title: Elucidation of tRNA-cytochrome c interactions through hydrogen/deuterium exchange mass spectrometry.
abstract: Cytochrome c (cyt c) is a mitochondrial protein responsible for transferring electrons between electron transport chain complexes III and IV. The release of cyt c from the mitochondria has been considered as a commitment step in intrinsic apoptosis. Transfer RNA (tRNA) has recently been found to interact with the released cyt c to prevent the formation of the apoptosome complex, thus preventing cell apoptosis. To understand the molecular basis of tRNA-cyt c interactions, we applied hydrogen/deuterium exchange mass spectrometry (HDXMS) to analyze the interactions between tRNA and cyt c. tRNAPhebinding to cyt c reduced the deuteration level of cyt c in all analyzed regions, indicating that tRNA binding blocks the solvent-accessible regions and results in the formation of a more compact conformation. Substitution of the tRNAPhewith the total tRNA from brewer's yeast in the HDXMS experiment significantly reduced deuteration in the N-terminus and the region 18-32 residue of cyt c, where all tRNAs are bound. To clarify the cause of binding, we used synthesized single-stranded oligonucleotides of 12-mer dA and dT to form complexes with cyt c. The exchange of the nucleotide bases between adenine and thymine did not affect the deuteration level of cyt c. However, the regions 1-10 and 65-82 showed minor decreases after unstructured dA or dT DNA binding. Collectively, these results reveal that cyt c maintains its globular structure to interact with tRNA. The region 18-32 selectively interacts with tRNA, and N-terminal 1-10 interacts with oligonucleotides electrostatically.
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Lo
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Hung-Wei
authors.initials: HW
authors.lastname: Huang
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Yi-Chuan
authors.initials: YC
authors.lastname: Huang
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Jui-Fen
authors.initials: JF
authors.lastname: Chan
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan; Biological Science Center, Tunghai University, Taichung, Taiwan. Electronic address: howardhsu@thu.edu.tw.
authors.forename: Yuan-Hao Howard
authors.initials: YH
authors.lastname: Hsu
mesh_terms: D017209:Apoptosis / Q000235:genetics; D053478:Apoptosomes / Q000737:chemistry / Q000235:genetics; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics; D013058:Mass Spectrometry; D008928:Mitochondria / Q000737:chemistry* / Q000235:genetics; D009711:Nucleotides / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000737:chemistry / Q000235:genetics
chemical_list: D053478:Apoptosomes; D009711:Nucleotides; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; D045304:Cytochromes c; D012343:RNA, Transfer; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III
keywords: Cardiolipin; Cytochrome c; Hydrogen/deuterium exchange; Mass spectrometry; Transfer RNA (tRNA)