Final Report Summary - SFAP2 (Structural and functional analysis of photosystem 2 from Nicotiana tabacum)
Photosynthesis is a biochemical process whereby light energy is converted into chemical energy associated to the chemical bonds of organic molecules. In order to work photosynthesis, it requires specific proteins called photosystems (PS) that have the function to harvest the light; PS are of two kinds: PSI and PSII. PSII is the subject of this project.
PSII not only captures the sunlight but also provide the splitting of the water molecule. By the coupling of those reactions PSII provides energy and electrons that feeds photosynthesis producing sugars and discharge the molecular oxygen to the atmosphere. The structural features of PSII from plants are still poorly understood. This project consisted in developing a procedure for studying PSII by X-ray diffraction a technique through which it is possible to define the structural feature of this multi-protein-complex at atomic level.
The work started with growing the candidate organism for those experiments. In our case we have choose the Nicotiana tabacum (tobacco) because it grows in relative short time and it is well characterised genetically and physiologically. The plants are grown under controlled conditions of temperature, light and humidity and finally the leafs are removed when the plants reach the age of 8 - 12 weeks. The leaf represents the starting material from which, through several steps of purification, we get pure samples of PSII. The first step consists in the harvest of the leafs and the extraction of the functional part into which is localised PSII: the thylakoid membranes. The thylakoid membranes are insoluble because of their strong lipid component so that the thylakoid samples extracted are in fact a suspension. The following step is the solubilis ation of the thylakoid membranes using specific detergents. This first group of steps is particularly important because the integrity of the thylakoids must be preserved as much as possible in order to avoid the destabilisation of PSII. For this reason this first part of the work has been under improvement during all the project with the aim to improve the sample quality. The subsequent steps consists in the purification of PSII from the solubilis ed thylakoids through specific chromatography techniques. In particular we have used three different steps of chromatography: affinity chromatography, ionic exchange chromatography and finally size exclusion chromatography. Those steps were required in order to increase as much as possible the purity of the sample in good compromise with preserving the functionality of PSII. During this project, several procedures of purification were developed and from those procedure was selected the one that had the best compromise between purity, quantity, stability and integrity of the final PSII sample obtained. The quality of thylakoids and PSII samples was analysed in every step by several techniques. The polyacrylamide gel electrophoresis (PAGE) under native conditions was used for studying the oligomeric composition of the complexes in the thylakoid and in the PSII samples. Moreover, the PAGE under denaturing condition was used for checking the polypeptide composition in the PSII sample. The integrity of thylakoids and PSII samples was constantly monitored by measuring the rates of oxygen evolution as an indicator of the PSII activity and stability.
In the following step, the purified samples are used for crystallisation trials. The crystallisation consists in a regular packing of the PSII molecule that can be studied by means of interaction with the X-rays. The diffraction that originates from the interaction between the crystal and the X-rays has specific characteristics that are directly related with the structure of PSII. This last part of the project was the most unpredictable and it consisted in getting crystals of good quality. The quality of the crystals is depending on the crystallisation condition used and on the quality of the PSII samples. During this project, we have tested more than 2000 crystallisation conditions and out of it we have found 52 conditions that induce PSII crystallisation. Of those 52 conditions, none of them gave adequate diffraction to achieve structural determination. However crystallisation experiments are continuing by improving the PSII quality and the crystallisation conditions.
By this project, several technical and physiological results have been achieved. The technical findings are related with the issues of samples and crystals quality both critical aspects for a successful crystallography. Regarding the sample quality, during this project we have developed specific procedures for processing the thylakoids samples and the PSII samples making them suitable for native electrophoresis. This can be considered an important objective since it allows for a constant check of the quality in every step reaching in this way a high standardisation of the samples that finally will be used for crystallisation. Moreover, another technical finding is the surprising amount of crystallisation conditions. We have found about 50 conditions that are able to induce PSII crystallisation but none of those crystals conditions, even after several steps of improvement, gave satisfactory diffraction. This fact and other evidences would suggest that the peculiar heterogeneity associated to PSII from higher plants is a mirror of the organisation and the topology of the thylakoid membranes.
During this project, several procedures of PSII purification were developed. The quality of the samples obtained by those procedures is given by the homogeneity of the samples meaning with this the presence of samples constituted only of monomer or dimers. Our best procedure has samples constituted mainly of monomers. Moreover those monomeric samples retain the subunit S of PSII (PsbS) an important polypeptide involved in the photo-protection of PSII. PsbS so far was never directly proven to be attached to PSII monomer in this work we observe a direct connection between PSII monomers and PsbS. We consider this an important finding with physiological implications.
In conclusion, we think that by this project was possible to give an important contribution toward the finding of good diffracting crystals suitable for structural analysis. For this reason this work will be continued with new funds in order to get this final aim. When the final aim will be reached it will have important socio-economic effects. In fact, the availability of a detailed structural description of PSII from higher plants would make possible to select cultivars or genetically modify varieties able to use the water in a more efficient way or create modifications in the polypeptide and cofactor properties in order to increase the conversion efficiency of the light energy. Furthermore, in a similar way it could help in developing more resistant and efficient cultivars that could grow in pre-desert areas. From the technological point of view these results would be of immense interest to help in developing artificial photosynthetic systems in order to product 'green energy'.
PSII not only captures the sunlight but also provide the splitting of the water molecule. By the coupling of those reactions PSII provides energy and electrons that feeds photosynthesis producing sugars and discharge the molecular oxygen to the atmosphere. The structural features of PSII from plants are still poorly understood. This project consisted in developing a procedure for studying PSII by X-ray diffraction a technique through which it is possible to define the structural feature of this multi-protein-complex at atomic level.
The work started with growing the candidate organism for those experiments. In our case we have choose the Nicotiana tabacum (tobacco) because it grows in relative short time and it is well characterised genetically and physiologically. The plants are grown under controlled conditions of temperature, light and humidity and finally the leafs are removed when the plants reach the age of 8 - 12 weeks. The leaf represents the starting material from which, through several steps of purification, we get pure samples of PSII. The first step consists in the harvest of the leafs and the extraction of the functional part into which is localised PSII: the thylakoid membranes. The thylakoid membranes are insoluble because of their strong lipid component so that the thylakoid samples extracted are in fact a suspension. The following step is the solubilis ation of the thylakoid membranes using specific detergents. This first group of steps is particularly important because the integrity of the thylakoids must be preserved as much as possible in order to avoid the destabilisation of PSII. For this reason this first part of the work has been under improvement during all the project with the aim to improve the sample quality. The subsequent steps consists in the purification of PSII from the solubilis ed thylakoids through specific chromatography techniques. In particular we have used three different steps of chromatography: affinity chromatography, ionic exchange chromatography and finally size exclusion chromatography. Those steps were required in order to increase as much as possible the purity of the sample in good compromise with preserving the functionality of PSII. During this project, several procedures of purification were developed and from those procedure was selected the one that had the best compromise between purity, quantity, stability and integrity of the final PSII sample obtained. The quality of thylakoids and PSII samples was analysed in every step by several techniques. The polyacrylamide gel electrophoresis (PAGE) under native conditions was used for studying the oligomeric composition of the complexes in the thylakoid and in the PSII samples. Moreover, the PAGE under denaturing condition was used for checking the polypeptide composition in the PSII sample. The integrity of thylakoids and PSII samples was constantly monitored by measuring the rates of oxygen evolution as an indicator of the PSII activity and stability.
In the following step, the purified samples are used for crystallisation trials. The crystallisation consists in a regular packing of the PSII molecule that can be studied by means of interaction with the X-rays. The diffraction that originates from the interaction between the crystal and the X-rays has specific characteristics that are directly related with the structure of PSII. This last part of the project was the most unpredictable and it consisted in getting crystals of good quality. The quality of the crystals is depending on the crystallisation condition used and on the quality of the PSII samples. During this project, we have tested more than 2000 crystallisation conditions and out of it we have found 52 conditions that induce PSII crystallisation. Of those 52 conditions, none of them gave adequate diffraction to achieve structural determination. However crystallisation experiments are continuing by improving the PSII quality and the crystallisation conditions.
By this project, several technical and physiological results have been achieved. The technical findings are related with the issues of samples and crystals quality both critical aspects for a successful crystallography. Regarding the sample quality, during this project we have developed specific procedures for processing the thylakoids samples and the PSII samples making them suitable for native electrophoresis. This can be considered an important objective since it allows for a constant check of the quality in every step reaching in this way a high standardisation of the samples that finally will be used for crystallisation. Moreover, another technical finding is the surprising amount of crystallisation conditions. We have found about 50 conditions that are able to induce PSII crystallisation but none of those crystals conditions, even after several steps of improvement, gave satisfactory diffraction. This fact and other evidences would suggest that the peculiar heterogeneity associated to PSII from higher plants is a mirror of the organisation and the topology of the thylakoid membranes.
During this project, several procedures of PSII purification were developed. The quality of the samples obtained by those procedures is given by the homogeneity of the samples meaning with this the presence of samples constituted only of monomer or dimers. Our best procedure has samples constituted mainly of monomers. Moreover those monomeric samples retain the subunit S of PSII (PsbS) an important polypeptide involved in the photo-protection of PSII. PsbS so far was never directly proven to be attached to PSII monomer in this work we observe a direct connection between PSII monomers and PsbS. We consider this an important finding with physiological implications.
In conclusion, we think that by this project was possible to give an important contribution toward the finding of good diffracting crystals suitable for structural analysis. For this reason this work will be continued with new funds in order to get this final aim. When the final aim will be reached it will have important socio-economic effects. In fact, the availability of a detailed structural description of PSII from higher plants would make possible to select cultivars or genetically modify varieties able to use the water in a more efficient way or create modifications in the polypeptide and cofactor properties in order to increase the conversion efficiency of the light energy. Furthermore, in a similar way it could help in developing more resistant and efficient cultivars that could grow in pre-desert areas. From the technological point of view these results would be of immense interest to help in developing artificial photosynthetic systems in order to product 'green energy'.