MAS solid-state NMR studies on the multidrug transporter EmrE

We study the uniformly ¹³C,¹⁵N isotopically enriched Escherichia coli multidrug resistance transporter EmrE using MAS solid-state NMR. Solid-state NMR can provide complementary structural information as the method allows studying membrane proteins in their native environment as no detergent is required for reconstitution. We compare the spectra obtained from wildtype EmrE to those obtained from the mutant EmrE-E14C. To resolve the critical amino acid E14, glutamic/aspartic acid selective experiments are carried out. These experiments allow to assign the chemical shift of the carboxylic carbon of E14. In addition, spectra are analyzed which are obtained in the presence and absence of the ligand TPP+.     

NMR structure of the integral membrane protein OmpX

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(delta1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel beta-barrel, with successive pairs of beta-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded beta-sheet beyond the height of the barrel. This protruding beta-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.     

Towards high-resolution solid-state NMR on large uniformly 15N- and [13C,15N]-labeled membrane proteins in oriented lipid bilayers

Based on exact numerical simulations, taking into account isotropic and conformation-dependent anisotropic nuclear spin interactions, we systematically analyse the prospects for high-resolution solid-state NMR on large isotope-labeled membrane proteins macroscopically oriented in phospholipid bilayers. Using the known X-ray structures of rhodopsin and porin as models for large membrane proteins with typical -helical and -barrel structural motifs, the analysis considers all possible one- to six-dimensional spectra comprised of frequency dimensions with evolution under any combination of amide ¹H, amide ¹⁵N, and carbonyl ¹³C chemical shifts as well as ¹H-¹⁵N dipole-dipole couplings. Under consideration of typical nuclear spin interaction and experimental line-shape parameters, the analysis provides new insight into the resolution capability and orientation-dependent transfer efficiency of existing experiments as well as guidelines as to improved experimental approaches for the study of large uniformly ¹⁵N- and [¹³C,¹⁵N]-labeled membrane proteins. On basis of these results and numerical optimizations of coherence-transfer efficiencies, we propose several new high-resolution experiments for sequential protein backbone assignment and structure determination.     

Selective and extensive 13C labeling of a membrane protein for solid-state NMR investigations

The selective and extensive 13C labeling of mostly hydrophobic amino acid residues in a 25 kDa membrane protein, the colicin Ia channel domain, is reported. The novel 13C labeling approach takes advantage of the amino acid biosynthetic pathways in bacteria and suppresses the synthesis of the amino acid products of the citric acid cycle. The selectivity and extensiveness of labeling significantly simplify the solid-state NMR spectra, reduce line broadening, and should permit the simultaneous measurement of multiple structural constraints. We show the assignment of most 13C resonances to specific amino acid types based on the characteristic chemical shifts, the 13C labeling pattern, and the amino acid composition of the protein. The assignment is partly confirmed by a 2D homonuclear double-quantum-filter experiment under magic-angle spinning. The high sensitivity and spectral resolution attained with this 13C-labeling protocol, which is termed TEASE for ten-amino acid selective and extensive labeling, are demonstrated.     

NMR structures of polytopic integral membrane proteins

Abstract

Membrane proteins represent up to 30% of the proteins in all organisms, they are involved in many biological processes and are the molecular targets for around 50% of validated drugs. Despite this, membrane proteins represent less than 1% of all high-resolution protein structures due to various challenges associated with applying the main biophysical techniques used for protein structure determination. Recent years have seen an explosion in the number of high-resolution structures of membrane proteins determined by NMR spectroscopy, especially for those with multiple transmembrane-spanning segments. This is a review of the structures of polytopic integral membrane proteins determined by NMR spectroscopy up to the end of the year 2010, which includes both β-barrel and α-helical proteins from a number of different organisms and with a range in types of function. It also considers the challenges associated with performing structural studies by NMR spectroscopy on membrane proteins and how some of these have been overcome, along with its exciting potential for contributing new knowledge about the molecular mechanisms of membrane proteins, their roles in human disease, and for assisting drug design.     

Resonance assignment of 13C/15N labeled solid proteins by two- and three-dimensional magic-angle-spinning NMR

The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of ¹³C and ¹⁵ N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to ¹³C and ¹⁵N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the ¹³C and ¹⁵N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields N_i-C_i–1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a ¹³C spin diffusion experiment, which exhibits ¹³C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive ¹³ C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive ¹³C labeling approach.     

Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments

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Structural constraints for the Crh protein from solid-state NMR experiments

We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Automated protein structure calculation from NMR data

Current software is almost at the stage to permit completely automatic structure determination of small proteins of <15 kDa, from NMR spectra to structure validation with minimal user interaction. This goal is welcome, as it makes structure calculation more objective and therefore more easily validated, without any loss in the quality of the structures generated. Moreover, it releases expert spectroscopists to carry out research that cannot be automated. It should not take much further effort to extend automation to ca 20 kDa. However, there are technological barriers to further automation, of which the biggest are identified as: routines for peak picking; adoption and sharing of a common framework for structure calculation, including the assembly of an automated and trusted package for structure validation; and sample preparation, particularly for larger proteins. These barriers should be the main target for development of methodology for protein structure determination, particularly by structural genomics consortia.

Estimation of protein secondary structure content directly from NMR spectra using an improved empirical correlation with averaged chemical shift

We have recently shown that the averaged chemical shift (ACS) of a nucleus in the protein backbone correlates well empirically to its secondary structure content (SSC). This allows the estimation of SSC directly from the NMR spectrum without the time intensive process of chemical shift assignment. Here, we present an empirical correlation that accounts both for contributions to the relevant protein and chemical shift databases made subsequent to the original analysis, and for missing or inconsistently referenced resonances. Our results affirm that this method provides a significant tool for initial structural prediction from NMR data prior to complete chemical shift assignment.     

Solution NMR structure determination of proteins revisited

This 'Perspective' bears on the present state of protein structure determination by NMR in solution. The focus is on a comparison of the infrastructure available for NMR structure determination when compared to protein crystal structure determination by X-ray diffraction. The main conclusion emerges that the unique potential of NMR to generate high resolution data also on dynamics, interactions and conformational equilibria has contributed to a lack of standard procedures for structure determination which would be readily amenable to improved efficiency by automation. To spark renewed discussion on the topic of NMR structure determination of proteins, procedural steps with high potential for improvement are identified.     

Spectral editing of two-dimensional magic-angle-spinning solid-state NMR spectra for protein resonance assignment and structure determination

Several techniques for spectral editing of 2D ¹³C?C¹³C correlation NMR of proteins are introduced. They greatly reduce the spectral overlap for five common amino acid types, thus simplifying spectral assignment and conformational analysis. The carboxyl (COO) signals of glutamate and aspartate are selected by suppressing the overlapping amide N?CCO peaks through ¹³C?C¹⁵N dipolar dephasing. The sidechain methine (CH) signals of valine, lecuine, and isoleucine are separated from the overlapping methylene (CH₂) signals of long-chain amino acids using a multiple-quantum dipolar transfer technique. Both the COO and CH selection methods take advantage of improved dipolar dephasing by asymmetric rotational-echo double resonance (REDOR), where every other ??-pulse is shifted from the center of a rotor period t_r by about 0.15 t_r. This asymmetry produces a deeper minimum in the REDOR dephasing curve and enables complete suppression of the undesired signals of immobile segments. Residual signals of mobile sidechains are positively identified by dynamics editing using recoupled ¹³C?C¹H dipolar dephasing. In all three experiments, the signals of carbons within a three-bond distance from the selected carbons are detected in the second spectral dimension via ¹³C spin exchange. The efficiencies of these spectral editing techniques range from 60?% for the COO and dynamic selection experiments to 25?% for the CH selection experiment, and are demonstrated on well-characterized model proteins GB1 and ubiquitin.     

Three-dimensional solid-state NMR spectroscopy of a peptide oriented in membrane bilayers

Summary A three-dimensional ¹H chemical shift/¹H-¹⁵N dipolar coupling/¹⁵N chemical shift correlation spectrum was obtained on a sample of specifically ¹⁵N-labeled magainin peptides oriented in lipid bilayers between glass plates in a flat-coil probe. The spectrum showed complete resolution of the resonances from two labeled amide sites in all three dimensions. The three orientationally dependent frequencies associated with each resonance enabled the orientation of the peptide planes to be determined relative to the direction of the applied magnetic field. These results demonstrate the feasibility of multiple-pulse spectroscopy in a flat-coil probe, the ability to measure three spectral parameters from each site in a single experiment, and the potential for resolving among many labeled sites in oriented membrane proteins.     

Protein dynamics detected in a membrane-embedded potassium channel using two-dimensional solid-state NMR spectroscopy

We report longitudinal ¹⁵N relaxation rates derived from two-dimensional (¹⁵N, ¹³C) chemical shift correlation experiments obtained under magic angle spinning for the potassium channel KcsA-Kv1.3 reconstituted in multilamellar vesicles. Thus, we demonstrate that solid-state NMR can be used to probe residue-specific backbone dynamics in a membrane-embedded protein. Enhanced backbone mobility was detected for two glycine residues within the selectivity filter that are highly conserved in potassium channels and that are of core relevance to the filter structure and ion selectivity.     

Structural descriptions of ligands in their binding site of integral membrane proteins at near physiological conditions using solid-state NMR

Using solid-state NMR approaches, it is now possible to define the structure and dynamics of binding for a small, isotopically (²H, ¹³C, ¹⁹F, ¹⁵N) labelled ligand, prosthetic group or solute in its binding site of a membrane-bound protein at near physiological conditions in natural membrane fragments or in reconstituted complexes. Studies of oriented membranes permit the orientational bond vectors of labelled groups to be determined to good precision, as shown for retinal in bacteriorhodopsin and bovine rhodopsin. Using novel magic angle spinning NMR methods on membrane dispersions, high-resolution NMR spectra can be obtained. Dipolar couplings can be reintroduced into the spectrum of labelled ligands in their binding sites of membrane-bound proteins to give interatomic distances to high precision (±0.5 Å). Relaxation and cross-polarization data give estimates for the kinetics for on-off rates for binding. In addition, chemical shifts can be measured directly to help provide details of the binding environment for a bound ligand, as shown for analogues of drugs used in peptic ulcer treatment in the gastric ATPase, and for acetylcholine in the acetylcholine receptor.     

Probing membrane protein unfolding with pulse proteolysis

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and, in many cases, may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose Halobacterium halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A₅₆₀). In this work, we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (C_m) shows excellent agreement with that determined by A₅₆₀. The C_m values determined by pulse proteolysis for M56A and Y57A bRs are also consistent with the measurements made by A₅₆₀. Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with Western blotting may allow the investigation of membrane protein unfolding in situ without overexpression or purification.     

Oligomeric structure, dynamics, and orientation of membrane proteins from solid-state NMR

Solid-state NMR is a versatile and powerful tool for determining the dynamic structure of membrane proteins at atomic resolution. I review the recent progress in determining the orientation, the internal and global protein dynamics, the oligomeric structure, and the ligand-bound structure of membrane proteins with both alpha-helical and beta sheet conformations. Examples are given that illustrate the insights into protein function that can be gained from the NMR structural information.     

Principal components analysis of protein structure ensembles calculated using NMR data

One important problem when calculating structures of biomolecules from NMR data is distinguishing converged structures from outlier structures. This paper describes how Principal Components Analysis (PCA) has the potential to classify calculated structures automatically, according to correlated structural variation across the population. PCA analysis has the additional advantage that it highlights regions of proteins which are varying across the population. To apply PCA, protein structures have to be reduced in complexity and this paper describes two different representations of protein structures which achieve this. The calculated structures of a 28 amino acid peptide are used to demonstrate the methods. The two different representations of protein structure are shown to give equivalent results, and correct results are obtained even though the ensemble of structures used as an example contains two different protein conformations. The PCA analysis also correctly identifies the structural differences between the two conformations.