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Implementation of Enzymatic Activity in a Naïve, de novo Designed Protein Scaffold by Rational Design and Laboratory Evolution

Total Cost €


EC-Contrib. €






 TIMEnzyme project word cloud

Explore the words cloud of the TIMEnzyme project. It provides you a very rough idea of what is the project "TIMEnzyme" about.

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Project "TIMEnzyme" data sheet

The following table provides information about the project.


Organization address
address: Raemistrasse 101
postcode: 8092

contact info
title: n.a.
name: n.a.
surname: n.a.
function: n.a.
email: n.a.
telephone: n.a.
fax: n.a.

 Coordinator Country Switzerland [CH]
 Total cost 175˙419 €
 EC max contribution 175˙419 € (100%)
 Programme 1. H2020-EU.1.3.2. (Nurturing excellence by means of cross-border and cross-sector mobility)
 Code Call H2020-MSCA-IF-2015
 Funding Scheme MSCA-IF-EF-ST
 Starting year 2016
 Duration (year-month-day) from 2016-03-01   to  2018-02-28


Take a look of project's partnership.

# participants  country  role  EC contrib. [€] 


 Project objective

The proposed research project aims at implementing enzymatic activity in a de novo designed, unbiased protein scaffold. First, simplified active site arrangements deduced from two previously evolved model enzymes (Kemp eliminase and retro-aldolase) will be implanted in the scaffold. This will allow evaluating the extent to which a fully computationally designed and naïve protein can be functionalized and evolved. A recently established fluorescence-based microfluidics setup will be utilized to screen large DNA libraries of several million clones per round of laboratory evolution. The artificial protein scaffold, kindly provided by Prof. David Baker (University of Washington), has been designed to adopt a minimalist TIM barrel fold, which is the most abundant and diversely evolved protein fold found for natural enzymes. Here, the substrate binding pocket is usually formed by an extended loop region on one side of the scaffold, which is not yet present in the naïve, designed variant. Thus, in the second step, I will generate a randomized library of loop fragments, insert into the scaffold and screen for improved activity. This approach can be extended from the retro-aldolase model reaction towards synthetic aldolases that catalyze the stereospecific formation of a new carbon-carbon bond. Ultimately, the aim is to evaluate whether loop libraries are an appropriate tool to broaden the substrate scope of these enzymes. Furthermore, the proposal contains a second, independent approach to equip the artificial scaffold with novel enzymatic functionality. Here, I plan to design a covalent dimer of two artificial TIM barrels carrying a cofactor-dependent active site in the dimeric interface. This work will not only generate fundamental insight into the evolution of catalytic activity, it also has great potential to contribute to the development of general strategies for creating enzymes with novel functionality, and thus, prospective applications in industry or medicine.


year authors and title journal last update
List of publications.
2016 Richard Obexer, Moritz Pott, Cathleen Zeymer, Andrew D. Griffiths, Donald Hilvert
Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting
published pages: 355-366, ISSN: 1741-0126, DOI: 10.1093/protein/gzw032
Protein Engineering Design and Selection 29/9 2019-06-13
2017 Cathleen Zeymer, Reinhard Zschoche, Donald Hilvert
Optimization of Enzyme Mechanism along the Evolutionary Trajectory of a Computationally Designed (Retro-)Aldolase
published pages: 12541-12549, ISSN: 0002-7863, DOI: 10.1021/jacs.7b05796
Journal of the American Chemical Society 139/36 2019-06-13

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