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
Son, M. van; Schilder, J.T.; Di Savino, A.; Blok, A.J.; Ubbink, M.; Huber, M.I. 2020
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
