A screening method for binding synthetic metallo-complexes to haem proteins

Analytical Biochemistry, vol. 653, pp. 114788

Abstract

The introduction of a second coordination sphere, in the form of a protein scaffold, to synthetic catalysts can be beneficial for their reactivity and substrate selectivity. Here we present semi-native polyacrylamide gel electrophoresis (semi-native PAGE) as a rapid screening method for studying metal complex-protein interactions. Such a screening is generally performed using electron spray ionization mass spectrometry (ESI-MS) and/or UV-Vis spectroscopy. Semi-native PAGE analysis has the advantage that it does not rely on spectral changes of the metal complex upon protein interaction and can be applied for high-throughput screening and optimization of complex binding. In semi-native PAGE non-denatured protein samples are loaded on a gel containing sodium dodecyl sulphate (SDS), leading to separation based on differences in structural stability. Semi-native PAGE gel runs of catalyst-protein mixtures were compared to gel runs obtained with native and denaturing PAGE. ESI-MS was additionally realised to confirm protein-complex binding. The general applicability of semi-native PAGE was investigated by screening the binding of various cobalt- and ruthenium-based compounds to three types of haem proteins.

Authors 7

1. Laura V. Opdam Aachen SSNMR/BPOC

   Leiden University

   Affiliation as printed

   SSNMR/BPOC, Einsteinweg 55, 2333 CC, Leiden, the Netherlands

2. Ehider A. Polanco Aachen MCBIM Departments

   Leiden University

   Affiliation as printed

   MCBIM Departments, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, the Netherlands

3. Boyd de Regt Aachen SSNMR/BPOC

   Leiden University

   Affiliation as printed

   SSNMR/BPOC, Einsteinweg 55, 2333 CC, Leiden, the Netherlands

4. Nicole Lambertina Aachen SSNMR/BPOC

   Leiden University

   Affiliation as printed

   SSNMR/BPOC, Einsteinweg 55, 2333 CC, Leiden, the Netherlands

5. Cas Bakker Aachen SSNMR/BPOC

   Leiden University

   Affiliation as printed

   SSNMR/BPOC, Einsteinweg 55, 2333 CC, Leiden, the Netherlands

6. Sylvestre A. Bonnet Aachen MCBIM Departments

   Leiden University

   Affiliation as printed

   MCBIM Departments, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, the Netherlands

7. Anjali Pandit corresponding Aachen SSNMR/BPOC

   Leiden University

   Affiliation as printed

   SSNMR/BPOC, Einsteinweg 55, 2333 CC, Leiden, the Netherlands. Electronic address: a.pandit@lic.leidenuniv.nl

   SSNMR/BPOC, Einsteinweg 55, 2333 CC, Leiden, the Netherlands