Project Details
Projekt
Long-range electrostatic interactions contribute to the target specificity and reactivity of thioredoxin family proteins
Subject Area
Biochemistry
Cell Biology
Cell Biology
Term
from 2014 to 2021
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 251869040
The spatio-temporal reduction and oxidation of protein thiols is an essential mechanism in signal transduction in all kingdoms of life. Thioredoxin (Trx) family proteins efficiently catalyse thiol-disulphide exchange reactions and the proteins are widely recognized for their importance in the operation of thiol switches. Trx family proteins have a broad and at the same time very distinct substrate specificity - a prerequisite for redox switching. Despite of multiple efforts, the basis for this specificity is still unclear. Our previous work suggests that thermodynamic parameters, such as the redox potential, do not determine specificity nor reactivity of the redoxins. Instead, the catalytic efficiency correlated strongly to a specific electrostatic field pattern of the redoxins [Chem. Sci. 6:7049-7058, 2015]. Efficient reaction rates rely on a sufficiently low activation energy barrier and a high frequency of effective collisions. Hence, we propose the following model: (1) The recognition of the proteins from a distance and their proper (pre-)orientation, dominated by long-range electrostatic interactions. (2) Attraction of the proteins towards each other. (3) Direct short-range molecular interactions, leading to the formation of the encounter complex. (4) The thiol-disulphide exchange reaction, subjected to a number of thermodynamic restrictions. (5) The dissociation of the complex. The primary aim of this project is to determine the importance of specific electrostatic interactions in the early phases (1-2) of the reaction. Most of all, we aim to establish the contributions of these forces to the specificity and overall rate constants of the reaction. The major focus of our research programme is the biochemical and biophysical characterisation of protein-protein interactions guided by computational analyses and predictions. As models, we propose to analyse the catalytic disulphide in E coli 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase by Grxs, the redox switch in human collapsin response mediator protein 2 2 (CRMP2) and, in cooperation with other members of the SPP 1710 consortium, other thiol switches of interest. We suggest to compute the electrostatic properties of the proteins using experimentally determined or modelled structures. Guided by these, we will analyse the interactions by enzyme kinetics, fluorescence quenching spectroscopy, surface plasmon resonance, and atomic force spectroscopy. We will determine the contribution of the association rates to the overall rate constants. Moreover, we aim to engineer specific interactions, for instance to turn E. coli Grx3 into an efficient reductant for PAPS reductase and to optimise the electron transfer between Grxs and the roGFP redox sensor. We have no doubt that the mechanisms that control the substrate specificity of Trx family proteins are key to the understanding of thiol switching.
DFG Programme
Priority Programmes
