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Helicase-Like Functions in Phosphate Loop Containing Beta-Alpha Polypeptides
Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.07.30.228619v1?rss=1
Authors: Vyas, P., Trofimyuk, O., Longo, L. M., Deshmukh, F. K., Sharon, M., Tawfik, D. S.
Abstract:
The P-loop Walker A motif underlies hundreds of essential enzyme families that bind NTPs and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple {beta}repeat proteins bind NTPs, but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one {beta}-(P-loop)- motif. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. These tantalizing resemblances to extant P-loop helicases and recombinases suggest that these engineered P-loop prototypes comprise a plausible description of the sequence, structure and function of the earliest P-loop NTPases. These prototypes also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.
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Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2020.07.30.228619v1?rss=1
Authors: Vyas, P., Trofimyuk, O., Longo, L. M., Deshmukh, F. K., Sharon, M., Tawfik, D. S.
Abstract:
The P-loop Walker A motif underlies hundreds of essential enzyme families that bind NTPs and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple {beta}repeat proteins bind NTPs, but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one {beta}-(P-loop)- motif. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. These tantalizing resemblances to extant P-loop helicases and recombinases suggest that these engineered P-loop prototypes comprise a plausible description of the sequence, structure and function of the earliest P-loop NTPases. These prototypes also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.
Copy rights belong to original authors. Visit the link for more info