Protein-protein interaction dynamics in photosynthetic membrane: biochemical and in silico comparative analysis

SUMMARY In higher plants, algae and cyanobacteria the photosynthesis depends on the activity of two protein supercomplexes, the photosystem I (PSI core and its antenna LHCI) and the photosystem II (PSII core and its antenna LHCII). Several photosystem subunits bind chromophores to absorb sun light and transfer excitation energy to the reaction centers, where charge separation and the main photochemical reactions occur. Photosynthetic complexes are localized in thylakoids membranes, folded so that two distinct domains are formed: stacked grana domains, connected to each other by the second type of membrane domain, the stroma lamellae. PSII and its antenna are mainly located in stacked grana, while the faster PSI in stroma lamellae. These structures can vary in dimensions and organization, depending on factors such as light intensity, as there is a continuous need for balancing the absorbed energy with the formation of dangerous free radicals. The non-photochemical quenching of energy excess in the form of heat (NPQ) is the fastest response to the photooxidative stress. PSII antenna proteins, being able to assume a light-harvesting or a dissipative configuration are involved in the NPQ mechanism. This thesis presents the results of the study of the role of PSII organization in the regulation of photoprotection and NPQ; the analysis is a biochemical and in silico comparative analysis with the intent of clarifying the relation between thylakoids membrane organization and the light harvesting regulation. Part I is focused on the photosynthetic membrane organization and on its determining factors, delving into the meaning of the reversible dissociation of a complex comprised in PSII and into its correlation with the NPQ phenomenon. In Chapter 2 the organization of thylakoids membrane is discussed along with the factors that determine the separation of PSI and PSII in its compartments and the meaning of the dynamic distribution of the membrane among them. In Chapter 3 the factors that contribute to the morphology of thylakoid are discussed. The specific cases of mutants missing PSII antennae (chlorina-f2) or the PSII core (viridis-115) are considered to clarify their contribution in the morphology. Chapter 4 focuses on the importance of the interactions among PSII components in determining the grana membrane organization. The mutant viridis zb63, lacking PSI, a drastic reduction of PSII antenna size occurs. In electron microscopy investigation of the organization of the minimal PSII configuration under extreme stress, appearing as an ordered array, singular composing proteins are identified by single particle analysis. Subsequently, a study focused on specific PSII minor antennae knockouts (CP24-, and CP26- CP24/26-) is presented. CP26 appears to be less important for the organization and more involved in photoprotection. The absence of CP24, conversely, leads to the formation of ordered arrays on grana membranes similar to the figure observed in viridis zb63. Finally, in mutant CP24/26- the formation of short chains of PSII is observed along with microdomains enriched either in PSII or LHCII. The photosynthetic parameters are influenced in various ways by the deletion of minor antennas, starting from the rate of electronic transport and NPQ (especially limited in mutant CP24-), to the reversible migration of the PSII antenna to PSI (faster in mutants in which CP24 and CP24 - / 26 -). In Chapter 5 the dependence between the dissociation of the membrane complex called B4C (formed by the minor antennas CP24, CP29 and a LHCII trimer), and NPQ was studied by correlating the two phenomena: the PsbS protein is essential for the NPQ and controls the association/dissociation of the B4C. Conversely when the B4C is present its dissociation is necessary for the onset of the NPQ. The mutant lacking CP24 and CP29 have a reduced level of NPQ and do not present the B4C. Direct observation by electron microscopy allowed to directly correlate the dissociation and association of B4C to the reduction of the space between PSII core upon high light exposure, and to its increase upon successive recovery in darkness. The mutant npq4, which lacks PsbS and therefore NPQ, does not undergo reorganization upon light exposure. We conclude that PsbS interacts with components of B4C to induce the reorganization of the photosynthetic membrane to adapt to changes in light intensity on both short and long term. In Part II, the phenomenon of reversible dissociation of B4C is the subject of an in silico study to determine whether a change in the strength of interactions between proteins can be an important factor for the onset of the reorganization in grana membranes. In Chapters 6 and 7 two key issues for a simulation of protein-protein interactions in biological membranes are discussed. Movement in eukaryotic cells is based primarily on the presence of the cytoskeleton; its fibers interacts with molecular motors, characterized by directionality and by the consumption of ATP. The prokaryotic cell contains many molecules homologue to those which allow movement in eukaryotic cells and several biological processes occur in a similar, but passive, way exploiting concentration and polarity. Chapter 7, on the basis of the principles outlined in the previous chapter, outlines the possible strategies for creating a computational model of protein-protein interactions at a mesoscopic level, abstracting from atomic details. In Chapter 8 I presents my work in the simulation of the dynamics of the interactions between proteins of PSII in the reversible dissociation of B4C. The software Meredys, suitably adapted, was used to simulate at a mesoscopic level the association and dissociation of protein complex and supercomplexes on grana membranes, with a particular reference to the phenomenon of the dynamics of B4C complex, activated by energy dissipation mechanisms. Each element of the simulation represents a protein or a protein complex and the attention is mainly focused on the bond formation and dissociation with rates consistent with experimental evidences. The process of formation of the more abundant configuration (C2S2M2) of the supercomplexes, where two cores, 4 major antennas trimers and 6 minor antennas are regularly combined, is simulated starting from PSII cores and light harvesting complexes monomers. The simulation volume is divided between an area corresponding to the grana membrane in the center, in communication with another that represents the edge to the stroma lamellae, suitably populated. The dynamic is discussed, in conditions corresponding to the dark adapted sample and to the high light exposed one. The simulation reproduce in orientations and distances distributions between nearest neighbors PSII cores, the experimental results assessed in B4C investigation and in others, based on electron microscopy. The change of the parameters related to the interactions putatively affected by the action of PsbS, has triggered a change in distances similar to that measured in the passage of the dark adapted sample under high light. Moreover, the diffusion coefficient of LHCII-M trimers associated in B4C is lower in conditions leading to its dissociation, in agreement with the hypothetical formation of microdomains in which the separated antenna is segregated as opposed to other enriched in PSII with reduced antenna. These results support the hypothesis that protein-protein interactions are a key factor for the organization of the whole grana thylakoids and B4C is one of the most important sites for interactions that regulate stress response by NPQ.