Protein in-cell NMR spectroscopy at 1.2\xa0GHz

Enrico Luchinat,Matteo Cremonini,Letizia Barbieri,Lucia Banci

doi:10.1007/s10858-021-00358-w

Abstract

In-cell NMR spectroscopy provides precious structural and functional information on biological macromolecules in their native cellular environment at atomic resolution. However, the intrinsic low sensitivity of NMR imposes a big limitation in the applicability of the methodology. In this respect, the recently developed commercial 1.2 GHz NMR spectrometer is expected to introduce significant benefits. However, cell samples may suffer from detrimental effects at ultrahigh fields, that must be carefully evaluated. Here we show the first in-cell NMR spectra recorded at 1.2 GHz on human cells, and we compare resolution and sensitivity against those obtained at 900 and 950 MHz. To evaluate the effects of different spin relaxation rates, SOFAST-HMQC and BEST-TROSY spectra were recorded on intracellular α-synuclein and carbonic anhydrase. Major improvements are observed at 1.2 GHz when analyzing unfolded proteins, such as α-synuclein, while the TROSY scheme improves the resolution for both globular and unfolded proteins.

Highlights

In-cell NMR is an application of biomolecular NMR spectroscopy to the characterization of the structure and dynamics of biological macromolecules inside living cells (Luchinat and Banci 2017; Siegal and Selenko 2019; Ito et al 2020)
Such limitation derives from the intrinsic insensitivity of NMR spectroscopy, due to the small energy difference between the nuclear spin states compared to the thermal fluctuations at physiological temperatures, and is further exacerbated by the limited number of molecules of interest in the sample—with volume-averaged concentrations usually in the order of 5–500 μM—and by the short lifetime of the cells in the instrument
The acquisition parameters were optimized to account for the higher resolution of lysate NMR compared to in-cell NMR (Table S1)

Summary

Introduction

In-cell NMR is an application of biomolecular NMR spectroscopy to the characterization of the structure and dynamics of biological macromolecules inside living cells (Luchinat and Banci 2017; Siegal and Selenko 2019; Ito et al 2020). Several examples of in-cell solution NMR both in Despite its huge potential, a major drawback of in-cell NMR compared to other cellular techniques is the low sensitivity Such limitation derives from the intrinsic insensitivity of NMR spectroscopy, due to the small energy difference between the nuclear spin states compared to the thermal fluctuations at physiological temperatures, and is further exacerbated by the limited number of molecules of interest in the sample—with volume-averaged concentrations usually in the order of 5–500 μM—and by the short lifetime of the cells in the instrument. On the other hand, increasing the static magnetic field strength (B0) increases proportionally the energy splitting and the spin population difference, and increases the signalto-noise ratio as a function of B03/2, positively affecting both the resolution and the sensitivity of the technique

Methods

Results

Conclusion