Optimal charge distribution is considered to be important for efficient formation of protein complexes. Electrostatic interactions guide encounter complex formation that precedes the formation of... Show moreOptimal charge distribution is considered to be important for efficient formation of protein complexes. Electrostatic interactions guide encounter complex formation that precedes the formation of an active protein complex. However, disturbing the optimized distribution by introduction of extra charged patches on cytochrome c peroxidase does not lead to a reduction in productive encounters with its partner cytochrome c. To test whether a complex with a high population of encounter complex is more easily affected by suboptimal charge distribution, the interactions of cytochrome c mutant R13A with wild-type cytochrome c peroxidase and a variant with an additional negative patch were studied. The complex of the peroxidase and cytochrome c R13A was reported to have an encounter state population of 80%, compared to 30% for the wild-type cytochrome c. NMR analysis confirms the dynamic nature of the interaction and demonstrates that the mutant cytochrome c samples the introduced negative patch. Kinetic experiments show that productive complex formation is fivefold to sevenfold slower at moderate and high ionic strength values for cytochrome c R13A but the association rate is not affected by the additional negative patch on cytochrome c peroxidase, showing that the total charge on the protein surface can compensate for less optimal charge distribution. At low ionic strength (44 mm), the association with the mutant cytochrome c reaches the same high rates as found for wild-type cytochrome c, approaching the diffusion limit. Show less
Protein–protein complexes are formed via transient states called encounter complexes that greatly influence the formation of the stereospecific complex. Electrostatic charges on the protein... Show moreProtein–protein complexes are formed via transient states called encounter complexes that greatly influence the formation of the stereospecific complex. Electrostatic charges on the protein surfaces play a major role in encounter complexes of electron transfer proteins. The complex formed by cytochrome c (Cc) and cytochrome c peroxidase (CcP) has been studied intensively because it is an excellent model to explore the properties of transient protein-protein interactions. PRE experiments previously described the encounter complex formed by the two proteins in detail. In this thesis we tested to what degree the electrostatic patch on CcP is optimized to enhance the rate of the formation of the stereospecific complex. Using paramagnetic NMR in combination with Monte Carlo simulations and stopped flow spectrophotometry, we investigate several CcP mutants with reengineered charged patches to create new encounter complexes and measure their effects on electron transfer from Cc to CcP. The results indicate that the interactions with Cc are affected more by the total charge of CcP surface than the specific distribution of the charges, bringing into question the concept of electrostatic patches being highly optimized by evolution. Show less
Di Savino, A.; Foerster, J.M.; Ullmann, G.M.; Ubbink, M. 2021
Paramagnetic NMR methods are excellently suited for the study of protein–protein complexes in solution. Intermolecular pseudocontact shifts (PCSs), residual dipolar couplings (RDCs) and... Show moreParamagnetic NMR methods are excellently suited for the study of protein–protein complexes in solution. Intermolecular pseudocontact shifts (PCSs), residual dipolar couplings (RDCs) and paramagnetic relaxations enhancements (PREs) can be used, ideally in combination, for docking proteins and determining their orientation in the complex. PCSs can be used for breaking the structure symmetry in dimer complexes. PCSs also can be applied to detect structural differences in proteins and protein complexes in solution in comparison to crystal structures. RDCs are sensitive to the degree of alignment of both partners in a protein complex and are thus very useful to detect dynamics within complexes. PREs can detect states in which nuclei approach a paramagnetic centre closely, even if it exists only for a small fraction of the time. Thus, PREs are used to detect minor states and characterize ensembles. PRE studies have been the foundation for characterizing encounter states and the process of protein complex formation. In weak complexes, such as are found in electron transfer chains, proteins can be in an encounter state for a large fraction of the complex lifetime. Paramagnetic NMR tools thus have found many applications for studying protein complexes, and more may be on the horizon. Show less
