NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Summary

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR ¹⁵N R₁ and R₂ relaxation and {¹H}-¹⁵N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

Abstract

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on ¹⁵N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard ¹⁵N relaxation measurements overview a protein's internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR ¹⁵N R₁, R₂, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

Introduction

The function of a protein is determined not only by its three-dimensional structure but also by its structural dynamics, encompassing its internal flexibility and structural transitions between different conformations the protein will adopt. Nuclear magnetic resonance (NMR) spectroscopy can investigate the structural dynamics of proteins in solution^1,2,3. Recent developments in proton-detected solid-state NMR also allow for the characterization of protein dynamics in a less soluble state, such as, e.g., a lipid bilayer membrane^4,

Protocol

1. NMR sample preparation

NOTE: Isotope labeling of the proteins is performed for higher-dimensional NMR and advanced NMR experiments. When protein expression in E. coli and protein purification had been established using rich media (e.g., Luria-Bertani [LB] or 2x yeast extract tryptone medium [2YT]) with a yield of several milligrams per liter, preparing an isotopically labeled NMR sample is usually relatively straightforward.

1. For isotope labeling, use M9.......

Discussion

This protocol described the setup of NMR ¹⁵N relaxation experiments by Lakomek et al.⁶⁹ and Stief et al.⁷⁰. We focused on NMR pulse sequences using a sensitivity-enhanced HSQC detection scheme. The ¹⁵N R₁ and R_1ρ experiments are implemented as described in detail by Stief et al.⁷⁰, and the hetNOE experiment is described by Lakomek et al.⁶⁹.

When setti.......

Materials

Name	Company	Catalog Number	Comments
Bruker 600 MHz AVANCE III HD spectrometer	Bruker	https://www.bruker.com/en/products-and-solutions/mr/nmr/avance-nmr-spectrometer.html	NMR experiments conducted