RuBisCO has an active site (binding pocket) that binds ribulose-1,5-bisphosphate (RuBP) and catalyzes the reaction between RuBP and CO2 or O2. In the figure, the two large RuBisCO subunits (blue and cyan) sandwich an RuBP molecule (orange) in the active site. The site is gated by the C-terminus (yellow), lysine 128 (purple), and loop 6 (green), which undergo periodic conformational changes that open or close the site. Reactants enter and products escape while it is in an open state, and carbon- fixation reactions occur during the closed state. Simulations of this gating mechanism allow predictions of the gating rate, which can be linked to RuBisCO performance characteristics.
GTL research teams led by Sandia National Laboratories and Oak Ridge National Laboratory are developing new experimental and computational tools to investigate carbon-sequestration behavior in marine cyanobacteria, in particular, Synechococcus and Synechocystis. These abundant marine microbes are known to play an important role in the global carbon cycle. Whole-cell imaging using a newly developed 3D hyperspectral microscope enabled researchers to detect the distribution of photosynthetic pigments in individual Synechocystis cells. The GTL team also improved the quality and information content in DNA microarray technology by combining hyperspectral imaging technology and patented multivariate statistical analysis. The new system collects a full fluorescence emission spectrum at each pixel, as compared to the single bands of a spectrum collected by current scanners. All relevant wavelengths of light thus are measured at each point across a surface rather than simply at predefined bands of wavelengths. This approach enables the identification, modeling, and correction of gene expressions for unknown and unanticipated emissions; increases throughput by accommodating many spectrally overlapped labels in a single scan; and improves sensitivity, accuracy, dynamic range, and reliability. The scanner is being modified to allow 3D imaging of many fluorescently tagged molecules in cells and tissues. New massively parallel modeling and simulation tools also developed by the team have yielded structural insight into the specificity of RuBisCO, an enzyme central to photosynthetic carbon fixation. The team also developed the computational capability to track spatial and temporal variations in protein species concentrations in realistic cellular geometries for important cyanobacterial subcellular processes. These tools include the Large-Scale Atomic/Molecular Massively Parallel Simulator (LAMMPS, http://lammps.sandia.gov/), a molecular simulation tool; and ChemCell, a whole-cell modeling tool that captures those and other results into a spatially realistic metabolic pathway simulation. LAAMPS enables investigations of the protein-sequence effect on different RuBisCO specificities and reaction rates in various species. Using this tool, researchers discovered that mutations in RuBisCO’s amino acid sequence substantially altered the free-energy barrier for gating the binding pocket. This result provided a molecular-level explanation for the experimentally observed species variations in RuBisCO performance (see illustration). LAMMPS was released as open-source software in September 2004 and was downloaded over 4000 times to June 2005. Via 3D simulations of diffusion and reaction in realistic geometries, ChemCell captured the carbon-fixation process carried out by RuBisCO in the carboxysome, a subcellular organelle. [Grant Heffelfinger, Sandia National Laboratories.]
Reference: M. B. Sinclair et al., “Design, Construction, Characterization, and Application of a Hyperspectral Microarray Scanner,” Appl. Optics 43, 2079–88 (2004).
Credit or Source: Paul Crozier, Sandia National Labs
US DOE. 2005. Genomics:GTL Roadmap, DOE/SC-0090, U.S. Department of Energy Office of Science. (p. 66) (website)