We present a strategy for rapidly gaining structural information about a protein from crosslinks formed by genetically encoded unnatural amino acids. We applied it to ISWI, a chromatin remodeling enzyme involved in chromatin assembly, DNA replication and transcription. ISWI is part of the vast Snf2 family of helicase-related proteins, many of which constitute the catalytic cores of chromatin remodeling complexes. Structural information about this family is scarce, hampering our mechanistic understanding of chromatin remodeling. Making use of cells that harbor a special tRNA/aminoacyl-tRNA synthetase pair, several residues within the ATPase domain of ISWI were individually substituted with the UV-reactive unnatural amino acid p-benzoyl-p-phenylalanine. Intramolecular crosslinks could be mapped with amino acid precision by high resolution tandem mass spectrometry and the novel bioinformatic tool "Crossfinder." Most crosslinks were fully consistent with published crystal structures of ISWI-related ATPases. A subset of crosslinks, however, disagreed with the conformations previously captured in crystal structures. We built a structural model using the distance information obtained from the crosslinks and the structure of the closest crystallized relative, Chd1. The model shows the ATPase lobes strongly rotated against each other, a movement postulated earlier to be necessary to achieve a catalytically competent state. The minimal requirements for solubility and protein amounts make our approach ideal for studying structures and conformations of proteins that are not amenable to conventional structural techniques.
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http://dx.doi.org/10.1074/mcp.M111.012088 | DOI Listing |
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Center for Computational Molecular Biology, Brown University, Providence, RI, USA.
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Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea.
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View Article and Find Full Text PDFPLoS One
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Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, 443002, China.
Long non-coding RNA (lncRNA) is a type of non-coding RNA distinguished by a length exceeding 200 nucleotides. Recent studies indicated that lncRNAs participate in various biological processes, such as chromatin remodeling, transcriptional and post-transcriptional regulation, and the modulation of cell proliferation, death, and differentiation, hence influencing gene expression and cellular function. ADAMTS9-AS1, an antisense long non-coding RNA situated on human chromosome 3p14.
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