De novo design of a multifunctional thermostable protein catalyst with a novel fold

Activity: Talk or presentationPoster presentationScience to science

Description

The design of a stable de novo protein scaffold which could be functionalized to catalyze a wide range of different reactions is a promising but still a challenging task. This study aims to use the Rosetta software to parametrically design a novel thermostable helix bundle protein with the ability to accommodate an active site. Our initial design (6H5L) forms a helical barrel structure with a hydrophobic channel and is comprised of six antiparallel straight helices connected by loops (Figure 1). As a first reaction model we designed several variants to catalyze the retro-aldol reaction, which has extensive application in biocatalysis and could be extended to perform different non-native carboligation reactions. All variants are readily produced in E. coli and characterized using biochemical and biophysical methods including circular dichroism spectroscopy (CD), UV-Vis and fluorescence spectroscopy, SAXS analysis and X-ray crystallography studies. The CD spectra of the designs confirm their alpha-helical fold and, moreover, a high thermal stability upon heating to 95°C with only minimal loss of signal at high temperatures and complete refolding after cooling back to 20°C. Furthermore, we performed a fluorescence-based binding assay with 1,6-Diphenyl-1,3,5-hexatriene (DPH) to probe the barrel shape of our designs. Alphafold2 showed a high predicted IDDT score of over 90 for the designed sequence and most variants. To test retro-aldolase activity, we performed an inhibition reaction assay with a naphthalene diketone derivative which reacts with a nucleophilic lysine residue and forms an imine intermediate that can be photometrically detected. The retro-aldol reaction of our designs with 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone (methodol) as a substrate was monitored by measuring the increase of fluorescence upon product formation. 6H5L and all its versions showed noticeable retro-aldolase activity. To increase the activity, we used rational and computational design approaches. The best variant showed 10-fold activity increase compared to the initial design (Figure 2). SAXS measurements of all variants gave an overall good fit between the measured and the calculated scattering profiles with low chi² values. We were able to determine the crystal structures of the apo design (2.2 Å) and a variant with the covalently bound ligand (naphthalene diketone derivative, 3.0 Å) To demonstrate the multifunctional use of our designs, we assayed them for other reactions and found some variants which showed a notable Michael addition activity and good PLP binding affinity. We are currently working on improving all catalytic activities.
Period18 Apr 202319 Apr 2023
Event titleNext Generation Biocatalysis 2023
Event typeConference
LocationGraz, AustriaShow on map
Degree of RecognitionInternational

Keywords

  • Protein Design

Fields of Expertise

  • Human- & Biotechnology

Treatment code (Nähere Zuordnung)

  • Application
  • Experimental

Cooperations

  • NAWI Graz