title: Influence of Electrostatics on Protein-Protein Interactions of Photosynthetic Proteins
Creators: name: Förster, Johannes M.
Publisher: name: University of Bayreuth
Year: 2018
Subjects: subject: Transient complex
subject: Encounter complex
subject: Electrostatics
subject: Paramagnetic NMR
subject: Protein-protein interactions
subject: Plastocyanin
subject: Cytochrome f
subject: 004
subject: 500
subject: 570

description: Protein-protein interactions play a central role in many cellular processes, such as signal transduction, gene regulation and molecular bioenergetics. A wide variety of complexes is needed to fulfill the entity of interaction types ranging from strong binding complexes to weakly interacting ones like transient complexes. Transient protein complexes are particularly found in photosynthesis, facilitating electron transfer reactions. A rapid complex formation coupled to a fast reaction and protein dissociation is crucial to ensure the electron transfer not being limited in turnover. The association process of such dynamic complexes can be described by a two-step model. Initially, proteins are separated, attracting each other only by means of long-range, electrostatic interactions. Proteins approach each other to form the so-called encounter complex. In this state the interaction partners can assume different orientations within the complex prior to the formation of the active complex. The proteins then sample each others surface with the objective to form the well-defined complex, where the reaction can take place. This second step of association is dominated by short-range interactions. In order to study the influence of charge-charge interactions on complex formation, the analysis of the encounter complex is fundamental. A combination of chemical shift perturbation (CSP), paramagnetic NMR experiments, ensemble docking as well as Monte Carlo (MC) docking simulations is used to investigate and to visualize the complex orientations at the encounter state. In paramagnetic NMR experiments a protein is labeled with a spin label, that causes paramagnetic relaxation enhancements (PREs) on nuclei in its direct vicinity. This method is highly dependent on the distance and enables the visualization of lowly populated states, making it most suitable for studying the encounter complexes. Within this work the program MontyDock—a useful tool to simulate the first state of the association process—is presented. MontyDock is a rigid protein docking program, which evaluates the protein interactions solely by considering electrostatic interactions. The program is demonstrated, using the well-established complex of cytochrome c and cytochrome c peroxidase. The analyzed complexes differ widely in the strength of electrostatic interactions. First, several plastocyanins (Pcs)—distinct in their surface charge distribution—were analyzed in complex with short, strongly charged, synthetic peptides marked with a paramagnetic spin label. The association of the peptides to the Pcs show a high dependency to electrostatic interactions. The NMR measurements of Pcs from Populus nigra and Dryopteris crassirhizoma reveal a good agreement with the docking results of MontyDock. In the visualized encounter complex the main ensembles are present at the highly charged regions of Pc, located at the eastern patch. Subsequently, the influence of a decrease in electrostatic stabilization is investigated with the complex of cytochrome f (Cytf) and Pc from the cyanobacterium Nostoc sp. PCC 7119. Here the MC docking results do not fit the experimental measurements very well. Only a subordinate accordance suggests a partial reorientation of Pc to Cytf based on electrostatic interactions. The dynamics of the complex and the binding orientation of Pc suggest hydrophobic interactions being the major stabilizing factor in the formation of this complex. Electrostatic interactions, however, still contribute a small part to the complex stabilization. The common description of the encounter complex by three distinct states is blurred by these findings and can be better represented by a smooth transition between the different states. This theory is emphasized by the analysis of the cross complex of Phormidium laminosum Pc and Nostoc Cytf. For Phormidium Pc, electrostatic interactions are even of less importance than for the Nostoc Pc. The cross complex shows a decreased affinity and a more dynamic encounter complex compared to the Nostoc wild type. Nevertheless, the still-observable dependency on ionic strength is an evidence that the complex is influenced to some extent by electrostatic attraction. As a result, a model is proposed where the charges orient Pc to Cytf in order to bring the hydrophobic regions in the vicinity of each other. Additionally, the cytochrome c6-Cytf complex from Nostoc was compared analytically to the Pc-Cytf complex. The ensemble fitting indicates that the ensemble distribution cannot be described by a single, well-defined complex but by a pure encounter state. The different types of complexes show that an encounter complex formation can only partially be described by electrostatic interactions alone. An interaction model is suggested where the interplay of hydrophobic interactions and electrostatic interactions regulates the dynamics and the specificity, resulting in indistinct states. This leads to an existence of various possibilities to conduct an electron transfer between transiently interacting proteins.