Dynamic Imaging of Structurally Heterogeneous Protein Complexes
EMSL Project ID
47484
Abstract
Our long-term goal is to identify biological design principles that will enable synthetic biology approaches for bioenergy applications. We hypothesize that the targeted engineering of selected interfaces within supramolecular protein complexes will enable the control of metabolic flux to optimize desired metabolic pathways. Central to our experimental strategy is the ability to use advanced fluorescence imaging, dynamic transmission electron microscopy (DTEM), and stimulated Raman spectroscopy to monitor time-dependent changes in protein structure and dynamics. In conjunction with established omics measurements and chemical imaging probes, proposed high-resolution imaging measurements will reveal fundamental design principles in biology. Focused measurements will target RNA polymerase (RNAP), formate-hydrogen lyase (FDH), and an metal-reductase complex (OMC). These supramolecular protein complexes respectively catalyze DNA-dependent transcription, hydrogen production/carbon fixation, and electron transport to extracellular metal oxides. Each protein complex has substantial importance to an ability to reprogram cellular metabolism for bioenergy applications. Specifically, an ability to target RNAP to specific promoters can be used to reprogram cellular metabolism at a global level to enhance desired pathways involving energy production at the expense of cellular growth. Adjusting the catalytic bias of FDH will permit enhanced rates of carbon dioxide fixation to form liquid fuels. Optimization of the maturation pathways of OMC will enable design of biofuel cells for energy storage. Collectively, these measurements will provide important fundamental insights regarding the assembly and molecular actions of supramolecular protein machines and interfacial electron transfer mechanisms important to the identification of design principles that will enable metabolic bioengineering approaches. This work will support four complementary projects supported by OBER, OBES, and DTRA that collectively seek to understand the functional regulation of protein complexes to control metabolic pathways, with applications to synthetic biology and bioenergy.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2012-10-01
End Date
2014-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Fu N, D Su, JR Cort, B Chen, Y Xiong, W Qian, A Konopka, DJ Bigelow, and TC Squier. 2013. "Synthesis and Application of an Environmentally Insensitive Cy3-Based Arsenical Fluorescent Probe to Identify Adaptive Microbial Responses Involving Proximal Dithiol Oxidation." Journal of the American Chemical Society 135(9):3567-3575. doi:10.1021/ja3117284
Fu N, Y Xiong, and TC Squier. 2012. "Synthesis of a Targeted Biarsenical Cy3-Cy5 Affinity Probe for Superresolution Fluorescence Imaging." Journal of the American Chemical Society 134(45):18530-18533. doi:10.1021/JA308503x
Fu N, Y Xiong, and TC Squier. 2013. "Optimized Design and Synthesis of Cell Permeable Biarsenical Cyanine Probe for Imaging Tagged Cytosolic Bacterial Proteins ." Bioconjugate Chemistry 24(2):251-259. doi:10.1021/bc300619m
Hua X, MJ Marshall, Y Xiong, X Ma, Y Zhou, AE Tucker, Z Zhu, S Liu, and XY Yu. 2015. "Two-dimensional and three-dimensional dynamic imaging of live biofilms in a microchannel by time-of-flight secondary ion mass spectrometry." Biomicrofluidics 9(3):Article No. 031101. doi:10.1063/1.4919807
Smith DMA, K Danyal, S Raugei, and LC Seefeldt. 2014. "Substrate Channel in Nitrogenase Revealed by a Molecular Dynamics Approach." Biochemistry 53(14):2278-2285. doi:10.1021/bi401313j