Analysis of protein–nucleic acid interactions by photochemical cross‐linking and mass spectrometry

Steen, Hanno; Jensen, Ole N.

doi:10.1002/mas.10024

Cited by 62 publications

(61 citation statements)

References 81 publications

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“…This result is in agreement with recently published structural data [29,30]. The exact location of the crosslink within the tryptic peptide was not further investigated, although the feasibility of doing this has been demonstrated with ESI MS [36,37].…”

Section: Discussionsupporting

confidence: 88%

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Kersten

Possling

Blaesing

et al. 2004

Analytical Biochemistry

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To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interactions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein-DNA interactions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate binding site, the DnaA box. Interactions of DnaA domain 4 with a high-aYnity DnaA box (R4) and with a low-aYnity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433-442 of Escherichia coli DnaA. This fragment contains six residues that were identiWed as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein-DNA interactions.  2004 Elsevier Inc. All rights reserved.

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Section: Discussionsupporting

confidence: 88%

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Kersten

Possling

Blaesing

et al. 2004

Analytical Biochemistry

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“…To further examine 1bD21 contact with the nascent RNA, we used positive-ion-mode mass spectrometry in an attempt to identify peptides that are not in contact with RNA (Steen and Jensen 2002). The negative charges of the RNA should bias against the RNA-contacting peptides once they are covalently linked with formaldehyde.…”

Section: Resultsmentioning

confidence: 99%

Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase

Vaughan

Fan²,

You³

et al. 2012

RNA

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Understanding how the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) interacts with nascent RNA would provide valuable insight into the virus's mechanism for RNA synthesis. Using a peptide mass fingerprinting method and affinity capture of peptides reversibly cross-linked to an alkyn-labeled nascent RNA, we identified a region below the D1 loop in the fingers domain of the HCV RdRp that contacts the nascent RNA. A modification protection assay was used to confirm the assignment. Several mutations within the putative nascent RNA binding region were generated and analyzed for RNA synthesis in vitro and in the HCV subgenomic replicon. All mutations tested within this region showed a decrease in primer-dependent RNA synthesis and decreased stabilization of the ternary complex. The results from this study advance our understanding of the structure and function of the HCV RdRp and the requirements for HCV RNA synthesis. In addition, a model of nascent RNA interaction is compared with results from structural studies.

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“…The advantage of using substituted photoactivating analogs is that activation can be achieved at wavelengths above 300 nm, thereby avoiding many problems associated with UV-induced protein degradation [9]. Golden and colleagues demonstrated the utility of using a substituted DNA in conjunction with MS to characterize protein-DNA interfaces [12].…”

Section: Protein-dna Interactionmentioning

confidence: 99%

“…Developing drugs to treat disease states involving these proteins necessitates a thorough understanding of how they interact with nucleic acids in the cell. NMR spectroscopy and X-ray diffraction are two of the most common traditional analytical methodologies for studying protein-DNA interactions [7,8], but recently MS has emerged as a promising new technique in this field [9]. Inducing covalent linkage between proteins and DNA through photo-activation of intrinsic photosensitive functional groups following complex formation has the advantage of stabilizing the often weak noncovalent interactions involved without greatly perturbing the binding interface.…”

Section: Protein-dna Interactionmentioning

confidence: 99%

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

Robinette¹,

Neamati²,

Tomer³

et al. 2006

Expert Review of Proteomics

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Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.Expert Rev. Proteomics 3(4), 399-408 (2006) In photoaffinity labeling (PAL), a ligand possessing a UV-photoactivating group is incubated with its receptor to form, first, a noncovalently bound ligand-receptor complex in which the ligand binds specifically to the binding site or pocket in the receptor due to its affinity. Some ligands, such as oligonucleotides, contain intrinsic photoactivating groups; alternatively, these groups can be introduced by synthesis into ligands for peptides and small molecules, such as drugs. It is important to establish that the incorporated photoactivating groups do not significantly alter the binding affinity of the ligand to its receptor and its functionality, compared with the nonderivatized ligand. Subsequent to the complex formation, UV irradiation triggers the photoactivating group in the ligand and leads to the conversion of the noncovalent binding into a covalent link between the ligand and its receptor at the binding site. A covalent ligand-receptor complex makes the study of the complex easier, since it permits the use of more rigorous but harsher analysis tools, such as SDS-PAGE, HPLC and mass spectrometry (MS), which would typically lead to the dissociation of the complex with information regarding the binding sites no longer being obtainable.Photoaffinity labeling with radioactively labeled ligands is a widely used method to determine the competitive binding affinities of other molecules for the binding sites in the binding pocket occupied by the radioactively labeled ligand and, as such, is a valuable tool for drug screening. When photoaffinity techniques are combined with modern MS, this approach can provide additional structural and mechanistic information regarding the protein complex, such as the ligand-receptor stoichiometry, a map of the binding pocket, and even the exact identification of the amino acid residues, which are involved in the ligand-receptor interaction. The data obtained can be used to create a 3D model of the ligand-binding pocket that might provide better insight into the nature of the ligand-receptor interaction. This knowledge can be v...

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Analysis of protein–nucleic acid interactions by photochemical cross‐linking and mass spectrometry

Cited by 62 publications

References 81 publications

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Identification and functional characterization of the nascent RNA contacting residues of the hepatitis C virus RNA-dependent RNA polymerase

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics

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