Progress 08/01/03 to 07/31/06
Outputs
Photosystem I (PS I) is a large membrane protein complex that catalyzes the first step of light reactions in photosynthesis. The molecular structure of this complex is solved to atomic resolution (2.5A). Ferredoxin acts as the natural electron acceptor of Photosystem I and mediates the electron transfer from Photosystem I to the FNR, where finally NADP+ is reduced to NADPH. The aim of our studies was to unravel the interaction between Photosystem I and ferredoxin at atomic detail by co-crystallization of Photosystem I with ferredoxin. The trimer of Photosystem I has a MW of 1,056 kDa compared with 10 kDa for Ferredoxin. Therefore co-crystallizing PSI and Ferredoxin for X-ray structure analysis is a very challenging project. Improvement of the crystallization conditions led to a new crystal type which differs from all previous grown PSI-Fd cocrystals. The space group has been determined to be primitive monoclinic P21 with a=214.5 A, b=235.6 A, c=261.2 A, alpha=90.0
beta=100.47 gamma=90.0 . Native data sets have been collected and are 99% complete at 4.0A and 63% complete at 3.5 A resolution. From the native diffraction data obtained at ALS (Beamline 5.0.2) at Berkeley we calculated a Electron density map using phases from the already solved structures of Photosystem I and Ferredoxin by molecular replacement. The first model was refined with the combination of molecular replacemnet and phase improvement by heavy metal modified crystals measured at APS Beamline ID-19 in Argonne and ALS Beamline 8.2.2 in Berkeley. The density map reveals that the unit cell contains two trimers of Photosystem I in the unt cell. The organization is a twofold trimer where all stromal Iron sulfur clusters are oriented to the outside. Therefore the trimer complexes are arranged face to face in one asymmetric unit, which corresponds to 78 proteinsubunits(chains) , 764 cofactors, 13,500 residues and a total of about 146,000 atoms. The complex we describe herein has a
total molecular weight of above 2 million Dalton. This huge PSI -Fd sandwich is a completely new challenge for the phase determination by molecular replacement. The electron density map indicates that the three stromal subunits(C,D,E) may undergo significant structural changes upon ferredoxin binding to the membrane extrinsic subunits of Photosystem I. In contrast the transmembrane part of Photosystem I is structural unaffected comparing the native Photosystem I and in the Ferredoxin/Photosystem I cocrystals. From the actual map we have assigned one Ferredoxin to the binding place to each Photosystem I. The assigned Ferredoxin position has been proofed within the whole structure and refined.
Impacts
The elucidation of the structure and function of membrane proteins, which represent 30% of all proteins and are the key enzymes in photosynthesis, respiration, cell recognition, transport and energy storage is one of the most challenging goals in biology. Whereas more than 25, 000 structures of soluble proteins have already been determined, less than 60 different membrane protein structures have been unravelled so far and only two of them are complexes of a membrane protein with a soluble protein. To studies these interactions on an atomic level is very important, because most of the membrane protein complexes do not act on their own but interact with soluble proteins, being involved in a complex network that maintains and regulates essential cell functions in plant, cyanobacteria an algae. The elucidation of the structure and the increase in knowledge about the dynamics of the electron transfer between Photosystem I and ferredoxin will be a breakthrough in
understanding of these complex processes. Thereby, the project has a high impact on the development of artificial photosynthesis as well as impact on the optimization of the natural photosynthetic efficiency which may lead in the future to a higher efficiency of crop and plant productivity.
Publications
- Fromme, P., Yu, H.Q., DeRuyter, Y.S., Jolley, C., Chauhan, D.K., Melkozernov, A. and Grotjohann, I. (2006) Structure of photosystems I and II. Comptes Rendus Chimie, 9, 188-200.
- Jolley, C., Ben-Shem, A., Nelson, N. and Fromme, P. (2005) Structure of plant photosystem I revealed by theoretical modeling. Journal of Biological Chemistry, 280, 33627-33636.
- Fromme, P., Yu, H., Bukman, Y., Ni, D., Varco-Merth, B., Chauhan, D., Vanselow, C., Jolley, C., Fromme, R. and Grotjohann, I. (2005) New Insigths into the structure and function of photosystem I and II. Febs Journal, 272, 451.
- Fromme, R., Yu, H., Grotjohann, I. and Fromme, P. (2005) Cocrystals of photosystem I with its soluble natural electron acceptor ferredoxin at 4 A resolution. Febs Journal, 272, 449.
- Grotjohann, I., Jolley, C. and Fromme, P. (2004) Evolution of photosynthesis and oxygen evolution: Implications from the structural comparison of Photosystems I and II. Physical Chemistry Chemical Physics, 6, 4743-4753.
- Fromme, P. and Mathis, P. (2004) Unraveling the Photosystem I reaction center: a history, or the sum of many efforts. Photosynthesis Research, 80, 109-124.
- Fromme, P., Yu, H., Bukman, Y., Yolley, C., Chauhan, D.K., Melkozernov, A. and Grotjohann, I. (2004) Structure and function of photosystem I and II. Biochimica Et Biophysica Acta-Bioenergetics, 1658, 77.
- Fromme, P., Melkozernov, A., Jordan, P. and Krauss, N. (2003) Structure and function of photosystem I: interaction with its soluble electron carriers and external antenna systems. Febs Letters, 555, 40-44.
- Antonkine, M.L., Jordan, P., Fromme, P., Krauss, N., Golbeck, J.H. and Stehlik, D. (2003) Assembly of protein subunits within the stromal ridge of photosystem - I. Structural changes between unbound and sequentially PSI-bound polypeptides and correlated changes of the magnetic properties of the terminal iron sulfur clusters. Journal of Molecular Biology, 327, 671-697.
- Fromme,Raimund Grotjohann, Ingo and Petra Fromme (2006) in: Structural Biology of Membrane proteins (S. Buchanan, R. Grisshammer eds.) Royal Society of Chemistry (Cambridge, UK) Structure of Photosystems I and II,ISBN 0 85404 361 6, pp. 320-348
- Fromme, P.and Grotjohann, I (2006) in: Photosystem I: the Ligth Driven, Plastocyanin:Ferredoxin Oxidoreductase Chapter 6, Series: Advances in Photosynthesis in Respiration by Kluwer Academic Publishers, B.V. (J.H. Golbeck ed.) in press Structural Analysis of Cyanobacterial Photosystem I
- Subramanyam, R., Jolley, C., Brune, D.C., Fromme, P. and Webber, A.N. (2006) Characterization of a novel photosystem I-LHCI supercomplex isolated from Chlamydomonas reinhardtii under anaerobic (State II) conditions. Febs Letters, 580, 233-238.
- Workshop on "Membranes, Membrane Proteins and Membrane Associated Molecular Maschines, Howard Hughes Medical Institute Headquaters, (2004), Chevy Chase, MA, USA,Crystallization of Photosystem I and II
- Invitedtalks:International conference on Porphyrins and Phthalocyanines ICCP-4 Rome 2-7, 2006 Rome, Italy Structure and function of Porphyrins in Photosystem I and II
- Workshop on Crystallization of Membrane Proteins, NSLS User Meeeting, Brookhaven, (2004) Brookhaven, USA, Overcoming the crystallization Problems of Photosystem I an II
- Annual Department of Energy Solar Photochemistry Research Conference, (2004), Airline, USA, Structure and Function of Photosystem I and II
- Martin Kamen Memorial Lecture, University of California, San Diego, (2004), San Diego, USA, From sunlight to electron transfer: structure and function of Photosystem I and II
- 48th Meeting of the Biophysical Society, Petra Fromme(2004), Baltimore, USA, Structure and Function of Photosystem I and II
- Nobel Symposium on "Membrane Proteins: Structure, Function and Assembly" August 2003 Stockholm, Sweden:Structure and Function of Photosystem I and II
- 29th Midwestern Photosynthesis Meeting (2003) Turkey Run, Indiana USA, Structure and Function of Photosystem I and II
- Petra Fromme(2003),6th International Symposium of the Volkswagen Stiftung on Intra- and Intermolecular Electron Transfer, Walberberg, Germany, Structure and Function of Photosystem I and II
- University of Kentucky, Department of Chemsitry and Biochemistry, Departmental seminar, March 24, 2006 From sun-light to electron transfer: Structure and function of Photosystem I and II
- 10th Annual Meeting of the Swedish Structural Biology Network, June 16-19, 2006, Tallberg, Sweden, New insigths into the structure and function of Photosystem I and II
- 3rd International Conference on Structure, Dynamics and Function of Proteins in Biological Membranes, May 14-19, 2006 Monte Verita, Switzerland: Structure and Function of Photosystem I
- Oxygen meeting of the Agouron Institute, (2006) April 6-10 Santa Fe, USA Water oxidation in Photosystem II: unsolved Questions based on the current status of X-ray Crystallography and Spectroscopy
- Petra Fromme(2005)Iowa State University: Department of Biochemistry and Biophysics, Departmental seminar, December 15, From sun-light to electron transfer: Structure and function of Photosystem I and II
- Workshop on Biological Membranes: Structure and Function" at The Ohio Center for Theoretical Science (OCTS), The Ohio State University. October 8-11, 2005, Ohio, USA From light to electron transfer: New insights into the structure and function of Photosystem I"
- GDCh-Jahrestagung (2005), September 11-14, 2005, Duesseldorf, Germany Eine faszinierende Reise in die Welt der Photosynthese
- Petra Fromme (2005) 30th FEBS Congress and 9th IUBMB Conference, The Protein World: Proteins and Peptides - Structure, Function and Organization, July 2-7, Budapest, Hungary
- Workshop on Crystallization: focus on optimization techniques, soluble and membrane proteins NSLS Brookhaven, June 6-9 (2005) Brookhaven, USA "Phase Diagrams: A Way for the Rational Design of Membrane Protein Crystallization
- ESRF Conference Molecular Bioenergetics of Cyanobacteria, 21-26 May 2005 Sant Feliu de Guixols, Spain:New insights into the structure and function of Photosystem I and II
- First International Symposium on Chloroplast Bioengineering, May 2-7, (2005)University of Illinois UI, USA :Molecular insights into the structure and function of photosystem I and II
- Departmental seminar, March 24, (2005), The University of Alabama, Tuscaloosa, Alabama, USA:From sun-light to electron transfer: Structure and function of Photosystem I and II
- University of California Riverside, (2005),Seminar in Biochemistry and Molecular Biology, Riverside, CA, USA:From sun-light to electron transfer: Structure and function of Photosystem I and II
- Western Photosynthesis Conference, Asilomar, USA ,(2005), Overview of the current state of PS II crystallography and advances that may be expected during the next five years
- 13th International Congress of Photosynthesis,(2004), Montreal, Canada, Structure and Function of Photosystem I and II
- 8th Cyanobacterial Molecular Biology Workshop,(2004), Quebec, Canada, Structure and Function of Photosystem I and II
- International Satelite Meeting "Photosynthesis and Post-Genomic era: From Biophysics to Molecular Biology, a Path in the Research of Photosystem II, (2004), Trois-Rivieres, Canada Structure and Function of Photosystem II
- European Bioenergetics Conference, (2004), Pisa, Italy Structure and Function of Photosystem I and II
- 15th International Conference on Photochemical Conversion and Storage of Solar Energy July 2004, Paris, France Structure and Function of Photosystem I and II
|
Progress 10/01/04 to 09/30/05
Outputs
The research conducted during the second funding period of this project from 10/1/2004 to 9/30/2005 was centered on the determination of the structure of a the complex between Photosystem I and its natural electron acceptor ferredoxin by X-ray structure analysis and the further improvement of the cocrystals. Photosystem I is a large membrane protein complex that catalyzes the first step of light reactions in photosynthesis. Ferredoxin acts as the natural electron acceptor of Photosystem I and mediates the electron transfer from Photosystem I to the FNR, where finally NADP+ is reduced to NADPH. The aim of our studies is to unravel the interaction between Photosystem I and ferredoxin at atomic detail by co-crystallization of Photosystem I with ferredoxin. The trimer of Photosystem I has a MW of 1 056 kDa compared with 10 kDa for Ferredoxin. Therefore co-crystallizing PSI and Ferredoxin and the X-ray structure analysis of this complex is a very challenging project.
Improvement of the crystallization conditions leads to a new crystal type which differs from all previous grown PSI-Fd cocrystals. The space group has been determined to be P21 with a=214.5 A, b=235.6 A, c=261.2 A, alpha=90.0 beta=100.47 gamma=90.0 . The data are 99% complete at 4.0A and 63% complete at 3.5 A. From the diffraction data obtained at ALS Beamline 5.0.2 at Berkeley we calculated an Electron density map using phases from the already solved structures of Photosystem I and Ferredoxin and a phase improvement by heavy metal modified crystals measured at APS Beamline ID-19 in Argonne and ALS Beamline 8.2.2 in Berkeley. From the density map we are able to fit six complete Photosystem I molecules in the unit cell. The organization is a twofold trimer where the trimers face each other with the lumenal sides and all stromal subunits with the iron sulfur clusters are oriented to the outside of the sandwich. Therefore the trimeric complexes arranged face to face in one asymmetric
unit, which corresponds to 72 proteins , 764 cofactors, 13,500 residues and a total of about 146,000 atoms. The complex we describe herein has a total molecular weight of above 2 million Dalton. This huge PSI-Fd sandwich is a completely new challenge for the phase determination by molecular replacement, as present MR programs had to be modified so that 72 protein chains and more than 700 cofactors could be handled. The phases were further improved by experimental phase information from X-ray data sets that were collected at the Fe-absorption edge. The present electron density map indicates that the three stromal subunits may undergo significant structural changes upon ferredoxin binding in contrast to the membrane intrinsic subunits of Photosystem I that are nearly identical in the native Photosystem I and in the cocrystals. The structural arrangement of the ferredoxin molecules is under refinement. From the actual map we have assigned one ferredoxin to a binding place, which was
unbiased calculated by molecular replacement. The ferredoxin is docked to PSI at the stromal side of the membrane at a distance of 15 A to FX. The refinement of the structure is currently in progress.
Impacts
Membrane proteins are important for many major functions in the cell like photosynthesis, respiration, cell communication and transport processes. The elucidation of the structure and function of membrane proteins is one of the most challenging goals in biology. Most of these membrane proteins interact with soluble molecules and proteins. Despite the great importance of the membrane proteins and their interaction partners, the knowledge on their structure is limited to less than 60 different membrane protein structures that have been unravelled so far. Only two of them are complexes of a membrane protein with a soluble protein. The aim of our studies is to unravel the interaction of Photosystem I with its natural electron donor ferredoxin. Ferredoxin is a very important regulatory protein in cells of plants, algae and cyanobacteria. The elucidation of the structure and the increase in knowledge about the dynamics of the electron transfer between Photosystem I and
ferredoxin will be a breakthrough in understanding of these complex processes. The project will increase our knowledge on then natural system and thereby help to development artificial photosynthesis. Furthermore the project will make significant contributions to the optimization of the natural photosynthetic efficiency. Finally, the understanding of the major processes on Photosynthesis may lead in the future to a higher efficiency of crop and plant productivity.
Publications
- Fromme,R. Grotjohann, I. and Petra Fromme (2006) in: Structural Biology of membrane proteins (S. Buchanan, R. Grisshammer eds) Royal Society of Chemistry (Cambridge, UK) in press. Structure of Photosystems I and II
- Fromme, P.and Grotjohann, I (2006) in:Photosystem I: the Light Driven, Plastocyanin:Ferredoxin Oxidoreductase Chapter 6, Series: Advances in Photosynthesis in Respiration by Kluwer Academic Publishers, B.V. (J.H. Golbeck ed.) in press Structural Analysis of Cyanobacterial Photosystem I
- Petra Fromme, HongQi Yu, Yana Bukman, Craig Jolley, Devendra K. Chauhan and Alexander Melkozernov and Ingo Grotjohann (2006) Comptes Rendus Chimie Volume 9 ( 2 ) 188-200 Structure of Photosystem I and II
- Grotjohann I, Fromme P (2005) Photosynth Res 85: 51-72, Structure of cyanobacterial photosystem I
- Structure and function of Photosystem I and II Workshop on Crystallization: focus on optimization techniques, soluble and membrane proteins NSLS Brookhaven, June 6-9 2005 Brookhaven, USA "Phase Diagrams: A Way for the Rational Design of Membrane Protein Crystallization
- ESRF Conference Molecular Bioenergetics of Cyanobacteria, 21-26 May 2005 Sant Feliu de Guixols, Spain
- New insights into the structure and function of Photosystem I and II First International Symposium on Chloroplast Bioengineering,Petra Fromme, May 2-7 2005, University of Illinois UI, USA
- Molecular insights into the structure and function of photosystem I and II Departmental seminar, March 24, 2005 The University of Alabama, Tuscaloosa, Alabama, USA From sunlight to electron transfer: Structure and function of Photosystem I and II
- University of California Riverside, Seminar in Biochemistry and Molecular Biology, Riverside, CA, USA, Petra Fromme(2005) From sunlight to electron transfer: Structure and function of Photosystem I and II
- The preliminary data of the project have been presented in invited lectures at the following national and international conferences in 2005 as well as presentations at universities in the US: Iowa State University: Department of Biochemistry and Biophysics, Departmental seminar, December 15., 2005 From sun-light to electron transfer: Structure and function of Photosystem I and II
- Workshop on Biological Membranes: Structure and Function" at The Ohio Center for Theoretical Science (OCTS), The Ohio State University. October 8-11, 2005, Ohio, USA From light to electron transfer: New insights into the structure and function of Photosystem I
- GDCh- Jahrestagung 2005, September 11-14, 2005, Duesseldorf, Germany Eine faszinierende Reise in die Welt der Photosynthese
- 30th FEBS Congress and 9th IUBMB Conference, The Protein World: Proteins and Peptides - Structure, Function and Organization, July 2-7, 2005 Budapest, Hungary
|
Progress 10/01/03 to 09/30/04
Outputs
The research conducted during the first funding period of this project from 10/1/2003 to 9/30/2004 was centered on the investigation of the interaction of Photosystem I with its natural electron acceptor ferredoxin with special focus on the co-crystallization of both proteins and the optimization of the crystals. Photosystem I is a large membrane protein complex which functions as a bio-solar energy converter by capturing the light-energy of the sun and converting it into electrical energy by performing a transmembrane charge separation finally leading to the reduction of ferredoxin. The optimization of the crystallization of the functionally active complex between Photosystem I and ferredoxin is the major requirement for the determination of the structure of the complex by X-ray structure analysis and the elucidation of the mechanism of protein docking and inter protein electron transfer. During this first year of funding, we achieved a major breakthrough in the
co-crystallization of Photosystem I with ferredoxin. Our work based on crystals that were quite large (up to 0.5 mm) diffracting X-rays to 7 to 8 Angstrom resolution. However these crystals were not useful for X-ray structure analysis due to the large internal disorder (mosaic spread) of more than 2-5 degree. Systematic screening of the maximization of the interaction of Photosystem I with ferredoxin (including an intensive screen of additives) combined with the determination of phase diagrams and the introduction of seeding techniques lead to the recent discovery of a new crystal form with shows a dramatic increase of quality. Even though the crystals are still quite small (0.1 mm) they show improved X-ray diffraction to a resolution of 3.8 Angstrom and a dramatic decrease of internal disorder by a factor of 8 to less than 0.7 degree. Complete native data sets and a partial data set at the Fe absorption edge have been collected at the Advanced Light Source at Lawrence National Lab
Berkeley, at the Advanced Photon Source at Argonne National Lab and at the National Synchrotron Light Source at Brookhaven National Lab. Data have been evaluated and are complete to 4 Angstrom resolution. Preliminary phase determination with molecular replacement already shows the main structure elements of PS I (as alpha helices and 4Fe4S clusters) as well as density that many indicate the location of the 2Fe2S cluster of ferredoxin. The present work focuses on the determination of experimental phases, which will lead to a further dramatic improvement of the structural information on the location of ferredoxin in the single crystals as well as the optimization of the X-ray diffraction quality of the crystals towards the determination of the atomic structure of the Photosystem I-ferredoxin complex. The structural work will be complemented by spectroscopic studies on the single crystals with EPR and optical spectroscopy. We have already designed a special cuvette for single crystal
optical spectroscopy, which will further be used to further study mechanism of electron transfer between Photosystem I and ferredoxin.
Impacts
The elucidation of the structure and function of membrane proteins, which represent 30% of all proteins and are the key enzymes in photosynthesis, respiration, cell recognition, transport and energy storage is one of the most challenging goals in biology. Whereas more than 25 000 structures of soluble proteins have already been determined, less than 60 different membrane protein structures have been unravelled so far and only two of them are complexes of a membrane protein with a soluble protein. To studies these interactions on an atomic level is very important, because most of the membrane protein complexes do not act on their own but interact with soluble proteins, being involved in a complex network that maintains and regulates essential cell functions in plant, cyanobacteria an algae. The elucidation of the structure and the increase in knowledge about the dynamics of the electron transfer between Photosystem I and ferredoxin will be a breakthrough in
understanding of these complex processes. Thereby, t he project has a high impact on the development of artificial photosynthesis as well as impact on the optimization of the natural photosynthetic efficiency which may lead in the future to a higher efficiency of crop and plant productivity.
Publications
- The publication of the improved crystallization and structure determination of the PSI-ferredoxin complex is in progress. The preliminary data of the project have been presented in invited lectures at the following national and international conferences in 2003 and 2004:
- Fromme P, Yu H, Grotjohann I, Bukhman Y, Jolley C, Chauhan DK, and Melkozernov A (2004), Structure and function of Photosystem I and II, Plenary Lecture, 13th International Congress of Photosynthesis, August 2004, Montreal, Canada
- Fromme, P (2004) Structure and function of Photosystem I and II Keynote Talk, 8th Cyanobacterial Molecular Biology Workshop, August 2004, Quebec, Canada
- Fromme P, Yu H, Bukhman Y, Jolley C, Chahan D, Melkozernov A and Grotjohann I (2004) Structure and function of Photosystem I and II, Plenary Lecture, European Bioenergetics Conference, August 2004, Pisa, Italy
- Yu H, Grotjohann I, Bukhman Y, Yolley C, Chauhan DK, Melkozernov A and Fromme P, (2004) Structure and Function of Photosystem I and II, 15th International Conference on Photochemical Conversion and Storage of Solar Energy July 2004, Paris, France
- Yu H, Grotjohann I, Bukhman Y, Jolley C, Chauhan DK and Melkozernov A and Fromme P (2004) Structure and Function of Photosystems I and II, Plenary Lecture, Annual Department of Energy Solar Photochemistry Research Conference, June 2004, Airline, USA
- Fromme, P (2004) Overcoming the Crystallization Problems of Photosystem I and II, Invited Lecture and Practicals, NSLS User's Meeting Brookhaven National Laboratory, May 2004, Brookhaven, USA
- Fromme,P (2004) Structure and Function of Photosystem I and II, Invited Lecture, 48th Meeting of the Biophysical Society, February 2004, Baltimore, USA
- Fromme P, Melkozernov A, Jordan P, Zouni A, Kern J, Biesiadka J, Loll B Saenger W, Krauss N and Witt, HT (2003) Structure and Function of Photosystem I and II, Invited Talk, 29th Midwestern Photosynthesis Meeting Oktober, 2003 Turkey Run, Indiana USA
- Fromme P, Melkozernov A, Jordan P, Zouni A, Kern J, Biesiadka J, Loll B Saenger W, Krauss N and Witt, HT (2003) Structure and Function of Photosystem I and II, 6th International Symposium of the Volkswagen Stiftung on Intra- and Intermolecular Electron Transfer, October 2003 Walberberg, Germany
|