Deuterated Background Research Articles

Temperature-Dependent Rotation of Protonated Methyl Groups in Otherwise Deuterated Proteins Modulates DEER Distance Distributions

AbstractTemperature-dependent DEER effects are observed as a function of methyl rotation by either leucine- or nitroxide-specific protonated methyl groups in an otherwise deuterated background. Both species induce a site-specific enhancement in the apparent Tm relaxation of the paramagnetic nitroxide label. The presence of a single protonated methyl group in close proximity (4–10 Å) to only one of the two nitroxide rotamer ensembles in AviTagged immunoglobulin-binding B domain of protein A results in a selective and substantial decrease in Tm, manifested by differential decay of the peak intensities in the bimodal P(r) distance distribution as a function of the total dipolar evolution time, temperature, or both. The temperature-dependent differential decay of the individual distance components was globally analyzed by fitting the DEER dipolar time traces to a three-site jump model that is defined by the activation energy of leucine- or nitroxide-specific methyl rotation. Temperature-assisted Tm filtering will capture the DEER structural analysis of biomolecular systems heterogenic conformations, including complexes involving multimeric proteins.

Towards cost-effective side-chain isotope labelling of proteins expressed in human cells.

Side chain isotope labelling is a powerful tool to study protein structure and interactions by NMR spectroscopy. 1H,13C labelling of side-chain methyl groups in a deuterated background allows studying large molecules, while side-chain aromatic groups are highly sensitive to the interaction with ligands, drugs, and other proteins. In E. coli, side chain labelling is performed by substituting amino acids with isotope-labelled precursors. However, proteins that can only be produced in mammalian cells require expensive isotope-labelled amino acids. Here we provide a simple and cost-effective method to label side chains in mammalian cells, which exploits the reversible reaction catalyzed by endogenous transaminases to convert isotope-labelled α-ketoacid precursors. We show by in-cell and in-lysate NMR spectroscopy that replacing an amino acid in the medium with its cognate precursor is sufficient to achieve selective labelling without scrambling, and how this approach allows monitoring conformational changes such as those arising from ligand binding.

Methyl probes in proteins for determining ligand binding mode in weak protein–ligand complexes

Structures of protein–ligand complexes provide critical information for drug design. Most protein–ligand complex structures are determined using X-ray crystallography, but where crystallography is not able to generate a structure for a complex, NMR is often the best alternative. However, the available tools to enable rapid and robust structure determination of protein–ligand complexes by NMR are currently limited. This leads to situations where projects are either discontinued or pursued without structural data, rendering the task more difficult. We previously reported the NMR Molecular Replacement (NMR2) approach that allows the structure of a protein–ligand complex to be determined without requiring the cumbersome task of protein resonance assignment. Herein, we describe the NMR2 approach to determine the binding pose of a small molecule in a weak protein–ligand complex by collecting sparse protein methyl-to-ligand NOEs from a selectively labeled protein sample and an unlabeled ligand. In the selective labeling scheme all methyl containing residues of the protein are protonated in an otherwise deuterated background. This allows measurement of intermolecular NOEs with greater sensitivity using standard NOESY pulse sequences instead of isotope-filtered NMR experiments. This labelling approach is well suited to the NMR2 approach and extends its utility to include larger protein–ligand complexes.

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Improved strategy for isoleucine 1H/13C methyl labeling in Pichia pastoris

Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein 1H/13C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins. The yeast Pichia pastoris (P. pastoris) is a commonly used host for expression of eukaryotic proteins, and site-specific methyl labeling of perdeuterated eukaryotic proteins has recently been achieved with this system. However, the practical utility of methyl labeling and deuteration in P. pastoris is limited by high costs. Here, we describe an improved method for 1H/13C-labeling of the δ-methyl group of isoleucine residues in a perdeuterated background, which reduces the cost by ≥ 50% without compromising the efficiency of isotope enrichment. We have successfully implemented this method to label actin and a G-protein coupled receptor. Our approach will facilitate studies of the structure and dynamics of eukaryotic proteins by NMR spectroscopy.

Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote Pichia pastoris.

(13)C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific (13)C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient (13)C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.

Structure refinement and membrane positioning of selectively labeled OmpX in phospholipid nanodiscs.

NMR structural studies on membrane proteins are often complicated by their large size, taking into account the contribution of the membrane mimetic. Therefore, classical resonance assignment approaches often fail. The large size of phospholipid nanodiscs, a detergent-free phospholipid bilayer mimetic, prevented their use in high-resolution solution-state NMR spectroscopy so far. We recently introduced smaller nanodiscs that are suitable for NMR structure determination. However, side-chain assignments of a membrane protein in nanodiscs still remain elusive. Here, we utilized a NOE-based approach to assign (stereo-) specifically labeled Ile, Leu, Val and Ala methyl labeled and uniformly (15)N-Phe and (15)N-Tyr labeled OmpX and calculated a refined high-resolution structure. In addition, we were able to obtain residual dipolar couplings (RDCs) of OmpX in nanodiscs using Pf1 phage medium for the induction of weak alignment. Back-calculated NOESY spectra of the obtained NMR structures were compared to experimental NOESYs in order to validate the quality of these structures. We further used NOE information between protonated lipid head groups and side-chain methyls to determine the position of OmpX in the phospholipid bilayer. These data were verified by paramagnetic relaxation enhancement (PRE) experiments obtained with Gd(3+)-modified lipids. Taken together, this study emphasizes the need for the (stereo-) specific labeling of membrane proteins in a highly deuterated background for high-resolution structure determination, particularly in large membrane mimicking systems like phospholipid nanodiscs. Structure validation by NOESY back-calculation will be helpful for the structure determination and validation of membrane proteins where NOE assignment is often difficult. The use of protein to lipid NOEs will be beneficial for the positioning of a membrane protein in the lipid bilayer without the need for preparing multiple protein samples.

Differential isotope-labeling for Leu and Val residues in a protein by E. coli cellular expression using stereo-specifically methyl labeled amino acids

The (1)H-(13)C HMQC signals of the (13)CH3 moieties of Ile, Leu, and Val residues, in an otherwise deuterated background, exhibit narrow line-widths, and thus are useful for investigating the structures and dynamics of larger proteins. This approach, named methyl TROSY, is economical as compared to laborious methods using chemically synthesized site- and stereo-specifically isotope-labeled amino acids, such as stereo-array isotope labeling amino acids, since moderately priced, commercially available isotope-labeled α-keto acid precursors can be used to prepare the necessary protein samples. The Ile δ1-methyls can be selectively labeled, using isotope-labeled α-ketobutyrates as precursors. However, it is still difficult to prepare a residue-selectively Leu and Val labeled protein, since these residues share a common biosynthetic intermediate, α-ketoisovalerate. Another hindering drawback in using the α-ketoisovalerate precursor is the lack of stereo-selectivity for Leu and Val methyls. Here we present a differential labeling method for Leu and Val residues, using four kinds of stereo-specifically (13)CH3-labeled [U-(2)H;(15)N]-leucine and -valine, which can be efficiently incorporated into a protein using Escherichia coli cellular expression. The method allows the differential labeling of Leu and Val residues with any combination of stereo-specifically isotope-labeled prochiral methyls. Since relatively small amounts of labeled leucine and valine are required to prepare the NMR samples; i.e., 2 and 10 mg/100 mL of culture for leucine and valine, respectively, with sufficient isotope incorporation efficiency, this approach will be a good alternative to the precursor methods. The feasibility of the method is demonstrated for 82 kDa malate synthase G.

Specific 12CβD212CγD2S13CεHD2 Isotopomer Labeling of Methionine To Characterize Protein Dynamics by 1H and 13C NMR Relaxation Dispersion

Protein dynamics on the micro- to millisecond time scale is increasingly found to be critical for biological function, as demonstrated by numerous NMR relaxation dispersion studies. Methyl groups are excellent probes of protein interactions and dynamics because of their favorable NMR relaxation properties, which lead to sharp signals in the 1H and 13C NMR spectra. Out of the six different methyl-bearing amino acid residue types in proteins, methionine plays a special role because of its extensive side-chain flexibility and the high polarizability of the sulfur atom. Methionine is over-represented in many protein–protein recognition sites, making the methyl group of this residue type an important probe of the relationships among dynamics, interactions, and biological function. Here we present a straightforward method to label methionine residues with specific 13CHD2 methyl isotopomers against a deuterated background. The resulting protein samples yield NMR spectra with improved sensitivity due to the essentially 100% population of the desired 13CHD2 methyl isotopomer, which is ideal for 1H and 13C spin relaxation experiments to investigate protein dynamics in general and conformational exchange in particular. We demonstrate the approach by measuring 1H and 13C CPMG relaxation dispersion for the nine methionines in calcium-free calmodulin (apo-CaM). The results show that the C-terminal domain, but not the N-terminal domain, of apo-CaM undergoes fast exchange between the ground state and a high-energy state. Since target proteins are known to bind specifically to the C-terminal domain of apo-CaM, we speculate that the high-energy state might be involved in target binding through conformational selection.

Methionine Scanning as an NMR Tool for Detecting and Analyzing Biomolecular Interaction Surfaces

Methyl NMR spectroscopy is a powerful tool for studying protein structure, dynamics, and interactions. Yet difficulties with resonance assignment and the low abundance of methyl groups can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy that overcomes these problems. We systematically substituted solvent-exposed residues with reporter methionines in the expected binding site and performed chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. We demonstrate the utility of this approach for the interaction between the HECT domain of the Rsp5p ubiquitin ligase and its cognate E2, Ubc4. Using these mutants, we could instantaneously assign all newly arising reporter methyl signals, determine the Ubc4 interaction surface on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl CSP experiments.

Detection of nanosecond time scale side-chain jumps in a protein dissolved in water/glycerol solvent

In solution, the correlation time of the overall protein tumbling, tau(R), plays a role of a natural dynamics cutoff-internal motions with correlation times on the order of tau ( R ) or longer cannot be reliably identified on the basis of spin relaxation data. It has been proposed some time ago that the 'observation window' of solution experiments can be expanded by changing the viscosity of solvent to raise the value of tau(R). To further explore this concept, we prepared a series of samples of alpha-spectrin SH3 domain in solvent with increasing concentration of glycerol. In addition to the conventional (15)N labeling, the protein was labeled in the Val, Leu methyl positions ((13)CHD(2) on a deuterated background). The collected relaxation data were used in asymmetric fashion: backbone (15)N relaxation rates were used to determine tau(R) across the series of samples, while methyl (13)C data were used to probe local dynamics (side-chain motions). In interpreting the results, it has been initially suggested that addition of glycerol leads only to increases in tau(R), whereas local motional parameters remain unchanged. Thus the data from multiple samples can be analyzed jointly, with tau(R) playing the role of experimentally controlled variable. Based on this concept, the extended model-free model was constructed with the intent to capture the effect of ns time-scale rotameric jumps in valine and leucine side chains. Using this model, we made a positive identification of nanosecond dynamics in Val-23 where ns motions were already observed earlier. In several other cases, however, only tentative identification was possible. The lack of definitive results was due to the approximate character of the model-contrary to what has been assumed, addition of glycerol led to a gradual 'stiffening' of the protein. This and other observations also shed light on the interaction of the protein with glycerol, which is one of the naturally occurring osmoprotectants. In particular, it has been found that the overall protein tumbling is controlled by the bulk solvent, and not by a thin solvation layer which contains a higher proportion of water.

Methyl-detected ‘out-and-back’ NMR experiments for simultaneous assignments of Alaβ and Ileγ2 methyl groups in large proteins

A set of sensitive methyl-detected 'out-and-back' NMR experiments for simultaneous assignments of Alabeta and Ilegamma2 methyl positions in large proteins is described. The developed methodology is applied to an 82-kDa enzyme Malate Synthase G. Complete alanine beta and isoleucine gamma2 1H-13C methyl chemical shift assignments could be obtained from the set of new methyl-detected 'out-and-back' 3D experiments. The described methodology for methyl assignments should be applicable to protein molecules within approximately 100-kDa molecular weight range irrespective of the labeling strategy chosen to produce selectively protonated Alabeta and Ilegamma2 13CH3 sites on a deuterated background.

A New Labeling Method for Methyl Transverse Relaxation-Optimized Spectroscopy NMR Spectra of Alanine Residues

The development of specific methyl labeling schemes and transverse relaxation-optimized spectroscopy (TROSY) has extended the molecular size range for the application of NMR spectroscopy to proteins. Generally, methyl groups of isoleucine, leucine, valine residues are specifically protonated in a highly deuterated background and 1H-13C correlation experiments provide a means to study structure and dynamics in multimeric complexes with molecular weights far in excess of 100 kDa. We have extended this approach to alanine residues which offers several potential advantages, including its high abundance and wide distribution in protein sequences together with a high tolerance to mutation. We have developed an efficient method for the synthesis and incorporation of l-alanine-3-13C,2-2H into protein sequences. We also demonstrate the usefulness of specific protonation of alanine residues in combination with methyl TROSY experiments on the 306 kDa fragment of the eukaryotic AAA-ATPase, p97, in complex with one of its many adaptor proteins.

Quantitative dynamics and binding studies of the 20S proteasome by NMR

The machinery used by the cell to perform essential biological processes is made up of large molecular assemblies. One such complex, the proteasome, is the central molecular machine for removal of damaged and misfolded proteins from the cell. Here we show that for the 670-kilodalton 20S proteasome core particle it is possible to overcome the molecular weight limitations that have traditionally hampered quantitative nuclear magnetic resonance (NMR) spectroscopy studies of such large systems. This is achieved by using an isotope labelling scheme where isoleucine, leucine and valine methyls are protonated in an otherwise highly deuterated background in concert with experiments that preserve the lifetimes of the resulting NMR signals. The methodology has been applied to the 20S core particle to reveal functionally important motions and interactions by recording spectra on complexes with molecular weights of up to a megadalton. Our results establish that NMR spectroscopy can provide detailed insight into supra-molecular structures over an order of magnitude larger than those routinely studied using methodology that is generally applicable.

Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background.

A general method for stable-isotope labeling of large proteins is introduced and applied for studies of the E. coli GroE chaperone proteins by solution NMR. In addition to enabling the residue-specific (15)N-labeling of proteins on a highly deuterated background, it is also an efficient approach for uniform labeling. The method meets the requirements of high-level deuteration, minimal cross-labeling and high protein yield, which are crucial for NMR studies of structures with sizes above 150 kDa. The results obtained with the new protocol are compared to other strategies for protein labeling, and evaluated with regard to the influence of external factors on the resulting isotope labeling patterns. Applications with the GroE system show that these strategies are efficient tools for studies of structure, dynamics and intermolecular interactions in large supramolecular complexes, when combined with TROSY- and CRINEPT-based experimental NMR schemes.

Aromatic and methyl NOEs highlight hydrophobic clustering in the unfolded state of an SH3 domain.

The N-terminal SH3 domain of Drosophila drk (drkN SH3 domain) exists in equilibrium between a folded (F(exch)) state and a relatively compact unfolded (U(exch)) state under nondenaturing conditions. Selectively labeled samples of the domain have been analyzed by NOESY NMR experiments to probe residual hydrophobic clustering in the U(exch) state. The labeling strategy included selective protonation of aromatic rings or delta-methyl groups on Ile and Leu residues in a highly deuterated background. Combined with long mixing times, the methods permitted observation of significant numbers of long-range interactions between hydrophobic side chains, providing evidence for multiple conformers involving non-native hydrophobic clusters around the Trp 36 indole. Comparison of these data with previously reported HN-HN NOEs yields structural insight into the diversity of structures within the U(exch) ensemble in the drkN SH3 domain. Many of the HN-HN NOEs are consistent with models containing compact residual nativelike secondary structure and greater exposure of the Trp 36 indole to solvent, similar to kinetic intermediates formed in the hierarchic condensation model of folding. However, the methyl and aromatic NOE data better fit conformations with non-native burial of the Trp indole surrounded by hydrophobic groups and more loosely formed beta-structure; these structural characteristics are more consistent with those of kinetic intermediates formed during the hydrophobic collapse mechanism of folding. This suite of NOE data provides a more complete picture of the structures that span the U(exch) state ensemble, from conformers with non-native structure but long-range contacts to those that are highly nativelike. Together, the results are also consistent with the folding funnel view involving multiple folding pathways for this molecule.

Stereospecific assignments of the isopropyl methyl groups of the membrane protein OmpX in DHPC micelles.

In NMR studies of large molecular structures, the number of conformational constraints based on NOE measurements is typically limited due to the need for partial deuteration. As a consequence, when using selective protonation of peripheral methyl groups on a perdeuterated background, stereospecific assignments of the diastereotopic methyl groups of Val and Leu can have a particularly large impact on the quality of the NMR structure determination. For example, 3D 15N- and 13C-resolved [1H,1H]-NOESY spectra of the E. Coli membrane protein OmpX in mixed micelles with DHPC, which have an overall molecular weight of about 60 kDa, showed that about 50% of all obtainable NOEs involve the diastereotopic methyl groups of Val and Leu. In this paper, we used biosynthetically-directed fractional 13C labeling of OmpX and [13C,1H]-HSQC spectroscopy to obtain stereospecific methyl assignments of Val and Leu in OmpX/DHPC. For practical purposes it is of interest that this data could be obtained without use of a deuterated background, and that combinations of NMR experiments have been found for obtaining the desired information either at a 1H frequency of 500 MHz, or with significantly reduced measuring time on a high-frequency instrument.

An approach for high-throughput structure determination of proteins by NMR spectroscopy.

An approach is described for rapidly determining protein structures by NMR that utilizes proteins containing 13C-methyl labeled Val, Leu, and Ile (delta1) and protonated Phe and Tyr in a deuterated background. Using this strategy, the key NOEs that define the hydrophobic core and overall fold of the protein are easily obtained. NMR data are acquired using cryogenic probe technology which markedly reduces the spectrometer time needed for data acquisition. The approach is demonstrated by determining the overall fold of the antiapoptotic protein, Bcl-xL, from data collected in only 4 days. Refinement of the Bcl-xL structure to a backbone rmsd of 0.95 A was accomplished with data collected in an additional 3 days. A distance analysis of 180 different proteins and structure calculations using simulated data suggests that our method will allow the global folds of a wide variety of proteins to be determined.