MAS NMR detection of hydrogen bonds for protein secondary structure characterization

Daniel Friedrich,Jacqueline Perodeau,Andrew J Nieuwkoop,Hartmut Oschkinat

doi:10.1007/s10858-020-00307-z

Abstract

Hydrogen bonds are essential for protein structure and function, making experimental access to long-range interactions between amide protons and heteroatoms invaluable. Here we show that measuring distance restraints involving backbone hydrogen atoms and carbonyl- or α-carbons enables the identification of secondary structure elements based on hydrogen bonds, provides long-range contacts and validates spectral assignments. To this end, we apply specifically tailored, proton-detected 3D (H)NCOH and (H)NCAH experiments under fast magic angle spinning (MAS) conditions to microcrystalline samples of SH3 and GB1. We observe through-space, semi-quantitative correlations between protein backbone carbon atoms and multiple amide protons, enabling us to determine hydrogen bonding patterns and thus to identify β-sheet topologies and α-helices in proteins. Our approach shows the value of fast MAS and suggests new routes in probing both secondary structure and the role of functionally-relevant protons in all targets of solid-state MAS NMR.

Highlights

One of the primary driving forces behind protein folding is the formation of hydrogen bonds characteristic for secondary structure (Dobson 2003; Jeffrey 1997; Pace et al 2014)
If protons involved in hydrogen bonds may be back-exchanged likewise, the situation seems ideal for determining which of these bonds are present in protein structure
To demonstrate the idea of cross polarization (CP)-based detection of long-range contacts between multiple amide protons and carbonyls, we acquired a proton-detected 2D (H)COH experiment (Supplementary Figure 2) at fast Magic angle spinning (MAS) on a 2H, 13C, 15N-labeled microcrystalline sample of the SH3 domain, back-exchanged in 70% 1H2O/30% 2H2O (Fig. 1a). This experiment consists of two 1H–13C CP transfers, both of which were set to a longer contact time (4 ms) than is traditionally used

Summary

Introduction

One of the primary driving forces behind protein folding is the formation of hydrogen bonds characteristic for secondary structure (Dobson 2003; Jeffrey 1997; Pace et al 2014). If protons involved in hydrogen bonds may be back-exchanged likewise, the situation seems ideal for determining which of these bonds are present in protein structure. This minimal bath of protons has additional benefits useful for acquiring structural restraints, in particular between amide protons and backbone carbons by utilizing cross polarization (CP), which we have designed an experiment to take advantage of. It has been shown earlier that protons in hydrogen bonds and hydroxyl protons can be detected by solid-state MAS NMR using CP (Agarwal et al 2010, 2013; Friedrich et al 2020)

Methods

Results

Conclusion