Site-selective <Superscript>13</Superscript>C labeling of proteins using erythrose

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with ¹³C and/or ¹H, which is achieved in the most general way by using site-selectively ¹³C-enriched glucose (1- and 2-¹³C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively ¹³C-enriched erythrose (1-, 2-, 3- and 4-¹³C) as a suitable precursor for ¹³C labeled aromatic side chains. We quantify ¹³C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the ¹³C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated ¹³C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective ¹³C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.     

Conformational exchange of aromatic side chains by <Superscript>1</Superscript>H CPMG relaxation dispersion

Aromatic side chains are attractive probes of protein dynamics on the millisecond time scale, because they are often key residues in enzyme active sites and protein binding sites. Further they allow to study specific processes, like histidine tautomerization and ring flips. Till now such processes have been studied by aromatic ¹³C CPMG relaxation dispersion experiments. Here we investigate the possibility of aromatic ¹H CPMG relaxation dispersion experiments as a complementary method. Artifact-free dispersions are possible on uniformly ¹H and ¹³C labeled samples for histidine δ2 and ε1, as well as for tryptophan δ1. The method has been validated by measuring fast folding–unfolding kinetics of the small protein CspB under native conditions. The determined rate constants and populations agree well with previous results from ¹³C CPMG relaxation dispersion experiments. The CPMG-derived chemical shift differences between the folded and unfolded states are in good agreement with those obtained directly from the spectra. In contrast, the ¹H relaxation dispersion profiles in phenylalanine, tyrosine and the six-ring moiety of tryptophan, display anomalous behavior caused by ³J ¹H–¹H couplings and, if present, strong ¹³C–¹³C couplings. Therefore they require site-selective ¹H/²H and, in case of strong couplings, ¹³C/¹²C labeling. In summary, aromatic ¹H CPMG relaxation dispersion experiments work on certain positions (His δ2, His ε1 and Trp δ1) in uniformly labeled samples, while other positions require site-selective isotope labeling.

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[15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N–1H, aromatic 13C–1H and side-chain 15N–1H2 correlations in large proteins

This paper describes a [¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide ¹⁵N–¹H groups, side chain ¹⁵N–¹H₂ groups and aromatic ¹³C–¹H groups in otherwise highly deuterated proteins. The ¹⁵N–¹H and ¹³C–¹H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the ¹H dimension. The side-chain ¹⁵N–¹H₂ groups, which are suppressed in conventional [¹⁵N,¹H)-TROSY, are observed with high sensitivity in the ¹⁵N–¹H subspectrum. [¹⁵N,¹H]/[¹³C,¹H]-TROSY was used as the heteronuclear correlation block in a 3D [¹H,¹H]-NOESY-[¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain ¹⁵N–¹H₂ groups.     

Heterologous Expression of Hen Egg White Lysozyme and Resonance Assignment of Tryptophan Side Chains in its Non-native States

A new protocol is described for the isotope (¹⁵N and ¹³C,¹⁵N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. ¹H,¹³C and ¹⁵N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the C^γ resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues.     

A novel method for the biosynthesis of deuterated proteins with selective protonation at the aromatic rings of Phe,Tyr and Trp

A novel biosynthetic strategy is described for the preparation of deuterated proteins containing protons at the ring carbons of Phe, Tyr and Trp, using the aromatic amino acid precursor shikimic acid. Specific protonation at aromatic side chains, with complete deuteration at C^/positions was achieved in proteins overexpressed in bacteria grown in shikimate-supplemented D₂O medium. Co-expression of a shikimate transporter in prototrophic bacteria resulted in protonation levels of 62–79%, whereas complete labeling was accomplished using shikimate auxotrophic bacteria. Our labeling protocol permits the measurement of important aromatic side chain derived distance restraints in perdeuterated proteins that could be utilized to enhance the accuracy of NMR structures calculated using low densities of NOEs from methyl selectively protonated samples.     

1H, 13C and 199Hg NMR characteristics of new amine-mercuric chloride complexes

Reduction of some N-alkylimines has been achieved with NaBH₄ to give the corresponding secondary amines with high yields (85–95%). These amines were characterized on the bases of their ¹H and ¹³C NMR spectra. The reaction of these amines with mercuric chloride to afford the corresponding complexes was found to occur through a weak dative bond between the nitrogen lone pair of electrons and the mercury atom to form HgCl₂L₂ complexes. The ¹H, ¹³C and ¹⁹⁹Hg NMR chemical shifts have been obtained as well as ¹J(¹³CH) and ²J(¹³CH) coupling constants. Labelling with nitrogen-15 revealed that there is a weak coupling between the nitrogen and the ¹⁹⁹Hg.     

1H-1H and 13C-13C vicinal coupling constants and amino acid side chain conformation in peptides

The vicinal coupling constants ¹³C′-¹³C^γ were measured in aspartic acid and phenylalanine (85 % ¹³C enrichment) as free amino acids and in the peptides Asp-Pro and Gly-Pro-Phe. These coupling constants used in connection with those between the α -and the β-protons provide the unambiguous assignment of rotamers I and II in the Asp and Phe side chains. The method is generally applicable to other amino acids and residues even in large peptides. A possible set of J_g^c,c and J_t^c,c values is proposed for the use of carbon 13-carbon 13 vicinal coupling constants in the side chain conformational studies of amino acid residues with a free carboxyl group.     

Determination of the glycosidic torsion angles in uniformly 13C-labeled nucleic acids from vicinal coupling constants 3J(C2)/4-H1' and 3J(C6)/8-H1'

A two-dimensional, quantitative J-correlation NMR experiment for precise measurements of the proton-carbon vicinal coupling constants ³J_C2/4–H1 and ³J_C6/8–H1 in uniformly ¹³C-labeled nucleic acids is presented. To reduce loss of signal due to ¹H -¹³C dipole-dipole relaxation, a multiple-quantum constant time experiment with appropriately incorporated band selective ¹H and ¹³C pulses was applied. The experiment is used to obtain the ³J_C2/4–H1 and ³J_C6/8–H1 coupling constants in a uniformly ¹³C, ¹⁵N-labeled [d(G₄T₄G₄)]₂ quadruplex. The measured values and glycosidic torsion angles in the G-quadruplex, obtained by restrained molecular dynamics with explicit solvent using the previously published restraints, along with selected data from the literature are used to check and modify existing parameters of the Karplus equations. The parameterizations obtained using glycosidic torsion angles derived from the original solution and recently determined X-ray structures are also compared.     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

Mapping the dynamics of ligand reorganization via 13CH3 and 13CH2 relaxation dispersion at natural abundance

Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain ¹³C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene ¹³CH₂ and methyl ¹³CH₃ groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of μs–ms conformational dynamics related to binding and catalysis. We perform these measurements at natural ¹³C abundance, in congruence with common pharmaceutical research settings. We illustrate these methods through a study of the interaction of a phosphopeptide ligand with the peptidyl-prolyl isomerase, Pin1. The results illuminate the side-chain moieties that undergo conformational readjustments upon complex formation. In particular, we find evidence that multiple exchange processes influence the side chain dispersion profiles. Collectively, our studies illustrate how side-chain relaxation dispersion can shed light on ligand conformational transitions required for activity, and thereby suggest strategies for its optimization.     

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an <Emphasis Type="Italic">E. coli</Emphasis> strain disabled in the oxidative pentose phosphate pathway

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (¹³C, ¹⁵N, and/or ²H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-¹³C-glycerol and ¹⁵N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, ¹³C-sodium formate combined with ¹³C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to ¹J_C5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of ¹³C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and ¹⁵N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.     

An NMR investigation of putative interresidue H-bonding in methyl α-cellobioside in solution

Methyl α-cellobioside (methyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranoside) was labeled with ¹³C at C4′ for use in NMR studies in DMSO-d₆ solvent to attempt the detection of a trans-H-bond J-coupling (^3hJ_CCOH) between C4′ and OH3. Analysis of the OH3 signal at 600 MHz revealed only the presence of two homonuclear J-couplings: ³J_H3,OH3 and a smaller, longer range J_HH. No evidence for ^3hJ_C4′,OH3 was found. The longer range J_HH was traced to ⁴J_H4,OH3 based on 2D ¹H–¹H COSY data and inspection of the H2 and H4 signal lineshapes. A limited set of DFT calculations was performed on a methyl cellobioside mimic to evaluate the structural dependencies of ⁴J_H2,O3H and ⁴J_H4,O3H on the H3–C3–O3–H torsion angle. Computed couplings range from about −0.7 to about +1.1 Hz, with maximal values observed when the C–H and O–H bonds are roughly diaxial.     

1H and 13C nmr studies of cyclic and linear dipeptides containing threonyl,seryl, aspartyl,and histidyl residues

The side-chain conformations of D - orL - Thr, D - or L -Ser, L -Asp, and L - His residues in cyclic and linear dipeptides in D₂O or in DMSO-d₆ are deduced from vicinal (¹H,¹H) and (¹³C, ¹H) coupling constants. Vicinal (¹³C, ¹³C) coupling constants strongly depend on substituents and cannot be used without a more sound analysis. In cyclic dipeptides, the Thr and Ser side chains are folded above the DKP ring, with χ¹ near 60°. The L -Asp side chain interacts more specifically with peptide bonds (χ¹ near 300°). The L - His side chain is more flexible and its conformation depends on the proximity of a second side chain and on solute-solvent interactions. In all cases, this side chain is not completely folded. In linear dipeptides, the conformation of a C-terminal L -His residue is mainly influenced by the end carboxylic group. On the other hand, a N-terminal L -His residue interacts more easily with a neighboring L -Asp residue. In aqueous solution, the imidazole pK_a depends on the proximity of terminal and lateral charged groups but does not reveal any specific interaction in cyclic dipeptides. A comparison between the conformations of cyclic peptides observed in solution, in the crystalline state and calculated by empirical methods, allows one to point out the discriminating role of the packing in crystals, and of solute-solvent interactions in solution.     

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

The ¹H–¹³C HMQC signals of the ¹³CH₃ 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 ¹³CH₃-labeled [U–²H;¹⁵N]-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.     

Backbone and Ile-δ1, Leu,Val methyl <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C,chemical shift assignments for <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> lipase

Lipase r27RCL is a 296-residue, 33 kDa monomeric enzyme with high ester hydrolysis activity, which has significant applications in the baking, paper and leather industries. The lipase gene proRCL from Rhizopus microsporus var. chinensis (also Rhizopus chinensis) CCTCC M201021 was cloned as a fusion construct C-terminal to a maltose-binding protein (MBP) tag, and expressed as MBP-proRCL in an Escherichia coli BL21 trxB (DE3) expression system with uniform ²H,¹³C,¹⁵N-enrichment and Ile-δ1, Leu, and Val ¹³CH₃ methyl labeling. The fusion protein was hydrolyzed by Kex2 protease at the recognition site Lys-Arg between residues ?29 and ?28 of the prosequence, producing the enzyme form called r27RCL. Here we report extensive backbone ¹H, ¹⁵N, and ¹³C, as well as Ile-δ1, Leu, and Val side chain methyl, NMR resonance assignments for r27RCL.     

CACA-TOCSY with alternate <Superscript>13</Superscript>C–<Superscript>12</Superscript>C labeling: a <Superscript>13</Superscript>C<Superscript>α</Superscript> direct detection experiment for mainchain resonance assignment,dihedral angle information,and amino acid type identification

We present a ¹³C direct detection CACA-TOCSY experiment for samples with alternate ¹³C–¹²C labeling. It provides inter-residue correlations between ¹³C^α resonances of residue i and adjacent C^αs at positions i − 1 and i + 1. Furthermore, longer mixing times yield correlations to C^α nuclei separated by more than one residue. The experiment also provides C^α-to-sidechain correlations, some amino acid type identifications and estimates for ψ dihedral angles. The power of the experiment derives from the alternate ¹³C–¹²C labeling with [1,3-¹³C] glycerol or [2-¹³C] glycerol, which allows utilizing the small scalar ³J_CC couplings that are masked by strong ¹J_CC couplings in uniformly ¹³C labeled samples.     

An Economical Method for Production of 2H,13CH3-Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome

NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby ¹³CH₃ methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-²H; β−²H;γ-¹³C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr ¹³C,¹H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[¹³C,¹H]-Thr.     

One-bond and two-bond J couplings help annotate protein secondary-structure motifs: J-coupling indexing applied to human endoplasmic reticulum protein ERp18

NMR coupling constants, both direct one‐bond (¹J) and geminal two‐bond (²J), are employed to analyze the protein secondary structure of human oxidized ERp18. Coupling constants collected and evaluated for the 18 kDa protein comprise 1268 values of ¹J_CαHα, ¹J_CαCβ, ¹J_CαC′, ¹J_C′N′, ¹J_N′Cα, ¹J_N′HN, ²J_CαN′, ²J_HNCα, ²J_C′HN, and ²J_HαC′. Comparison with ¹J and ²J data from reference proteins and pattern analysis on a per‐residue basis permitted main‐chain φ,ψ torsion‐angle combinations of many of the 149 amino‐acid residues in ERp18 to be narrowed to particular secondary‐structure motifs. J‐coupling indexing is here being developed on statistical criteria and used to devise a ternary grid for interpreting patterns of relative values of J. To account for the influence of the varying substituent pattern in different amino‐acid sidechains, a table of residue‐type specific threshold values was compiled for discriminating small, medium, and large categories of J. For the 15‐residue insertion that distinguishes the ERp18 fold from that of thioredoxin, the J‐coupling data hint at a succession of five isolated Type‐I β turns at progressively shorter sequence intervals, in agreement with the crystal structure. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

Correlation of 2J couplings with protein secondary structure

Geminal two‐bond couplings (²J) in proteins were analyzed in terms of correlation with protein secondary structure. NMR coupling constants measured and evaluated for a total six proteins comprise 3999 values of ²J_CαN′, ²J_C′HN, ²J_HNCα, ²J_C′Cα, ²J_HαC′, ²J_HαCα, ²J_CβC′, ²J_N′Hα, ²J_N′Cβ, and ²J_N′C′, encompassing an aggregate 969 amino‐acid residues. A seamless chain of pattern comparisons across the spectrum datasets recorded allowed the absolute signs of all ²J coupling constants studied to be retrieved. Grouped by their mediating nucleus, C′, N′ or C^α, ²J couplings related to C′ and N′ depend significantly on ?,ψ torsion‐angle combinations. β turn types I, I′, II and II′, especially, can be distinguished on the basis of relative‐value patterns of ²J_CαN′, ²J_HNCα, ²J_C′HN, and ²J_HαC′. These coupling types also depend on planar or tetrahedral bond angles, whereas such dependences seem insignificant for other types. ²J_HαCβ appears to depend on amino‐acid type only, showing negligible correlation with torsion‐angle geometry. Owing to its unusual properties, ²J_CαN′ can be considered a “one‐bond” rather than two‐bond interaction, the allylic analog of ¹J_N′Cα, as it were. Of all protein J coupling types, ²J_CαN′ exhibits the strongest dependence on molecular conformation, and among the ²J types, ²J_HNCα comes second in terms of significance, yet was hitherto barely attended to in protein structure work. Proteins 2010. © 2009 Wiley‐Liss, Inc.