Nucleic acid–protein interfaces studied by MAS solid-state NMR spectroscopy

Efficient polarizing agents for dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) NMR spectroscopy are of high current interest due to the potential to significantly boost NMR sensitivity. While most efforts have centered on cross-effect (CE) or Overhauser effect (OE) mechanisms, yielding enhancement factors up to ∼300 at 9.4 T, radicals yielding solid effect (SE) DNP have seen less development. Here we model the comparative performance of OE and SE mechanisms and then measure 1 H enhancement factors up to 500 from 1,3-bisdiphenylene-2-phenylallyl (BDPA) in an ortho-terphenyl (OTP) matrix at 9.4 T, 100 K, achieved via increased microwave power across the sample volume. The measured SE and OE performances are in good agreement with the predictions. We note that both the experimental and theoretical analyses indicate that SE DNP remains saturation limited, particularly at elevated temperatures, and we envisage that further improvements in microwave power will further increase SE DNP enhancement factors.

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Investigating Protein-Nucleic Acid Binding Interactions with Diethylpyrocarbonate Covalent Labeling–Mass Spectrometry

Kirsch,

Ashby,

Vachet

2024

J. Am. Soc. Mass Spectrom.

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Nucleic acids are important biomolecules that facilitate numerous cellular functions and have in recent years become promising candidates for treating disease. Consequently, there is a need for methods to characterize protein interactions with these molecules. Here, we demonstrate that diethylpyrocarbonate (DEPC) covalent labeling−mass spectrometry (CL-MS) can provide structural information for protein-nucleic acid binding by characterizing the binding sites of two DNA aptamers specific to thrombin. Reductions in thrombin labeling are observed at the pair's binding interfaces. Furthermore, we find that binding of the aptamers causes changes in labeling at residues in the thrombin active site and known exosites for each aptamer, showcasing the sensitivity of DEPC CL-MS to significant allosteric changes.

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Nucleic acid–protein interfaces studied by MAS solid-state NMR spectroscopy

Cited by 3 publications

References 169 publications

Structural studies of protein–nucleic acid complexes: A brief overview of the selected techniques

Structural studies of protein–nucleic acid complexes: A brief overview of the selected techniques

Solid Effect Dynamic Nuclear Polarization Enhancement of >500 at 9.4 T

Investigating Protein-Nucleic Acid Binding Interactions with Diethylpyrocarbonate Covalent Labeling–Mass Spectrometry

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