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Design of a Zinc-Finger Hydrolase with a Synthetic alpha beta beta Protein

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Title Design of a Zinc-Finger Hydrolase with a Synthetic alpha beta beta Protein
 
Creator SRIVASTAVA, KR
DURANI, S
 
Subject COMPUTATIONAL ENZYME DESIGN
DE-NOVO DESIGN
CARBONIC-ANHYDRASE
BINDING PROTEIN
COMPLEXES
MODEL
HYDROLYSIS
STABILITY
SELECTION
SEQUENCE
 
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 alpha beta beta 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.
 
Publisher PUBLIC LIBRARY SCIENCE
 
Date 2014-12-29T06:28:50Z
2014-12-29T06:28:50Z
2014
 
Type Article
 
Identifier PLOS ONE, 9(5)
1932-6203
http://dx.doi.org/10.1371/journal.pone.0096234
http://dspace.library.iitb.ac.in/jspui/handle/100/17311
 
Language English