TY - JOUR

T1 - Computational and Experimental Assessment of Backbone Templates for Computational Redesign of the Thioredoxin Fold

AU - Marin, Frederikke Isa

AU - Johansson, Kristoffer Enøe

AU - O'Shea, Charlotte

AU - Lindorff-Larsen, Kresten

AU - Winther, Jakob Rahr

PY - 2021

Y1 - 2021

N2 - Computational protein design has taken big strides in recent years; however, the tools available are still not at a state where a sequence can be designed to fold into a given protein structure at will and with high probability. We have applied here a recent release of Rosetta Design to redesign a set of structurally very similar proteins belonging to the thioredoxin fold. We used a genetic screening tool to estimate solubility/folding of the designed proteins in E. coli and to select the best hits from this for further biochemical characterization. We have previously used this set of template proteins for redesign and found that success was highly dependent on template structure, a trait which was also found in this study. Nevertheless, state-of-the-art design software is now able to predict the best template, most likely due to the introduction of an energy term that reports on stress in covalent bond lengths and angles. The template that led to the greatest fraction of successful designs was the same (a thioredoxin from spinach) as that identified in our previous study. Our previously described redesign of thioredoxin, which also used the spinach protein as a template, however also performed well as a template. In the present study, both of these templates yielded proteins with compact folded structures and enforced the conclusion that any design project must carefully consider different design templates. Fortunately, selecting designs based on energies appears to correctly identify such templates.

AB - Computational protein design has taken big strides in recent years; however, the tools available are still not at a state where a sequence can be designed to fold into a given protein structure at will and with high probability. We have applied here a recent release of Rosetta Design to redesign a set of structurally very similar proteins belonging to the thioredoxin fold. We used a genetic screening tool to estimate solubility/folding of the designed proteins in E. coli and to select the best hits from this for further biochemical characterization. We have previously used this set of template proteins for redesign and found that success was highly dependent on template structure, a trait which was also found in this study. Nevertheless, state-of-the-art design software is now able to predict the best template, most likely due to the introduction of an energy term that reports on stress in covalent bond lengths and angles. The template that led to the greatest fraction of successful designs was the same (a thioredoxin from spinach) as that identified in our previous study. Our previously described redesign of thioredoxin, which also used the spinach protein as a template, however also performed well as a template. In the present study, both of these templates yielded proteins with compact folded structures and enforced the conclusion that any design project must carefully consider different design templates. Fortunately, selecting designs based on energies appears to correctly identify such templates.

U2 - 10.1021/acs.jpcb.1c05528

DO - 10.1021/acs.jpcb.1c05528

M3 - Journal article

C2 - 34592819

VL - 125

SP - 11141

EP - 11149

JO - Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical

JF - Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical

SN - 1520-6106

IS - 40

ER -