Engineering protein dynamics is a challenging and unsolved problem in protein design. Loop transplantation or loop grafting has been previously employed to transfer dynamic properties between proteins. We recently released a LoopGrafter Web server to execute the loop grafting task, employing eight computational tools and one database. The LoopGrafter method relies on the prediction of the local dynamic behavior of the elements to be transplanted and has successfully reconstructed previously engineered sequences. However, it was unclear whether catalytically competitive previously uncharacterized designs could be obtained by this method. Here, we address this question, showing how LoopGrafter generates viable loop-grafted chimeras of luciferases, how these chimeras encompass the activity of interest and unique kinetic properties, and how all this process is done fully automatically and agnostic of any previous knowledge. All constructed designs proved to be catalytically active, and the most active one improved the activity of the template enzyme by 4 orders of magnitude. The computational details and parameter optimization of the sequence pairing step of the LoopGrafter workflow are revealed. The optimized and experimentally validated loop grafting workflow available as a fully automated Web server represents a powerful approach for engineering catalytically efficient enzymes by modification of protein dynamics.

• Publikační typ
• časopisecké články MeSH

Recent progress in engineering highly promising biocatalysts has increasingly involved machine learning methods. These methods leverage existing experimental and simulation data to aid in the discovery and annotation of promising enzymes, as well as in suggesting beneficial mutations for improving known targets. The field of machine learning for protein engineering is gathering steam, driven by recent success stories and notable progress in other areas. It already encompasses ambitious tasks such as understanding and predicting protein structure and function, catalytic efficiency, enantioselectivity, protein dynamics, stability, solubility, aggregation, and more. Nonetheless, the field is still evolving, with many challenges to overcome and questions to address. In this Perspective, we provide an overview of ongoing trends in this domain, highlight recent case studies, and examine the current limitations of machine learning-based methods. We emphasize the crucial importance of thorough experimental validation of emerging models before their use for rational protein design. We present our opinions on the fundamental problems and outline the potential directions for future research.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering is the unique dynamism that enables them to exert allosteric control over the catalytic function of enzymes. Thus, when engaging in a transplantation effort, such dynamics in the context of protein structure need consideration. A second practical challenge is identifying successful excision points for the transplantation or grafting. Here, we present LoopGrafter (https://loschmidt.chemi.muni.cz/loopgrafter/), a web server that specifically guides in the loop grafting process between structurally related proteins. The server provides a step-by-step interactive procedure in which the user can successively identify loops in the two input proteins, calculate their geometries, assess their similarities and dynamics, and select a number of loops to be transplanted. All possible different chimeric proteins derived from any existing recombination point are calculated, and 3D models for each of them are constructed and energetically evaluated. The obtained results can be interactively visualized in a user-friendly graphical interface and downloaded for detailed structural analyses.

• MeSH
• internet MeSH
• molekulární modely MeSH
• proteinové inženýrství MeSH
• proteiny * genetika   chemie   MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny * MeSH

Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.

• MeSH
• buňky NIH 3T3 MeSH
• katalýza MeSH
• kinetika MeSH
• konformace proteinů MeSH
• luciferasy renil chemie   genetika   metabolismus   MeSH
• luciferasy chemie   genetika   metabolismus   MeSH
• mutace MeSH
• mutageneze MeSH
• myši MeSH
• proteinové inženýrství * MeSH
• savci MeSH
• simulace molekulární dynamiky * MeSH
• stabilita enzymů MeSH
• teplota MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• luciferasy renil MeSH
• luciferasy MeSH