Proton‐Detected Solid‐State NMR Spectroscopy of a Zinc Diffusion Facilitator Protein in Native Nanodiscs

The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane‐mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co‐polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high‐resolution solid‐state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD.     

In‐Cell NMR and EPR Spectroscopy of Biomacromolecules

The dream of cell biologists is to be able to watch biological macromolecules perform their duties in the intracellular environment of live cells. Ideally, the observation of both the location and the conformation of these macromolecules with biophysical techniques is desired. The development of many fluorescence techniques, including superresolution fluorescence microscopy, has significantly enhanced our ability to spot proteins and other molecules in the crowded cellular environment. However, the observation of their structure and conformational changes while they attend their business is still very challenging. In principle, NMR and EPR spectroscopy can be used to investigate the conformation and dynamics of biological macromolecules in living cells. The development of in‐cell magnetic resonance techniques has demonstrated the feasibility of this approach. Herein we review the different techniques with a focus on liquid‐state in‐cell NMR spectroscopy, provide an overview of applications, and discuss the challenges that lie ahead.     

Proton‐Detected Solid‐State NMR Spectroscopy of Natural‐Abundance Peptide and Protein Pharmaceuticals

The natural way : A sensitive NMR spectroscopic method is developed to obtain well‐resolved two‐dimensional spectra (¹⁵N–¹H and ¹³C–¹H) for natural‐abundance (that is, without the need for isotopic enrichment) large‐molecule samples, such as biopharmaceuticals. This method gives structural insights on two lyophilized aprotinin samples and three insulin samples in lyophilized, microcrystalline suspension formulation (red; see picture) and fibril (green) forms.

     

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

¹H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for ¹H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water‐inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high‐resolution ¹H‐detected solid‐state NMR (ssNMR) studies of water‐inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K⁺ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K⁺ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.     

Conformation and Topology of Diacylglycerol Kinase in E.coli Membranes Revealed by Solid‐state NMR Spectroscopy

Solid‐state NMR is a powerful tool for studying membrane proteins in a native‐like lipid environment. 3D magic angle spinning (MAS) NMR was employed to characterize the structure of E.coli diacylglycerol kinase (DAGK) reconstituted into its native E.coli lipid membranes. The secondary structure and topology of DAGK revealed by solid‐state NMR are different from those determined by solution‐state NMR and X‐ray crystallography. This study provides a good example for demonstrating the influence of membrane environments on the structure of membrane proteins.     

Solid‐state NMR Spectroscopy of Quadrupolar Nuclei in Inorganic Chemistry

We here review the principles and applications of solid‐state NMR spectroscopy of quadrupolar nuclei, with a special emphasis on structural studies of inorganic solids. Most NMR‐observable nuclei have spin I > 1/2, and possess a quadrupole moment. The resulting quadrupolar interaction severely broadens the resonances, but also encapsulates valuable information about the symmetry of the electronic surroundings of the observed nucleus. The effect of the quadrupolar interaction, as well as that of the chemical shift and dipolar interaction, on solid‐state NMR spectra is examined in this article. To regain good resolution, specifically designed NMR techniques exist to remove the quadrupolar broadening, i.e. overtone and MQMAS spectroscopy, the principles of which are outlined here. In addition, the possibility of distance measurements via the dipolar interaction using the REDOR technique is discussed. The combined information derived from distance measurements, quadrupolar and chemical shift parameters can be helpful for determination of the crystal structure, or for detection of impurity phases, as illustrated by surveying a number of case studies covering spin I = 1, 3/2, 5/2 and 7/2.     

Functional Dynamics of Deuterated β2‐Adrenergic Receptor in Lipid Bilayers Revealed by NMR Spectroscopy

G‐protein‐coupled receptors (GPCRs) exist in conformational equilibrium between active and inactive states, and the former population determines the efficacy of signaling. However, the conformational equilibrium of GPCRs in lipid bilayers is unknown owing to the low sensitivities of their NMR signals. To increase the signal intensities, a deuteration method was developed for GPCRs expressed in an insect cell/baculovirus expression system. The NMR sensitivities of the methionine methyl resonances from the β₂‐adrenergic receptor (β₂AR) in lipid bilayers of reconstituted high‐density lipoprotein (rHDL) increased by approximately 5‐fold upon deuteration. NMR analyses revealed that the exchange rates for the conformational equilibrium of β₂AR in rHDLs were remarkably different from those measured in detergents. The timescales of GPCR signaling, calculated from the exchange rates, are faster than those of receptor tyrosine kinases and thus enable rapid neurotransmission and sensory perception.     

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.     

Solid‐State NMR Spectroscopy Reveals that E. coli Inclusion Bodies of HET‐s(218–289) are Amyloids

Protein deposition frequently occurs as inclusion bodies (IBs) during heterologous protein expression in E. coli. The structure of these E. coli IBs of the prion‐forming domain from the fungal prion HET‐s is the same as that previously determined for fibrils assembled in vitro, and show prion infectivity. These results demonstrate that the IBs of HET‐s(218–289) are amyloids.

     

Weak and Transient Protein Interactions Determined by Solid‐State NMR

Despite their roles in controlling many cellular processes, weak and transient interactions between large structured macromolecules and disordered protein segments cannot currently be characterized at atomic resolution by X‐ray crystallography or solution NMR. Solid‐state NMR does not suffer from the molecular size limitations affecting solution NMR, and it can be applied to molecules in different aggregation states, including non‐crystalline precipitates and sediments. A solid‐state NMR approach based on high magnetic fields, fast magic‐angle sample spinning, and deuteration provides chemical‐shift and relaxation mapping that enabled the characterization of the structure and dynamics of the transient association between two regions in an 80 kDa protein assembly. This led to direct verification of a mechanism of regulation of E. coli DNA metabolism.     

Intermolecular Interactions and Protein Dynamics by Solid‐State NMR Spectroscopy

Understanding the dynamics of interacting proteins is a crucial step toward describing many biophysical processes. Here we investigate the backbone dynamics for protein GB1 in two different assemblies: crystalline GB1 and the precipitated GB1–antibody complex with a molecular weight of more than 300 kDa. We perform these measurements on samples containing as little as eight nanomoles of GB1. From measurements of site‐specific ¹⁵N relaxation rates including relaxation dispersion we obtain snapshots of dynamics spanning nine orders of magnitude in terms of the time scale. A comparison of measurements for GB1 in either environment reveals that while many of the dynamic features of the protein are conserved between them (in particular for the fast picosecond–nanosecond motions), much greater differences occur for slow motions with motions in the >500 ns range being more prevalent in the complex. The data suggest that GB1 can potentially undergo a small‐amplitude overall anisotropic motion sampling the interaction interface in the complex.     

The G‐Protein‐Coupled Neuropeptide Y Receptor Type 2 is Highly Dynamic in Lipid Membranes as Revealed by Solid‐State NMR Spectroscopy

In spite of the recent success in crystallizing several G‐protein‐coupled receptors (GPCRs), a comprehensive biophysical characterization of these molecules under physiological conditions also requires the study of the molecular dynamics of these proteins. The molecular mobility of the human neuropeptide Y receptor type 2 reconstituted into dimyristoylphosphatidylcholine (DMPC) membranes was investigated by means of solid‐state NMR spectroscopy. Static ¹⁵N NMR spectra show that the receptor performs axially symmetric motions in the membrane, and several residues undergo large amplitude fluctuations. This was confirmed by quantitative measurements of the motional ¹H,¹³C order parameter of the CH, CH₂, and CH₃ groups. In directly polarized ¹³C NMR experiments, these order parameters showed astonishingly low values of S_CH=0.55, S=0.33, and S=0.17, which corresponds to segmental amplitudes of approximately 50° in the backbone and approximately 50–60° in the side chain. At physiological temperature, ²H NMR spectra of the deuterated receptor showed a narrow component that is indicative of molecular order parameters of S≤0.3 superimposed with a very broad spectrum that could stem from the transmembrane α‐helices. These results suggest that the crystal structures of GPCRs only represent a static snapshot of these highly mobile molecules, which undergo significant structural fluctuations with relatively large amplitudes in a liquid‐crystalline membrane at physiological temperature.     

An Efficient Labelling Approach to Harness Backbone and Side‐Chain Protons in 1H‐Detected Solid‐State NMR Spectroscopy

¹H‐detection can greatly improve spectral sensitivity in biological solid‐state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of ¹H‐detection by the loss of aliphatic side‐chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for ¹H‐detected ssNMR, and it gives high quality spectra for both side‐chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent ¹H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR.     

Solid‐State 73Ge NMR Spectroscopy of Simple Organogermanes

Germanium‐73 is an extremely challenging nucleus to examine by NMR spectroscopy due to its unfavorable NMR properties. Through the use of an ultrahigh (21.1 T) magnetic field, a systematic study of a series of simple organogermanes was carried out. In those cases for which X‐ray structural data were available, correlations were drawn between the NMR parameters and structural metrics. These data were combined with DFT calculations to obtain insight into the structures of several compounds with unknown crystal structures.