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.     

Chemical shift based editing of CH<Subscript>3</Subscript> groups in fractionally <Superscript>13</Superscript>C-labelled proteins using GFT (3, 2)D CT-H<Emphasis Type="Underline">CC</Emphasis>H-COSY: stereospecific assignments of CH<Subscript>3</Subscript> groups of Val and Leu residues

We propose a (3, 2)D CT-HCCH-COSY experiment to rapidly collect the data and provide significant dispersion in the spectral region containing (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues. This enables one to carry out chemical shift based editing and grouping of all the (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues in fractionally (10%) (13)C-labelled proteins, which in turn aids in the sequence-specific resonance assignments in general and side-chain resonance assignments in particular, in any given protein. Further, we demonstrate the utility of this experiment for stereospecific assignments of the pro-R and pro-S methyl groups belonging to the Leu and Val residues in fractionally (10%) (13)C-labelled proteins. The proposed experiment opens up a wide range of applications in resonance assignment strategies and structure determination of proteins.     

Fractional <Superscript>13</Superscript>C enrichment of isolated carbons using [1-<Superscript>13</Superscript>C]- or [2-<Superscript>13</Superscript>C]-glucose facilitates the accurate measurement of dynamics at backbone C<Superscript>α</Superscript> and side-chain methyl positions in proteins

A simple labeling approach is presented based on protein expression in [1-¹³C]- or [2-¹³C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C^α sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated ¹³C spins (i.e., no ¹³C–¹³C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at C^β positions for 17 of the common 20 amino acids and there are no cases for which ¹³C^α−¹³CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone ¹⁵N studies. The utility of the labeling is established by recording ¹³C R _1ρ and CPMG-based experiments on a number of different protein systems.     

High-resolution methyl edited GFT NMR experiments for protein resonance assignments and structure determination

Three-dimensional (3D) structure determination of proteins is benefitted by long-range distance constraints comprising the methyl groups, which constitute the hydrophobic core of proteins. However, in methyl groups (of Ala, Ile, Leu, Met, Thr and Val) there is a significant overlap of ¹³C and ¹H chemical shifts. Such overlap can be resolved using the recently proposed (3,2)D HCCH-COSY, a G-matrix Fourier transform (GFT) NMR based experiment, which facilitates editing of methyl groups into distinct spectral regions by combining their ¹³C chemical shifts with that of the neighboring, directly attached, ¹³C nucleus. Using this principle, we present three GFT experiments: (a) (4,3)D NOESY-HCCH, (b) (4,3)D ¹H-TOCSY-HCCH and (c) (4,3)D ¹³C-TOCSY-HCCH. These experiments provide unique 4D spectral information rapidly with high sensitivity and resolution for side-chain resonance assignments and NOE analysis of methyl groups. This is exemplified by (4,3)D NOESY-HCCH data acquired for 17.9 kDa non-deuterated cytosolic human J-protein co-chaperone, which provided crucial long-range distance constraints for its 3D structure determination.     

Classification of amino acid spin systems using PFG HCC(CO)NH-TOCSY with constant-time aliphatic 13C frequency labeling

Summary We have developed a useful strategy for identifying amino acid spin systems and side-chain carbon resonance assignments in small ¹⁵N-, ¹³C-enriched proteins. Multidimensional constant-time pulsed field gradient (PFG) HCC(CO)NH-TOCSY experiments provide side-chain resonance frequency information and establish connectivities between sequential amino acid spin systems. In PFG HCC(CO)NH-TOCSY experiments recorded with a properly tuned constant-time period for frequency labeling of aliphatic ¹³C resonances, phases of cross peaks provide information that is useful for identifying spin system types. When combined with ¹³C chemical shift information, these patterns allow identification of the following spin system types: Gly, Ala, Thr, Val, Leu, Ile, Lys, Arg, Pro, long-type (i.e., Gln, Glu and Met), Ser, and AMX-type (i.e., Asp, Asn, Cys, His, Phe, Trp and Tyr).     

Selective editing of Val and Leu methyl groups in high molecular weight protein NMR

The development of methyl-TROSY approaches and specific (13)C-(1)H labeling of Ile, Leu and Val methyl groups in highly deuterated proteins has made it possible to study high molecular weight proteins, either alone or in complexes, using solution nuclear magnetic resonance (NMR) spectroscopy. Here we present 2-dimensional (2D) and 3-dimensional (3D) NMR experiments designed to achieve complete separation of the methyl resonances of Val and Leu, labeled using the same precursor, α-ketoisovalerate or acetolactate. The 2D experiment can further select the methyl resonances of Val or Leu based on the C(α) or C(β) chemical shift values of Val or Leu, respectively. In the 3D spectrum, the methyl cross peaks of Val and Leu residues have opposite signs; thus, not only can the residue types be easily distinguished, but the methyl pairs from the same residue can also be identified. The feasibility of this approach, implemented in both 2D and 3D experiments, has been demonstrated on an 82 kDa protein, malate synthase G. The methods developed in this study will reduce resonance overlaps and also facilitate structure-guided resonance assignments.     

Silencing threonine deaminase and JAR4 in Nicotiana attenuata impairs jasmonic acid-isoleucine-mediated defenses against Manduca sexta

Threonine deaminase (TD) catalyzes the conversion of Thr to alpha-keto butyrate in Ile biosynthesis; however, its dramatic upregulation in leaves after herbivore attack suggests a role in defense. In Nicotiana attenuata, strongly silenced TD transgenic plants were stunted, whereas mildly silenced TD transgenic plants had normal growth but were highly susceptible to Manduca sexta attack. The herbivore susceptibility was associated with the reduced levels of jasmonic acid-isoleucine (JA-Ile), trypsin proteinase inhibitors, and nicotine. Adding [(13)C(4)]Thr to wounds treated with oral secretions revealed that TD supplies Ile for JA-Ile synthesis. Applying Ile or JA-Ile to the wounds of TD-silenced plants restored herbivore resistance. Silencing JASMONATE-RESISTANT4 (JAR4), the N. attenuata homolog of the JA-Ile-conjugating enzyme JAR1, by virus-induced gene silencing confirmed that JA-Ile plays important roles in activating plant defenses. TD may also function in the insect gut as an antinutritive defense protein, decreasing the availability of Thr, because continuous supplementation of TD-silenced plants with large amounts (2 mmol) of Thr, but not Ile, increased M. sexta growth. However, the fact that the herbivore resistance of both TD- and JAR-silenced plants was completely restored by signal quantities (0.6 mumol) of JA-Ile treatment suggests that TD's defensive role can be attributed more to signaling than to antinutritive defense.     

A simple strategy for <Superscript>13</Superscript>C,<Superscript>1</Superscript>H labeling at the Ile-γ2 methyl position in highly deuterated proteins

A straightforward approach for the production of highly deuterated proteins labeled with ¹³C and ¹H at Ile-γ2 methyl positions is described. The utility of the methodology is illustrated with an application involving the half proteasome (360 kDa). High quality 2D Ile ¹³C^γ2,¹H^γ2 HMQC data sets, exploiting the methyl-TROSY principle, are recorded with excellent sensitivity and resolution, that compare favorably with Ile ¹³C^δ1,¹H^δ1 spectra. This labeling scheme adds to a growing list of different approaches that are significantly impacting the utility of solution NMR spectroscopy in studies of supra-molecular systems.     

Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.     

An attenuating mutation in nsP1 of the Sindbis-group virus S.A.AR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.     

Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling

Summary Biosynthetically directed fractional incorporation of¹³C into proteins results in nonrandom¹³C-labeling patterns that can be investigated by analysis of the¹³C–¹³C scalar coupling fine structures in heteronuclear¹³C–¹H or homonuclear¹³C–¹³C correlation experiments. Previously this approach was used for obtaining stereospecific¹H and¹³C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the¹H spin-system identifications. Studies of the hydrolysates of fractionally¹³C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of¹³C–¹³C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear¹H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the - and -methyl groups of Ile. In addition to the methyl resonances, the CH₂ groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the -carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.Abbreviations NOE nuclear Overhauser effect - fractional¹³C labeling biosynthetically directed fractional¹³C-labeling - TOCSY total correlation spectroscopy - ROESY rotating frame Overhauser enhancement spectroscopy - [¹³C,¹H]-COSY two-dimensional¹³C–¹H correlation spectroscopy - isotopomer isotope isomer - P22 c2 repressor c2 repressor of the salmonella phage P22 consisting of a polypeptide chain with 216 residues - P22 c2(1-76) N-terminal domain of the P22 c2 repressor with residues 1–76     

Structure-based prediction of methyl chemical shifts in proteins

Protein methyl groups have recently been the subject of much attention in NMR spectroscopy because of the opportunities that they provide to obtain information about the structure and dynamics of proteins and protein complexes. With the advent of selective labeling schemes, methyl groups are particularly interesting in the context of chemical shift based protein structure determination, an approach that to date has exploited primarily the mapping between protein structures and backbone chemical shifts. In order to extend the scope of chemical shifts for structure determination, we present here the CH3Shift method of performing structure-based predictions of methyl chemical shifts. The terms considered in the predictions take account of ring current, magnetic anisotropy, electric field, rotameric type, and dihedral angle effects, which are considered in conjunction with polynomial functions of interatomic distances. We show that the CH3Shift method achieves an accuracy in the predictions that ranges from 0.133 to 0.198 ppm for ¹H chemical shifts for Ala, Thr, Val, Leu and Ile methyl groups. We illustrate the use of the method by assessing the accuracy of side-chain structures in structural ensembles representing the dynamics of proteins.     

Amino acid sequences of two tryptic peptides from D(-)-beta-hydroxybutyrate dehydrogenase radiolabeled at essential carboxyl and sulfhydryl groups

D(-)beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains essential thiol and carboxyl groups. A tryptic BDH peptide labeled at an essential thiol with [3H]N-ethylmaleimide (NEM), and another tryptic peptide labeled at an essential carboxyl with N,N'-dicyclohexyl [14C]carbodiimide (DCCD), were isolated and sequenced. The peptide labeled with [3H]NEM had the sequence Met.Glu.Ser.Tyr.Cys*.Thr.Ser. Gly.Ser.Thr.Asp.Thr.Ser.Pro.Val.Ile.Lys. The label was at Cys. The same peptide was isolated from tryptic digests of BDH labeled at its nucleotide-binding site with the photoaffinity labeling reagent, arylazido- -[3-3H] alanyl-NAD. These results suggest that the essential thiol of BDH is located at its nucleotide-binding site, and agree with our previous observation that NAD and NADH protect BDH against inhibition by thiol modifiers. The [14C]DCCD-labeled peptide had the sequence Glu.Val.Ala.Glu*.Val. Asn. Leu.Trp.Gly.Thr.Val.Arg. DCCD appeared to modify the glutamic acid residue marked by an asterisk. Sequence analogies between these peptides and other proteins have been discussed.     

Complete assignment of the methionyl carbonyl carbon resonances in switch variant anti-dansyl antibodies labeled with [1-13C]methionine

A 13C NMR study is reported of switch variant anti-dansyl antibodies developed by Dangl et al. [(1982) Cytometry 2, 395-401], who had used the fluorescence-activated cell sorter to select and clone these variants. These switch variant antibodies possess the identical VH, VL, and CL domains in conjunction with different heavy chain constant regions. In the present study, switch variant antibodies of IgG1, IgG2a, and IgG2b subclasses were used along with a short-chain IgG2a antibody, in which the entire CH1 domain is deleted. The switch variant antibodies were specifically labeled with [1-13C]methionine by growing hybridoma cells in serum-free medium. Assignments of all the methionyl carbonyl carbon resonances have been completed by using the intact antibodies along with their fragments and recombined proteins in which either heavy or light chain is labeled. A double labeling method [Kainosho, M., & Tsuji, T. (1982) Biochemistry 21, 6273-6279] has played a crucial role in the process of the spectral assignments. The strategy used for the assignments has been described in detail. In incorporating 15N-labeled amino acids into the antibodies for the double labeling, isotope dilution caused a serious problem except in the cases of [alpha-15N]lysine and [15N]threonine, both of which cannot become the substrate of transaminases. It was found that beta-chloro-L-alanine is most effective in suppressing the isotope scrambling. So far, spectral assignments by the double labeling method have been possible with 15N-labeled Ala, His, Ile, Lys, Met, Ser, Thr, Tyr, and Val.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids

RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.     

Contribution of Thr29 to the thermodynamic stability of goat alpha-lactalbumin as determined by experimental and theoretical approaches

The Thr29 residue in the hydrophobic core of goat alpha-lactalbumin (alpha-LA) was substituted with Val (Thr29Val) and Ile (Thr29Ile) to investigate the contribution of Thr29 to the thermodynamic stability of the protein. We carried out protein stability measurements, X-ray crystallographic analyses, and free energy calculations based on molecular dynamics simulation. The equilibrium unfolding transitions induced by guanidine hydrochloride demonstrated that the Thr29Val and Thr29Ile mutants were, respectively, 1.9 and 3.2 kcal/mol more stable than the wild-type protein (WT). The overall structures of the mutants were almost identical to that of WT, in spite of the disruption of the hydrogen bonding between the side-chain O-H group of Thr29 and the main-chain C=O group of Glu25. To analyze the stabilization mechanism of the mutants, we performed free energy calculations. The calculated free energy differences were in good agreement with the experimental values. The stabilization of the mutants was mainly caused by solvation loss in the denatured state. Furthermore, the O-H group of Thr29 favorably interacts with the C=O group of Glu25 to form hydrogen bonds and, simultaneously, unfavorably interacts electrostatically with the main-chain C=O group of Thr29. The difference in the free energy profile of the unfolding path between WT and the Thr29Ile mutant is discussed in light of our experimental and theoretical results.     

Theoretical 3D model of histamine N-methyltransferase: insights into the effects of a genetic polymorphism on enzymatic activity and thermal stability

Histamine N-methyltransferase (HNMT) catalyzes the N-methylation of histamine in mammals. The experimentally determined HNMT three-dimensional (3D) structure is not available. However, there is a common genetic polymorphism for human HNMT (Thr105Ile) that reduces enzymatic activity and is a risk factor for asthma. To obtain insights into mechanisms responsible for the effects of that polymorphism on enzymatic activity and thermal stability, we predicted the 3D structure of HNMT using the threading method and molecular dynamics simulations in water. Herein, we report a theoretical 3D model of human HNMT which reveals that polymorphic residue Thr105Ile is located in the turn between a beta strand and an alpha helix on the protein surface away from the active site of HNMT. Ile105 energetically destabilizes folded HNMT because of its low Chou-Fasman score for forming a turn conformation and the exposure of its hydrophobic side chain to aqueous solution. It thus promotes the formation of misfolded proteins that are prone to the clearance by proteasomes. This information explains, for the first time, how genetic polymorphisms can cause enhanced protein degradation and why the thermal stability of allozyme Ile105 is lower than that of Thr105. It also supports the hypothesis that the experimental observation of a significantly lower level of HNMT enzymatic activity for allozyme Ile105 than that with Thr105 is due to a decreased concentration of allozyme Ile105, but not an alternation of the active-site topology of HNMT caused by the difference at residue 105.     

VITAL NMR: using chemical shift derived secondary structure information for a limited set of amino acids to assess homology model accuracy

Homology modeling is a powerful tool for predicting protein structures, whose success depends on obtaining a reasonable alignment between a given structural template and the protein sequence being analyzed. In order to leverage greater predictive power for proteins with few structural templates, we have developed a method to rank homology models based upon their compliance to secondary structure derived from experimental solid-state NMR (SSNMR) data. Such data is obtainable in a rapid manner by simple SSNMR experiments (e.g., ¹³C–¹³C 2D correlation spectra). To test our homology model scoring procedure for various amino acid labeling schemes, we generated a library of 7,474 homology models for 22 protein targets culled from the TALOS+/SPARTA+ training set of protein structures. Using subsets of amino acids that are plausibly assigned by SSNMR, we discovered that pairs of the residues Val, Ile, Thr, Ala and Leu (VITAL) emulate an ideal dataset where all residues are site specifically assigned. Scoring the models with a predicted VITAL site-specific dataset and calculating secondary structure with the Chemical Shift Index resulted in a Pearson correlation coefficient (−0.75) commensurate to the control (−0.77), where secondary structure was scored site specifically for all amino acids (ALL 20) using STRIDE. This method promises to accelerate structure procurement by SSNMR for proteins with unknown folds through guiding the selection of remotely homologous protein templates and assessing model quality.     

A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins

A new method for stereospecific assignment of prochiral methyl groups in proteins is presented in which protein samples are produced using U-[¹³C]glucose and subsaturating amounts of 2-[¹³C]methyl-acetolactate. The resulting non-uniform labeling pattern allows proR and proS methyl groups to be easily distinguished by their different phases in a constant-time two-dimensional ¹H-¹³C correlation spectra. Protein samples are conveniently prepared using the same media composition as the main uniformly-labeled sample and contain higher levels of isotope-enrichment than fractional labeling approaches. This new strategy thus represents an economically-attractive, robust alternative for obtaining isotopically-encoded stereospecific NMR assignments of prochiral methyl groups.