skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Hydroxyl Radical Footprinting in vivo: Mapping Macromolecular Structures with Synchrotron Radiation

Authors:
; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
980246
Report Number(s):
BNL-93164-2010-JA
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nucleic Acids Research; Journal Volume: 34; Journal Issue: 8
Country of Publication:
United States
Language:
English
Subject:
national synchrotron light source

Citation Formats

Adilakshmi, T., Lease, R, and Woodson, S. Hydroxyl Radical Footprinting in vivo: Mapping Macromolecular Structures with Synchrotron Radiation. United States: N. p., 2006. Web. doi:10.1093/nar/gkl291.
Adilakshmi, T., Lease, R, & Woodson, S. Hydroxyl Radical Footprinting in vivo: Mapping Macromolecular Structures with Synchrotron Radiation. United States. doi:10.1093/nar/gkl291.
Adilakshmi, T., Lease, R, and Woodson, S. Sun . "Hydroxyl Radical Footprinting in vivo: Mapping Macromolecular Structures with Synchrotron Radiation". United States. doi:10.1093/nar/gkl291.
@article{osti_980246,
title = {Hydroxyl Radical Footprinting in vivo: Mapping Macromolecular Structures with Synchrotron Radiation},
author = {Adilakshmi, T. and Lease, R and Woodson, S},
abstractNote = {},
doi = {10.1093/nar/gkl291},
journal = {Nucleic Acids Research},
number = 8,
volume = 34,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • 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
  • Cited by 6
  • 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
  • Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein-protein and protein-DNA interactions. Using synchrotron radiolysis, exposure of proteins to a 'white' X-ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time-resolved approach, which would be amore » valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium-dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time-resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)-based method can be utilized for quantification of oxidized species, improving the signal-to-noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis-driven structural mass spectrometry experiments.« less