Design of a zinc-finger hydrolase with a synthetic αββ protein.

Recent advances in protein design have opened avenues for the creation of artificial enzymes needed for biotechnological and pharmaceutical applications. However, designing efficient enzymes remains an unrealized ambition, as the design must incorporate a catalytic apparatus specific for the desired...

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Autores principales:	Kinshuk Raj Srivastava, Susheel Durani
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b707d3a1720846c7b0f236163fe62276
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spelling	oai:doaj.org-article:b707d3a1720846c7b0f236163fe622762021-11-18T08:19:54ZDesign of a zinc-finger hydrolase with a synthetic αββ protein.1932-620310.1371/journal.pone.0096234https://doaj.org/article/b707d3a1720846c7b0f236163fe622762014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24816915/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Recent advances in protein design have opened avenues for the creation of artificial enzymes needed for biotechnological and pharmaceutical applications. However, designing efficient enzymes remains an unrealized ambition, as the design must incorporate a catalytic apparatus specific for the desired reaction. Here we present a de novo design approach to evolve a minimal carbonic anhydrase mimic. We followed a step-by-step design of first folding the main chain followed by sequence variation for substrate binding and catalysis. To optimize the fold, we designed an αββ protein based on a Zn-finger. We then inverse-designed the sequences to provide stability to the fold along with flexibility of linker regions to optimize Zn binding and substrate hydrolysis. The resultant peptides were synthesized and assessed for Zn and substrate binding affinity by fluorescence and ITC followed by evaluation of catalytic efficiency with UV-based enzyme kinetic assays. We were successful in mimicking carbonic anhydrase activity in a peptide of twenty two residues, using p-nitrophenyl acetate as a CO2 surrogate. Although our design had modest activity, being a simple structure is an advantage for further improvement in efficiency. Our approach opens a way forward to evolving an efficient biocatalyst for any industrial reaction of interest.Kinshuk Raj SrivastavaSusheel DuraniPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 5, p e96234 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kinshuk Raj Srivastava Susheel Durani Design of a zinc-finger hydrolase with a synthetic αββ protein.
description	Recent advances in protein design have opened avenues for the creation of artificial enzymes needed for biotechnological and pharmaceutical applications. However, designing efficient enzymes remains an unrealized ambition, as the design must incorporate a catalytic apparatus specific for the desired reaction. Here we present a de novo design approach to evolve a minimal carbonic anhydrase mimic. We followed a step-by-step design of first folding the main chain followed by sequence variation for substrate binding and catalysis. To optimize the fold, we designed an αββ protein based on a Zn-finger. We then inverse-designed the sequences to provide stability to the fold along with flexibility of linker regions to optimize Zn binding and substrate hydrolysis. The resultant peptides were synthesized and assessed for Zn and substrate binding affinity by fluorescence and ITC followed by evaluation of catalytic efficiency with UV-based enzyme kinetic assays. We were successful in mimicking carbonic anhydrase activity in a peptide of twenty two residues, using p-nitrophenyl acetate as a CO2 surrogate. Although our design had modest activity, being a simple structure is an advantage for further improvement in efficiency. Our approach opens a way forward to evolving an efficient biocatalyst for any industrial reaction of interest.
format	article
author	Kinshuk Raj Srivastava Susheel Durani
author_facet	Kinshuk Raj Srivastava Susheel Durani
author_sort	Kinshuk Raj Srivastava
title	Design of a zinc-finger hydrolase with a synthetic αββ protein.
title_short	Design of a zinc-finger hydrolase with a synthetic αββ protein.
title_full	Design of a zinc-finger hydrolase with a synthetic αββ protein.
title_fullStr	Design of a zinc-finger hydrolase with a synthetic αββ protein.
title_full_unstemmed	Design of a zinc-finger hydrolase with a synthetic αββ protein.
title_sort	design of a zinc-finger hydrolase with a synthetic αββ protein.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/b707d3a1720846c7b0f236163fe62276
work_keys_str_mv	AT kinshukrajsrivastava designofazincfingerhydrolasewithasyntheticabbprotein AT susheeldurani designofazincfingerhydrolasewithasyntheticabbprotein
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Design of a zinc-finger hydrolase with a synthetic αββ protein.

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