In photosynthetic organisms, photons are captured by light-harvesting antenna complexes, and energy is transferred to reaction centers where photochemical reactions take place. We describe here the isolation and characterization of a fully functional megacomplex composed of a phycobilisome antenna complex and photosystems I and II from the cyanobacterium Synechocystis PCC 6803. A combination of in vivo protein cross-linking, mass spectrometry, and time-resolved spectroscopy indicates that the megacomplex is organized to facilitate energy transfer but not intercomplex electron transfer, which requires diffusible intermediates and the cytochrome b6f complex. The organization provides a basis for understanding how phycobilisomes transfer excitation energy to reaction centers and how the energy balance of two photosystems is achieved, allowing the organism to adapt to varying ecophysiological conditions.

The high excitation energy-transfer efficiency demanded in photosynthetic organisms relies on the optimal pigment-protein binding orientation in the individual protein complexes and also on the overall architecture of the photosystem. In green sulfur bacteria, the membrane-attached Fenna-Matthews-Olson (FMO) antenna protein functions as a ''wire'' to connect the large peripheral chlorosome antenna complex with the reaction center (RC), which is embedded in the cytoplasmic membrane (CM). Energy collected by the chlorosome is funneled through the FMO to the RC. Although there has been considerable effort to understand the relationships between structure and function of the individual isolated complexes, the specific architecture for in vivo interactions of the FMO protein, the CM, and the chlorosome, ensuring highly efficient energy transfer, is still not established experimentally. Here, we describe a mass spectrometrybased method that probes solvent-exposed surfaces of the FMO by labeling solvent-exposed aspartic and glutamic acid residues. The locations and extents of labeling of FMO on the native membrane in comparison with it alone and on a chlorosome-depleted membrane reveal the orientation. The large differences in the modification of certain peptides show that the Bchl a #3 side of the FMO trimer interacts with the CM, which is consistent with recent theoretical predictions. Moreover, the results also provide direct experimental evidence to confirm the overall architecture of the photosystem from Chlorobaculum tepidum (C. tepidum) and give information on the packing of the FMO protein in its native environment.chemical labeling ͉ energy transfer ͉ FMO protein ͉ mass spectrometry ͉ protein footprinting P hotosynthesis is a fundamental biological process that harvests solar energy to power life on Earth (1). A diverse family of pigment-protein complexes and elegant architectures accomplish the necessary light-harvesting and energy-storage processes (2-5). In photosynthetic green sulfur bacteria, light absorbed by a large antenna complex known as a chlorosome (6-8) is transferred through a protein called Fenna-Matthews-Olson (FMO) (9) to the reaction center, which is embedded in the cytoplasmic membrane (CM). Together, they form a funnel-like architecture to facilitate energy transfer. The specific orientation of the critical linker, the FMO protein, however is unknown (Fig. 1A).The structure of the FMO protein was the first (bacterio)chlorophyll binding protein to be determined by X-ray crystallography. Structures of this protein from 2 species, Prosthecochloris aestuarii 2K (10, 11) and Chlorobaculum tepidum (12) are now available, and they show strong structural and spectral similarities. The FMO protein consists of 3 identical subunits of mass 40 kDa related by a 3-fold axis of symmetry. The 3 monomers form a disc with a C3 symmetry axis perpendicular to the disc plane (Fig. 1B). There are 7 BChl a molecules in a monomer, although an eighth pigment has been resolved in newly solved structures (13,14). Eac...

The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrometry and its coupling with native electrospray ionization (ESI) make the combination complementary to other biophysical methods of protein analysis. Protein assemblies with molecular masses up to MDa are now accessible by this approach. Most current approaches have used quadrupole/time-of-flight tandem mass spectrometry, sometimes coupled with ion mobility, to reveal stoichiometry, shape, and dissociation of protein assemblies. The amino-acid sequence of the subunits, however, still relies heavily on independent bottom-up proteomics. We describe here an approach to study protein assemblies that integrates electron-capture dissociation (ECD), native ESI, and FTICR mass spectrometry (12 Tesla). Flexible regions of assembly subunits of yeast alcohol dehydrogenase (147 kDa), concanavalin A (103 kDa), and photosynthetic Fenna-Matthews-Olson antenna protein complex (140 kDa) can be sequenced by ECD or “activated-ion” ECD. Furthermore, non-covalent metal-binding sites can also be determined for the ConA assembly. Most importantly, the regions that undergo fragmentation, either from one of the termini by ECD or from the middle of a protein, as initiated by CID, correlate well with the B-factor from X-ray crystallography of that protein. This factor is a measure of the extent an atom can move from its coordinated position as a function of temperature or crystal imperfections. The approach provides not only top-down proteomics information of the complex subunits but also structural insights complementary to those obtained by ion mobility.

The intact yeast alcohol dehydrogenase (ADH) tetramer of 147 kDa was introduced into a FTICR mass spectrometer by native electrospray. Electron capture dissociation of the entire 23ϩ to 27ϩ charge state distribution produced the expected charge-reduced ions and, more unexpectedly, 39 c-type peptide fragments that identified N-terminus acetylation and the first 55 amino acids. The results are in accord with the crystal structure of yeast ADH, which shows that the C-terminus is buried at the assembly interface, whereas the N-terminus is exposed, allowing ECD to occur. This remarkable observation shows promise that a top-down approach for intact protein assemblies will be effective for characterizing their components, inferring their interfaces, and obtaining both proteomics and structural biology information in one experiment. (J Am Soc Mass Spectrom 2010, 21, 1966 -1968 ECD and FT (Fourier transform) ion cyclotron resonance (ICR) MS combine as an appealing approach for study of protein assemblies because, in principle, the masses of the complex and of the subunits, as well as some sequence of the constituents can be acquired in a single experiment. Indeed, we report here a successful ECD-based FTICR MS top-down approach that affords the composition, stoichiometry, and partial sequence of a 147 kDa noncovalent protein assembly; namely, the yeast alcohol dehydrogenase (ADH) tetramer.

We describe here the analysis of Nanodisc complexes by using native mass spectrometry (MS) to characterize their molecular weight (MW) and polydispersity. Nanodiscs are nanoscale lipid bilayers that offer a platform for solubilizing membrane proteins. Unlike detergent micelles, Nanodiscs are native-like lipid bilayers that are well defined and potentially monodisperse. Their mass spectra allow peak assignment based on differences in the mass of a single lipid per complex. Resultant masses agree closely with predicted values and demonstrate conclusively the narrow dispersity of lipid molecules in the Nanodisc. Fragmentation with collisionally activated dissociation (CAD) or electron-capture dissociation (ECD) shows loss of a small number of lipids and eventual collapse of the Nanodisc with release of the scaffold protein. These results provide a foundation for future studies utilizing Nanodiscs as a platform for launching membrane proteins into the gas phase.