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Title: Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting

Authors:
; ; ; ; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1354498
Report Number(s):
BNL-113015-2016-JA
Journal ID: ISSN 1046-2023
DOE Contract Number:
SC00112704
Resource Type:
Journal Article
Resource Relation:
Journal Name: Methods; Journal Volume: 103
Country of Publication:
United States
Language:
English

Citation Formats

Hulscher, Ryan M., Bohon, Jen, Rappé, Mollie C., Gupta, Sayan, D’Mello, Rhijuta, Sullivan, Michael, Ralston, Corie Y., Chance, Mark R., and Woodson, Sarah A. Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting. United States: N. p., 2016. Web. doi:10.1016/j.ymeth.2016.03.012.
Hulscher, Ryan M., Bohon, Jen, Rappé, Mollie C., Gupta, Sayan, D’Mello, Rhijuta, Sullivan, Michael, Ralston, Corie Y., Chance, Mark R., & Woodson, Sarah A. Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting. United States. doi:10.1016/j.ymeth.2016.03.012.
Hulscher, Ryan M., Bohon, Jen, Rappé, Mollie C., Gupta, Sayan, D’Mello, Rhijuta, Sullivan, Michael, Ralston, Corie Y., Chance, Mark R., and Woodson, Sarah A. Fri . "Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting". United States. doi:10.1016/j.ymeth.2016.03.012.
@article{osti_1354498,
title = {Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting},
author = {Hulscher, Ryan M. and Bohon, Jen and Rappé, Mollie C. and Gupta, Sayan and D’Mello, Rhijuta and Sullivan, Michael and Ralston, Corie Y. and Chance, Mark R. and Woodson, Sarah A.},
abstractNote = {},
doi = {10.1016/j.ymeth.2016.03.012},
journal = {Methods},
number = ,
volume = 103,
place = {United States},
year = {Fri Jul 01 00:00:00 EDT 2016},
month = {Fri Jul 01 00:00:00 EDT 2016}
}
  • Cited by 6
  • No abstract prepared.
  • The structural models of the fibrils formed by the 40-residue amyloid-β (Aβ40) peptide in Alzheimer’s disease typically consist of linear polypeptide segments, oriented approximately perpendicular to the long axis of the fibril, and joined together as parallel in-register β-sheets to form filaments. However, various models differ in the number of filaments that run the length of a fibril, and in the topological arrangement of these filaments. In addition to questions about the structure of Aβ40 monomers in fibrils, there are important unanswered questions about their structure in prefibrillar intermediates, which are of interest because they may represent the most neurotoxicmore » form of Aβ40. To assess different models of fibril structure and to gain insight into the structure of prefibrillar intermediates, the relative solvent accessibility of amino acid residue side chains in fibrillar and prefibrillar Aβ40 preparations was characterized in solution by hydroxyl radical footprinting and structural mass spectrometry. A key to the application of this technology was the development of hydroxyl radical reactivity measures for individual side chains of Aβ40. When we combined mass-per-length measurements performed by dark-field electron microscopy, we determined that the results of our study were consistent with the core filament structure represented by two- and three-filament solid state nuclear magnetic resonance-based models of the Aβ40 fibril (such as 2LMN, 2LMO, 2LMP, and 2LMQ), with minor refinements, but they are inconsistent with the more recently proposed 2M4J model. Our results also demonstrate that individual Aβ40 fibrils exhibit structural heterogeneity or polymorphism, where regions of two-filament structure alternate with regions of three-filament structure. The footprinting approach utilized in this study will be valuable for characterizing various fibrillar and nonfibrillar forms of the Aβ peptide.« less
  • We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotronmore » X-ray beam can footprint the tertiary structure and protein contacts of RNA-protein complexes with nucleotide resolution in living cells.« less