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Title: Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc

Authors:
 [1];  [1];  [2];  [3];  [4];  [1]
  1. Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States; Photosynthetic Antenna Research Center, Washington University in St. Louis, St. Louis, Missouri 63130, United States
  2. Photosynthetic Antenna Research Center, Washington University in St. Louis, St. Louis, Missouri 63130, United States
  3. Photosynthetic Antenna Research Center, Washington University in St. Louis, St. Louis, Missouri 63130, United States; Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States
  4. Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States; Photosynthetic Antenna Research Center, Washington University in St. Louis, St. Louis, Missouri 63130, United States; Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130, United States
Publication Date:
Research Org.:
Energy Frontier Research Centers (EFRC) (United States). Photosynthetic Antenna Research Center (PARC)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1388787
DOE Contract Number:
SC0001035
Resource Type:
Journal Article
Resource Relation:
Journal Name: Analytical Chemistry; Journal Volume: 88; Journal Issue: 17; Related Information: PARC partners with Washington University in St. Louis (lead); University of California, Riverside; University of Glasgow, UK; Los Alamos National Laboratory; University of New Mexico; New Mexico Corsortium; North Carolina State University; Northwestern University; Oak Ridge National Laboratory; University of Pennsylvania; Sandia National Laboratories; University of Sheffield, UK
Country of Publication:
United States
Language:
English
Subject:
solar (fuels), photosynthesis (natural and artificial), biofuels (including algae and biomass), bio-inspired, charge transport, membrane, synthesis (novel materials), synthesis (self-assembly)

Citation Formats

Lu, Yue, Zhang, Hao, Niedzwiedzki, Dariusz M., Jiang, Jing, Blankenship, Robert E., and Gross, Michael L.. Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc. United States: N. p., 2016. Web. doi:10.1021/acs.analchem.6b01945.
Lu, Yue, Zhang, Hao, Niedzwiedzki, Dariusz M., Jiang, Jing, Blankenship, Robert E., & Gross, Michael L.. Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc. United States. doi:10.1021/acs.analchem.6b01945.
Lu, Yue, Zhang, Hao, Niedzwiedzki, Dariusz M., Jiang, Jing, Blankenship, Robert E., and Gross, Michael L.. 2016. "Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc". United States. doi:10.1021/acs.analchem.6b01945.
@article{osti_1388787,
title = {Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc},
author = {Lu, Yue and Zhang, Hao and Niedzwiedzki, Dariusz M. and Jiang, Jing and Blankenship, Robert E. and Gross, Michael L.},
abstractNote = {},
doi = {10.1021/acs.analchem.6b01945},
journal = {Analytical Chemistry},
number = 17,
volume = 88,
place = {United States},
year = 2016,
month = 8
}
  • The light-harvesting complex of photosystem I (LHCI) was isolated from wild-type cells of Chlamydomonas reinhardtii; the Chl a/b-protein complex contains four major polypeptides of approximately 27, 26, 24, and 20 kDa (polypeptides 14, 15, 17.2, and 22, respectively, in the nomenclature for Chlamydomonas thylakoid proteins). Antiserum against the 20-kDa subunit of LHCI was prepared and used to determine the membrane topology, subcellular site of synthesis, and cell-cycle regulation of this polypeptide. The results indicate that the 20-kDa subunit as well as the other major LHCI polypeptides are integral membrane proteins. Moreover, protease digestion experiments reveal that the 20-kDa polypeptide ismore » completely protected by the membrane bilayer but the 27- and 26-kDa LHCI polypeptides are exposed at the membrane surface. In vivo synthesis of the 20-kDa polypeptide is sensitive to cycloheximide but not to chloramphenicol; the form of the polypeptide recovered from in vitro translations of polyadenylated RNA is approximately 24 kDa, 4 kDa larger than the mature polypeptide. It is concluded that this LHCI polypeptide is nuclear encoded and synthesized in the cytoplasm as a higher molecular weight precursor. Synthesis of the 20-kDa polypeptide is restricted to the light period in light-dark synchronized cells. Translatable mRNA for this polypeptide accumulates during the light but levels are dramatically reduced during the dark period. Thus, synthesis of the 20-kDa subunit of LHCI appears to be transcriptionally regulated during the cell cycle.« less
  • The radiolabeled guanidinating reagent 2-S-({sup 14}C)thiuroniumethanesulfonate reacts with the {epsilon}-amino groups of accessible lysyl residues of membrane proteins under relatively mild labeling conditions, yielding labeled homoarginyl residues. Model studies have shown that the resulting homoarginyl residues do act as new cleavage sites for trypsin, but only at a very slow rate of hydrolysis. The reagent has been shown to be impermeable to the intracytoplasmic membranes of Rhodobacter sphaeroides: when cytoplasmic-side-out chromatophores were treated with the reagent, it reacted with all four of the light-harvesting proteins, all of which have one or more lysyl residues on the N-terminal sides of theirmore » hydrophobic regions. However, when periplasmic-side-out vesicles, prepared by cytochrome c affinity chromatography, were treated with the guanidinating reagent, three of the light-harvesting proteins (B850{alpha}, B850{beta}, and B870{beta}) were not labeled. The only light-harvesting protein to be labeled (B870{alpha}) was the only one of the four to have a lysyl residue on the C-terminal side of its hydrophobic region. The results have confirmed the asymmetric orientation of the light-harvesting proteins of R. sphaeroides, with their N-termini on the cytoplasmic side of the intracytoplasmic membrane. In the case of the B870{alpha} subunit, the protein has been shown to be transmembrane with its C-terminus on the periplasmic side of the membrane. The results have also demonstrated the effectiveness of the labeling reagent for future membrane-protein orientation studies.« less
  • Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp 117,more » Glu 221, and Glu 224 were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.« less