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

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