A Method to Selectively Determine Concentrations of Macromolecular Complexes: Overhauser-DNP Spectroscopy

28 August 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Dynamic nuclear polarization (DNP) is a process achieved by transferring the spin polarization from an unpaired electron to the half-spin nuclei in a substance of interest through microwave irradiation, effectively enhancing the nuclear magnetic resonance (NMR) signals of the substance. The polarization mechanism involved in Overhauser-DNP allows the process to be carried out in ambient conditions, thus facilitating its technological implementation. Here we show a method to quantify protein complexes in solution by exploiting the correlation among the concentration, the enhancement of the solvent NMR signals, the unpaired electrons’ motions, and the microwave irradiation frequency. As a proof of concept, we quantify the proteins avidin and C-reactive protein by means of adequate paramagnetic molecular probes (i.e., ligands), and we show that the method allows to estimate the dissociation constant for the C-reactive protein complex. Furthermore, we provide a full structural characterization of the avidin complex by X-ray crystallography (XRD) and cryogenic electron microscopy (cryo-EM).


Overhauser-DNP Spectroscopy
Dynamic Nuclear Polarization
Macromolecular Complex
Protein Complex
Stable Radical
C-Reactive Protein

Supplementary materials

Supporting Information
Theoretical considerations Biotin-TEMPO and Avidin-biotin-TEMPO complex (ODNP, EPR, XRD, Cryo-EM) Determination of KD for CRP-phosphonooxy-TEMPO Mass spectrum for Biotin-TEMPO


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