOLVEpro substantially enhances the efficiency and effectiveness of in silico protein evolution, surpassing current state-of-the-art methods and yielding proteins with up to 100-fold improvement of desired properties. We showcase EVOLVEpro for five proteins across three applications: T7 RNA polymerase for RNA production, a miniature CRISPR nuclease, a prime editor, and an integrase for genome editing, and a monoclonal antibody for epitope binding. These results demonstrate the advantages of few-shot active learning with small amounts of experimental data over zero-shot predictions. EVOLVEpro paves the way for broader applications of AI-guided protein engineering in biology and medicine.

Protein engineering enables a soakable crystal form of human CDK7 primed for high-throughput crystallography and structure-based drug design

Cyclin dependent kinase 7 (CDK7) is an important therapeutic kinase best known for its dual role in cell cycle regulation and gene transcription. Here, we describe the application of protein engineering to generate constructs leading to high resolution crystal structures of human CDK7 in both active and inactive conformations. The active state of the kinase was crystallized by incorporation of an additional surface residue mutation (W132R) onto the double phosphomimetic mutant background (S164D and T170E) that yielded the inactive kinase structure. A novel back-soaking approach was developed to determine crystal structures of several clinical and pre-clinical inhibitors of this kinase, demonstrating the potential utility of the crystal system for structure-based drug design (SBDD). The crystal structures help to rationalize the mode of inhibition and the ligand selectivity profiles versus key anti-targets. The protein engineering approach described here illustrates a generally applicable strategy for structural enablement of challenging molecular targets.

Endolysins: a new antimicrobial agent against antimicrobial resistance. Strategies and opportunities in overcoming the challenges of endolysins against Gram-negative bacteria.

Antibiotic-resistant bacteria are rapidly emerging, and the increasing prevalence of multidrug-resistant (MDR) Acinetobacter baumannii poses a severe threat to humans and healthcare organizations, due to the lack of innovative antibacterial drugs. Endolysins, which are peptidoglycan hydrolases encoded by a bacteriophage, are a promising new family of antimicrobials. Endolysins have been demonstrated as an effective therapeutic agent against bacterial infections of A. baumannii and many other Gram-positive and Gram-negative bacteria. Endolysin research has progressed from basic in vitro characterization to sophisticated protein engineering methodologies, including advanced preclinical and clinical testing. Endolysin are therapeutic agent that shows antimicrobial properties against bacterial infections caused by drug-resistant Gram-negative bacteria, there are still barriers to their implementation in clinical settings, such as safety concerns with outer membrane permeabilizers (OMP) use, low efficiency against stationary phase bacteria, and stability issues. The application of protein engineering and formulation techniques to improve enzyme stability, as well as combination therapy with other types of antibacterial drugs to optimize their medicinal value, have been reviewed as well. In this review, we summarize the clinical development of endolysin and its challenges and approaches for bringing endolysin therapies to the clinic. This review also discusses the different applications of endolysins.

SpyTag Peptide with Alkoxyl Aspartic Acids for pH-Dependent Activation of the SpyCatcher/Tag System.

The SpyCatcher/SpyTag system is a protein pair that forms a covalent isopeptide bond without an additional energy supply. The ability to connect fused proteins makes this system an attractive tool for several protein engineering applications. Conditional activation of the SpyCatcher/SpyTag complex formation further expands the use of this system. Here, we evaluated the pH activation of SpyTag using alkoxyaspartic acids in the isopeptide-forming residue. We found that a peptide with an ethoxy group can be activated by hydrolysis under high pH conditions. However, the hydrolysis induces isoaspartate (isoAsp) formation, which is confirmed by an isoAsp-inserted short peptide. We overcame this problem by changing the C-terminal side of the aspartic acid position to Pro, which does not form isoAsp under high pH conditions. The findings of this study provide fundamental knowledge of the synthetic construction of the modified SpyTag peptide.

Design of a recombinant asparaginyl ligase for site-specific modification using efficient recognition and nucleophile motifs

Asparaginyl ligases have been extensively utilized as valuable tools for site-specific bioconjugation or surface-modification. However, the application is hindered by the laborious and poorly reproducible preparation processes, unstable activity and ambiguous substrate requirements. To address these limitations, this study employed a structure-based rational approach to obtain a high-yield and high-activity protein ligase called OaAEP1-C247A-aa55-351. It was observed that OaAEP1-C247A-aa55-351 exhibits appreciable catalytic activities across a wide pH range, and the addition of the Fe3+ metal ion effectively enhances the catalytic power. Importantly, this study provides insight into the recognition and nucleophile peptide profiles of OaAEP1-C247A-aa55-351. The ligase demonstrates a higher recognition ability for the “Asn-Ala-Leu” motif and an N-terminus “Arg-Leu” as nucleophiles, which significantly increases the reaction yield. Consequently, the catalytic activity of OaAEP1-C247A-aa55-351 with highly efficient recognition and nucleophile motif, “Asn-Ala-Leu” and “Arg-Leu” under the buffer containing Fe3+ is 70-fold and 2-fold higher than previously reported OaAEP1-C247A and the most efficient butelase-1, respectively. Thus, the designed OaAEP1-C247A-aa55-351, with its highly efficient recognition and alternative nucleophile options, holds promising potential for applications in protein engineering, chemo-enzymatic modification, and the development of drugs.

Highly chromophoric fluorescent-labeled methionyl-initiator tRNAs applicable in living cells.

Fluorescent initiator tRNAs (tRNAi) play a crucial role in studying protein synthesis, yet generating highly fluorescent tRNAi complexes remains challenging. We present an optimized strategy to effectively generate highly fluorescent initiator-tRNA complexes in living cells. Our strategy allows the generation of Fluo-Met-tRNAiMet complexes. These complexes can have highly chromogenic N-terminal labeling. For generating such complexes, we use either purified fluorescent methionine (PFM) or non-purified fluorescently labeled methionine (NPFM). Furthermore, PFM promotes the active generation of endogenous tRNAi in cells, leading to highly efficient Fluo-Met-tRNAiMet complexes. Finally, PFM-tRNAiMet complexes also facilitate the visualization of native fluorescently labeled Tat binding to beads. This demonstrates the potential of our approach to advance precision protein engineering and biotechnology applications.

Fluctuation Relations to Calculate Protein Redox Potentials from Molecular Dynamics Simulations.

The tunable design of protein redox potentials promises to open a range of applications in biotechnology and catalysis. Here, we introduce a method to calculate redox potential changes by combining fluctuation relations with molecular dynamics simulations. It involves the simulation of reduced and oxidized states, followed by the instantaneous conversion between them. Energy differences introduced by the perturbations are obtained using the Kubo-Onsager approach. Using a detailed fluctuation relation coupled with Bayesian inference, these are postprocessed into estimates for the redox potentials in an efficient manner. This new method, denoted MD + CB, is tested on a de novo four-helix bundle heme protein (the m4D2 "maquette") and five designed mutants, including some mutants characterized experimentally in this work. The MD + CB approach is found to perform reliably, giving redox potential shifts with reasonably good correlation (0.85) to the experimental values for the mutants. The MD + CB approach also compares well with redox potential shift predictions using a continuum electrostatic method. The estimation method employed within the MD + CB approach is straightforwardly transferable to standard equilibrium MD simulations and holds promise for redox protein engineering and design applications.

Inteins-mechanism of protein splicing, emerging regulatory roles, and applications in protein engineering.

Protein splicing is a posttranslational process in which an intein segment excises itself from two flanking peptides, referred to as exteins. In the native context, protein splicing results in two separate protein products coupled to the activation of the intein-containing host protein. Inteins are generally described as either full-length inteins, mini-inteins or split inteins, which are differentiated by their genetic structure and features. Inteins can also be divided into three classes based on their splicing mechanisms, which differ in the location of conserved residues that mediate the splicing pathway. Although inteins were once thought to be selfish genetic elements, recent evidence suggests that inteins may confer a genetic advantage to their host cells through posttranslational regulation of their host proteins. Finally, the ability of modified inteins to splice and cleave their fused exteins has enabled many new applications in protein science and synthetic biology. In this review, we briefly cover the mechanisms of protein splicing, evidence for some inteins as environmental sensors, and intein-based applications in protein engineering.

Expanding the Genetic Code: Incorporation of Functional Secondary Amines via Stop Codon Suppression

AbstractEnzymes are attractive catalysts for chemical industries, and their use has become a mature alternative to conventional chemical methods. However, biocatalytic approaches are often restricted to metabolic and less complex reactivities, given the limited amount of functional groups present. This drawback can be addressed by incorporating non‐canonical amino acids (ncAAs) harboring new‐to‐nature chemical groups. Inspired by organocatalysis, we report the design, synthesis and characterization of a panel of ncAAs harboring functional secondary amines and their cellular incorporation into different protein scaffolds. D/L‐pyrrolidine‐ and D/L‐piperidine‐based ncAAs were successfully site‐specifically incorporated into proteins via stop codon suppression methodology. To demonstrate the utility of these ncAAs, the catalytic performance of the obtained artificial enzymes was investigated in a model Michael addition reaction. The incorporation of pyrrolidine‐ and piperidine‐ based ncAAs significantly expands the available toolbox for protein engineering and chemical biology applications.

Molecular analysis of the type III interferon complex and its applications in protein engineering

Type III interferons (IFNλs) are cytokines with critical roles in the immune system and are attractive therapeutic candidates due to their tissue-specific activity. Despite entering several clinical trials, results have demonstrated limited efficacy and potency, partially attributed to low-affinity protein-protein interactions (PPIs) responsible for receptor complex formation. Subsequently, structural studies of the native IFNλ signaling complexes remain inaccessible. While protein engineering can overcome affinity limitations, tools to investigate low-affinity systems like these remain limited. To provide insights into previous efforts to strengthen the PPIs within this complex, we perform a molecular analysis of the extracellular ternary complexes of IFNλ3 using both computational and experimental approaches. We first use molecular simulations and modeling to quantify differences in PPIs and residue strain fluctuations, generate detailed free energy landscapes, and reveal structural differences between an engineered, high-affinity complex, and a model of the wild-type, low-affinity complex. This analysis illuminates distinct behaviors of these ligands, yielding mechanistic insights into IFNλ complex formation. We then apply these computational techniques in protein engineering and design by utilizing simulation data to identify hotspots of interaction to rationally engineer the native cytokine-receptor complex for increased stability. These simulations are then validated by experimental techniques, showing that a single mutation at a computationally predicted site of interaction between the two receptors increases PPIs and improves complex formation for all IFNλs. This study highlights the power of molecular dynamics simulations for protein engineering and design as applied to the IFNλ family but also presents a potential tool for analysis and engineering of other systems with low-affinity PPIs.

A Chicken Tapasin ortholog can chaperone empty HLA-B∗37:01 molecules independent of other peptide-loading components

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC), and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange invitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in a stable form, independent of co-chaperones. chTapasin can bind the human HLA-B∗37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved β2m epitope on HLA-B∗37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B∗37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

Automated identification of chalcogen bonds in AlphaFold protein structure database files: is it possible?

Protein structure prediction and structural biology have entered a new era with an artificial intelligence-based approach encoded in the AlphaFold2 and the analogous RoseTTAfold methods. More than 200 million structures have been predicted by AlphaFold2 from their primary sequences and the models as well as the approach itself have naturally been examined from different points of view by experimentalists and bioinformaticians. Here, we assessed the degree to which these computational models can provide information on subtle structural details with potential implications for diverse applications in protein engineering and chemical biology and focused the attention on chalcogen bonds formed by disulphide bridges. We found that only 43% of the chalcogen bonds observed in the experimental structures are present in the computational models, suggesting that the accuracy of the computational models is, in the majority of the cases, insufficient to allow the detection of chalcogen bonds, according to the usual stereochemical criteria. High-resolution experimentally derived structures are therefore still necessary when the structure must be investigated in depth based on fine structural aspects.