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Investigation of the Interactions Leading to Phycobilisome Assembly

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Investigation of the Interactions Leading to Phycobilisome Assembly Investigation of the interactions leading to Phycobilisome assembly Ofir Tal Investigation of the interactions leading to Phycobilisome assembly Research thesis submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Ofir Tal Submitted to the Senate of The Technion – Israel Institute of Technology TISHREI, 5774 HAIFA OCTOBER 2013 This research thesis was done under the supervision of Prof. Noam Adir in the Schulich Faculty of Chemistry. The generous financial help of The Technion is gratefully acknowledged. Table of Contents Introduction ................................................................................................................. 1 The Phycobilisome .................................................................................................... 1 LPs and Minor Core Components ................................................................................ 4 Phycobilisome from an evolutionary point of view ........................................................ 5 Energy transfer in the Phycobilisome ........................................................................... 6 PBS models............................................................................................................... 8 Research goals ........................................................................................................... 12 Methods, Materials and Procedures ............................................................................. 14 Methods .................................................................................................................... 14 Transmission electron microscopy (TEM) and single particle reconstruction .................. 14 Peptides analysis by tandem mass spectrometry (MS/MS) ........................................... 16 Chemical cross-linking for complex structure investigation .......................................... 17 Protein structure prediction and protein-protein docking ............................................ 19 X-ray crystallography ............................................................................................... 21 Materials ................................................................................................................... 24 Cyanobacteria Growth Medium (BG11) ..................................................................... 24 PBS isolation ........................................................................................................... 24 Cross-linked complex Isolation and Purification Buffers ............................................... 24 SDS-PAGE ............................................................................................................... 24 Procedures ................................................................................................................ 25 Growth of cells and isolation of PBSs ......................................................................... 25 Cross-linking and complex isolation ........................................................................... 26 MS Analysis ............................................................................................................ 26 Fluorescence and absorbance measurements ............................................................ 27 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ........................ 27 2D gel .................................................................................................................... 27 Electron microscopy and Single Particle Reconstruction .............................................. 28 Crystallization and data collection ............................................................................. 28 Structure determination, refinement and analysis ...................................................... 28 Modeling and computational tools ............................................................................ 28 Results ...................................................................................................................... 30 PBS purification and characterization ........................................................................ 30 Cross-linking preparation, complex isolation and characterization ................................ 36 Glutaraldehyde cross-linking .................................................................................... 36 BS3 cross-linking ..................................................................................................... 37 HPLC ...................................................................................................................... 39 2D-Gel ................................................................................................................... 40 MS/MS analysis....................................................................................................... 41 Ultrafast Transient Absorption Spectroscopy .............................................................. 44 Protein – Protein Docking and Models filtration .......................................................... 46 Electron Microscopy - Single Particle Reconstruction ................................................... 56 X-ray crystallography ............................................................................................... 58 Structure determination of linker containing PC rods .................................................. 58 Proposed Models .................................................................................................... 62 Linkers - LR and CpcG ............................................................................................... 62 Full core model ....................................................................................................... 64 Discussion .................................................................................................................. 65 References ................................................................................................................. 76 List of Figures Figure 1: Structural similarity between PC and APC monomers, and the common phycocyanobilin they carry. ........................................................................................... 2 Figure 2: Association of PBP subunits and monomers into the larger aggregates. ................. 3 Figure 3: Model of three cylindrical PBS. .......................................................................... 4 Figure 4: Common PBS models. ...................................................................................... 9 Figure 5: Preparative sucrose gradient of PBSs. .............................................................. 30 Figure 6: Absorption spectra of sucrose gradient isolated bands. ..................................... 31 Figure 7: Fluorescence spectra of sucrose gradient isolated bands. .................................. 33 Figure 9: Centrifugation of PBSs in linear sucrose gradient and a series of phosphate buffer concentrations. .......................................................................................................... 34 Figure 10: Emission spectra (λex=540nm) of reconstituted PBSs in pH=6.0-8.0. ................... 35 Figure 11: Optimization of minimal GA cross-linking condition for isolation of PC-APC pairs.37 Figure 12: Optimization of minimal BS3 cross-linking condition for isolation of PC-APC pairs. ................................................................................................................................. 38 Figure 13: Isolation of functional PC-APC complexes. ...................................................... 39 Figure 14: Second purification step of the complexes by HPLC-AEC................................... 40 Figure 15: Separation of sub-fractions of the cross-linked PC-APC adducts by 2D gel electrophoresis. .......................................................................................................... 41 Figure 16: Schematic map of the Identified cross-linked residues. .................................... 44 Figure 17: Ultrafast transient absorption spectroscopy of the isolated complex. ................ 45 Figure 18: The rate of change in 655/620 nm ratio during time (log). ................................ 46 Figure 19: Filtration of protein-pair models based on the modeling limitations. ................. 48 Figure 20: Probability to have the combination of distances, which were detected on filtration results models, by chance is insignificant.......................................................... 50 Figure 21: Distributions of all virtual versus experimental distances on the complete core cylinder model............................................................................................................ 50 Figure 22: The proposed model of PC-APC interaction..................................................... 52 Figure 23: The complete model of a core hexamer containing the minor core components. 55 Figure 24: Fourier Shell Correlation curve of the final 3D density model. ........................... 57 Figure 25: Electron microscopy and 3D reconstruction calculated in cutoff resolution of 29Å of negatively stained isolated complexes. ...................................................................... 57 Figure 26: Crystals of purified complex in the crystallization drops. .................................. 58 Figure 27: Comparison between the isolated complex structure
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