13C-methyl Groups Research Articles

Cooperative dynamics across distinct structural elements regulate PTP1B activity

Protein-tyrosine phosphatase 1B (PTP1B) is the canonical enzyme for investigating how distinct structural elements influence enzyme catalytic activity. Although it is recognized that dynamics are essential for PTP1B function, the data collected thus far have not resolved whether distinct elements are dynamically coordinated or, alternatively, whether they fulfill their respective functions independently. To answer this question, we performed a comprehensive 13C-methyl relaxation study of Ile, Leu, and Val (ILV) residues of PTP1B, which, because of its substantially increased sensitivity, provides a comprehensive understanding of the influence of protein motions on different time scales for enzyme function. We discovered that PTP1B exhibits dynamics at three distinct time scales. First, it undergoes a distinctive slow motion that allows for the dynamic binding and release of its two most N-terminal helices from the catalytic core. Second, we showed that PTP1B 13C-methyl group side chain fast time-scale dynamics and 15N backbone fast time-scale dynamics are fully consistent, demonstrating that fast fluctuations are essential for the allosteric control of PTP1B activity. Third, and most importantly, using 13C ILV constant-time Carr-Purcell-Meiboom-Gill relaxation measurements experiments, we demonstrated that all four catalytically important loops-the WPD, Q, E, and substrate-binding loops-work in dynamic unity throughout the catalytic cycle of PTP1B. Thus, these data show that PTP1B activity is not controlled by a single functional element, but instead all key elements are dynamically coordinated. Together, these data provide the first fully comprehensive picture on how the validated drug target PTP1B functions.

Application of methyl-TROSY to a large paramagnetic membrane protein without perdeuteration: 13C-MMTS-labeled NADPH-cytochrome P450 oxidoreductase.

NMR spectroscopy of membrane proteins involved in electron transport is difficult due to the presence of both the lipids and paramagnetic centers. Here we report the solution NMR study of the NADPH-cytochrome P450 oxidoreductase (POR) in its reduced and oxidized states. We interrogate POR, first, in its truncated soluble form (70kDa), which is followed by experiments with the full-length protein incorporated in a lipid nanodisc (240kDa). To overcome paramagnetic relaxation in the reduced state of POR as well as the signal broadening due to its high molecular weight, we utilized the methyl-TROSY approach. Extrinsic 13C-methyl groups were introduced by modifying the engineered surface-exposed cysteines with methyl-methanethiosulfonate. Chemical shift dispersion of the resonances from different sites in POR was sufficient to monitor differential effects of the reduction-oxidation process and conformation changes in the POR structure related to its function. Despite the high molecular weight of the POR-nanodisc complex, the surface-localized 13C-methyl probes were sufficiently mobile to allow for signal detection at 600MHz without perdeuteration. This work demonstrates a potential of the solution methyl-TROSY in analysis of structure, dynamics, and function of POR, which may also be applicable to similar paramagnetic and flexible membrane proteins.

The equivalence of the methyl groups in puckered 3,3-dimethyloxetane

3,3-dimethyloxetane (DMO) has a nonplanar ring equilibrium configuration (ϕ=17.5°) and a double minimum potential function, with a barrier of 47cm−1, for ring-puckering vibration. In a previous work, the observation of endocyclic 13C and 18O monosubstituted isotopologues allowed to conclude that the ring is puckered, consistent with the observed ring-puckering potential function. However, an interesting feature was observed for the 13C substitutions at the methyl carbon atoms. While two different, axial and equatorial, 13C-methyl groups spectra are predicted from a rigid nonplanar ring DMO model, only one spectrum was found. This behavior evidences that the two methyl groups of DMO are equivalent as could be expected for a planar ring. In this work we show how consideration of the potential function and the dynamical path for ring-puckering motion allows to reproduce the experimental results.

Investigation of Structural Changes Upon Ligand Binding of the Methylated Neuropeptide Y Receptor Type 2

G protein-coupled receptors (GPCRs) are membrane spanning proteins, which represent very important drug targets. For any pharmacological interference, detailed knowledge about the structure and dynamics of the molecules are essential, in particular those structural changes are relevant that occur upon ligand binding to the receptor. Here, we have studied the neuropeptide Y receptor type 2 (Y2R), which belongs to the class A of GPCRs and has a wide variety in function.We are able to produce large amounts (35 mg/l minimal media) of the receptor in a prokaryotic expression system as inclusion bodies. These protein aggregates were isolated, solubilized in SDS-micelles and purified. Subsequently, the receptor was refolded into its functional state [1]. Our aim is to investigate ligand-specific conformational changes of the receptor by NMR spectroscopy. Therefore, we used the reductive methylation of lysine residues to introduce 13C-methyl groups [2]. Due to their favorable relaxation properties, these methyl groups allow for sensitive NMR-measurements. We detected 1H-13C HSQC NMR spectra, which provide some resolved NMR signals of the respective methyl groups. Chemical shift changes of some signals are observable, which are induced by ligand binding. These chemical shift changes could be related to alterations of salt bridges or ring current effects that occur due to structural changes induced by ligand binding. We are currently working on the assignment of the residues using site-directed mutagenesis.A very astonishing side effect is that the methylated receptor shows a dramatic increase in the stability.[1] P. Schmidt et al. (2009). Biotechnol. Prog. 25(6):1732-9.[2] M.P. Bokoch et al. (2010). Nature 463(7277):108-12.

ChemInform Abstract: N-Methylputrescine Oxidation During Cocaine Biosynthesis: Study of Prochiral Methylene Hydrogen Discrimination Using the Remote Isotope Method.

[structure: see text] The stereoselectivity of N-methylputrescine (3) oxidation to pyrrolinium ion 4 in Erythroxylum coca during cocaine (1) biosynthesis was studied. The remote isotope method was used to advantage. Each enantiomer of 4-monodeuterated N-methylputrescine served as a precursor for plant feeding. To facilitate mass-spectrometric analysis of products, a 2H3 13C-methyl group was also incorporated into the 4-deuterio-N-methylputrescines. Oxidative deamination of N-methylputrescine was found to be stereoselective; the pro-S hydrogen atom is removed with 6-10:1 selectivity.

2D-NMR reveals different populations of exposed lysine residues in the apoB-100 protein of electronegative and electropositive fractions of LDL particles

Several potentially atherogenic LDL subfractions present low affinity for the LDL receptor, which result in impaired plasma clearance. Electronegative LDL [LDL(-)] is one of these minor subfractions and the molecular basis for its reduced receptor affinity is not well understood. In the present study, high-resolution 2D-NMR spectroscopy has been employed to characterize the surface-exposed lysine residues of the apolipoprotein (apo)B-100 protein in both LDL(-) and LDL(+) subfractions. LDL(+) showed two populations of lysine residues, similar to those previously described in total LDL. "Normal" Lys have a pk(a) of 10.4 whereas "active" Lys have a pk(a) of 8.8 and have been suggested to be involved in receptor binding. In contrast to LDL(+), the LDL(-) subfraction presented a third type of Lys, named as "intermediate" Lys, with a different microenvironment and higher basicity (pk(a) 10.7). These intermediate Lys cannot be reliably identified by 1D-NMR. Because the abundance of normal Lys is similar in LDL(+) and LDL(-), the intermediate Lys in the apoB-100 molecule of LDL(-) should come from a group of active Lys in LDL(+) particles that have a less basic microenvironment in the LDL(-) particle. These differences between LDL(+) and LDL(-) are indicative of a distinct conformation of apoB-100 that could be related to loss of affinity of LDL(-) for the LDL receptor.

Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins

Reductive methylation of lysine residues in proteins offers a way to introduce 13C methyl groups into otherwise unlabeled molecules. The 13C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of 13C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca2+-bound Calmodulin, Lysozyme, Ca2+-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of 13C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment.

Localizing Fatty Acid Binding Sites on Human Serum Albumin by 2D-NMR

The most abundant protein in plasma, human serum albumin (HSA), is the principal carrier for endogenous lipophilic compounds and lipophilic drugs. Non-esterified long chain fatty acids (FA) are its primary physiological ligand, with multiple binding sites of varying affinities. Detailed structural understandings of HSA-ligand interactions are vital during the development of drugs. HSA ligand interactions have been studied by various biophysical methods: x-ray crystallography, fluorescence spectroscopy, and nuclear magnetic resonance (NMR). Numerous crystal structures of HSA, with and without drugs and/or FAs, have been published. Compared to crystallography, our solution state NMR studies are focused on the more physiologically relevant state. Previously, we identified three high affinity and several low affinity FA binding sites on HSA by 1D-NMR at low resolution. Now, we are correlating crystallographic data with high-resolution 2D-NMR spectra of 13C-methyl-labeled oleic acid (OA). Our 2D-HSQC spectra show the 13C-methyl group of OA bound to HSA. The unique microenvironments that each methyl group experiences in the binding pockets gives rise to different NMR signals of varying intensities. These correspond to the different affinities that HSA has for OA, and our 2D-NMR spectra clearly differentiate the three highest affinity-binding sites from six lower affinity-binding sites. We have identified Sudlow's Drug Sites by utilizing the known FA competitors and Sudlow's site binders: warfarin and diazepam. By expanding this drug competition strategy with ibuprofen and diflunisal, we have identified additional FA binding sites. The highest affinity FA binding sites do not have known competitors and these sites are identified with the use of site-directed mutagenesis of HSA. In summary, we are reporting an approach for studying the interactions of FAs with HSA and FA-competitive drugs in a site-specific manner through the use of state-of-the-art 2D-NMR techniques.

Detection of protein–ligand interactions by NMR using reductive methylation of lysine residues

We show that reductive methylation of proteins can be used for highly sensitive NMR identification of conformational changes induced by metal- and small molecule binding, as well as protein-protein interactions. Reductive methylation of proteins introduces two (13)C-methyl groups on each lysine in the protein of interest. This method works well even when the lysines are not actively involved in the interaction, due to changes in the microenvironments of lysine residues. Most lysine residues are located on the protein exterior, and the exposed (13)C-methyl groups may exhibit rapid localized motions. These motions could be faster than the tumbling rate of the molecule as a whole. Thus, this technique has great potential in the study of large molecular weight systems which are currently beyond the scope of conventional NMR methods.

Increased soil organic carbon sequestration through hydrophobic protection by humic substances

We studied the effect of humified organic matter (OM) on the mineralization of a representative labile organic compound in soil. In an incubation experiment, a 13C-labeled 2-decanol was added to soil either alone (2-dec ∗) or in mixture with two humic acids from compost (HAC ∗) and lignite (HAL ∗) which had different hydrophobic properties. Isotopic dilution ( δ 13C) showed that after 3 months of incubation about 58, 40 and 28% of the added 13C was retained in the whole soil treated with HAL ∗, HAC ∗ and 2-dec ∗, respectively. The higher the hydrophobicity of the employed humic material, the larger was the sequestration of organic carbon in soil. Fractionation of incubated samples revealed that the labeled carbon progressively accumulated in the finest particle-size fractions. However, the high hydrophobicity of the lignite HA favored accumulation of 13C also in the sand-sized fraction. The NMR spectra of humic extracts showed that the 13C-methyl group in the original 2-decanol had been oxidized to a 13C-carboxyl group during incubation for all treatments. This indicated that despite its hydrophilicity, the resulting carboxyl carbon was sequestered into the hydrophobic domains of the humic pool in soil. In fact, the residual 13C was larger in humic than in fulvic extracts for the control sample (2-dec ∗) and even more so in extracts from soil treated with both exogenous humic acids. Our results suggest that labile organic compounds may be effectively protected in soil by humified OM and their microbial mineralization substantially reduced. Innovative soil management practices employing hydrophobic humic substances may increase the biological stability of soil OM and thus contribute to significantly mitigate CO 2 emissions from agricultural soils.

High-Resolution Solid-State 13C NMR Studies of Poly[(R)-3-hydroxybutyric] Acid

Solid poly[(R)-3-hydroxybutyric] acid was examined by high-resolution 13C MAS NMR, differential scanning calorimetry and infrared spectroscopy. The 13C methyl group resonance consists of three components: the rigid amorphous phase, the crystalline phase and the mobile amorphous phase. Spectral deconvolution, using the Lorentz function, reveals the relative amounts as 62% crystalline and 38% amorphous at 333 K, and 42% crystalline, 40% pure amorphous and 18% β-orthorhombic at 413 K. NMR indicates a large difference in molecular mobility between the crystalline and amorphous regions of the sample. Infrared spectroscopy shows that the stretching at 1725 cm−1 (characteristic of the α-form) comes from the crystalline region, and the bands at 1744 cm−1 (characteristic of the β-form) and 1800 cm−1 come from the amorphous region.

N-methylputrescine oxidation during cocaine biosynthesis: study of prochiral methylene hydrogen discrimination using the remote isotope method.

[structure: see text] The stereoselectivity of N-methylputrescine (3) oxidation to pyrrolinium ion 4 in Erythroxylum coca during cocaine (1) biosynthesis was studied. The remote isotope method was used to advantage. Each enantiomer of 4-monodeuterated N-methylputrescine served as a precursor for plant feeding. To facilitate mass-spectrometric analysis of products, a 2H3 13C-methyl group was also incorporated into the 4-deuterio-N-methylputrescines. Oxidative deamination of N-methylputrescine was found to be stereoselective; the pro-S hydrogen atom is removed with 6-10:1 selectivity.

Cardiac troponin I induced conformational changes in cardiac troponin C as monitored by NMR using site-directed spin and isotope labeling.

Conformational changes in both free cardiac troponin C (cTnC) and in complex with a recombinant troponin I protein [cTnI(33-211), cTnI(33-80), or cTnI (86-211)] were observed by means of a combination of selective carbon-13 and spin labeling. The paramagnetic effect from the nitroxide spin label, MTSSL, attached to cTnC(C35S) at Cys 84 allowed measurement of the relative distances to the 13C-methyl groups of the 10 methionines of cTnC in the monomer or complex. All 10 1H-13C correlations in the heteronuclear single- and multiple-quantum coherence (HSMQC) spectrum of [13C-methyl] Met cTnC in the complex with cTnI(33-211) were previously assigned [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., & Rosevear, P. R. (1994) J. Biol. Chem. 269, 23731-23735]. In the presence of oxidized spin label, nine of the 10 Met methyl 1H-13C correlations of cTnC were significantly broadened in the cTnC(C35S) monomer. This suggests flexibility within the central helix, or interdomain D/E helical linker, bringing the N- and C-terminal domains in closer proximity than predicted from the crystallographic structure of TnC. In the spin-labeled cTnC(C35S). cTnI(33-211) complex only N-terminal Met methyl 1H-13C correlations of cTnC(C35S) were paramagnetically broadened beyond detection, whereas correlations for Met residues (103, 120, 137, and 157) in the C-terminal domain were not. Thus, complex formation with cTnI decreases interdomain flexibility and maintains cTnC in an extended conformation. This agrees with the recently published study suggesting that sTnC is extended when bound to sTnI [Olah, G. A., & Trewhella, J. (1994) Biochemistry 33, 12800-12806]. The recombinant N-terminal domain of cTnI, cTnI(33-80), gave similar results as observed with cTnI(33-211) when complexed with spin-labeled cTnC(C35S). However, complex formation with the C-terminal fragment, cTnI(86-211), which contains the inhibitory sequence, is insufficient to maintain cTnC extended to the amount observed with either cTnI(33-211) or cTnI(33-80); although compared to that observed in free cTnC, it does cause decreased flexibility in the interdomain linker. In the absence of the N-terminal domain of cTnI, there is a decrease in flexibility within the N-terminal domain of cTnC. Interestingly, the N-terminal domain of cTnC in the reduced spin-labeled complex with cTnI(86-211), in the presence of ascorbate, showed two distinct conformations which were not seen in the complex with cTnI(33-211).(ABSTRACT TRUNCATED AT 400 WORDS)

The Calmodulin-Binding Domain of Caldesmon Binds to Calmodulin in an .alpha.-Helical Conformation

The binding of calcium-calmodulin (CaM) to caldesmon (CaD) contributes to the regulation of smooth muscle contraction. It has been reported that a 17-residue synthetic peptide encompassing the residues Gly651-Ser667 of smooth muscle CaD constitutes its CaM-binding domain [Zhan, Q., Wong, S. S., & Wang, C.L.A. (1991) J. Biol. Chem. 266, 21810-21814]. This peptide does not share sequence homology with the CaM-binding domains of other proteins, and in addition, the binding of CaM to CaD is known to be relatively weak. Here we have investigated the properties of this atypical CaM-binding domain by NMR and circular dichroism (CD) spectroscopy. Two dimensional NMR studies performed in aqueous TFE mixture (75%/25%) showed that the peptide has the capacity to adopt an amphiphilic alpha-helical conformation. TRNOESY experiments and CD spectroscopy were used to determine that the CaD peptide binds in an alpha-helical conformation to CaM. The addition of TFE or the binding of the CaD peptide to CaM induces an alpha-helical structure only for the central 10 amino acid residues of the peptide. Titrations of CaM with the CaD peptide were followed by proton NMR and show the formation of a 1:1 complex and that the binding is calcium-dependent. The chemical shifts of 13C-methyl groups of specifically labeled Met residues and of the 15N backbone amide groups of CaM undergo changes upon addition of the CaD peptide; these data suggest that both domains and the central helix of CaM are involved in the binding of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

ChemInform Abstract: 13C NMR Studies of Substituted 2,3‐Diphenylindoles. Methyl and Nitro Derivatives.

The 13C nmr spectra of a series of methyl- and/or nitro-2,3-diphenylindole derivatives are reported. The dependence of a) 1H and 13C methyl group shieldings on molecular geometry and b) the possibility that 13C nmr might be a useful tool in determining photochemical reactivity are discussed briefly.

13C NMR studies of substituted 2,3‐diphenylindoles. Methyl and nitro derivatives

AbstractThe 13C nmr spectra of a series of methyl‐ and/or nitro‐2,3‐diphenylindole derivatives are reported. The dependence of a) 1H and 13C methyl group shieldings on molecular geometry and b) the possibility that 13C nmr might be a useful tool in determining photochemical reactivity are discussed briefly.

13C-NMR Investigation of the insertion of the bee venom melittin into lecithin vesicles

The orientation of melittin in lecithin membranes was investigated by means of 13C-NMR spectroscopy. Phospholipase-free melittin was labeled with 13C-methyl groups at the ω-amino side chains of lysine 7, 21, and 23. From the pH dependence of the corresponding 13C resonances, pK values of the lysine residues were derived that were different for free and membrane-bound melittin. The shift reagent Pr(NO3)3 induced shifts in the 13C resonance position of all three lysines when melittin and the shift reagent were added to a lecithin vesicle suspension, whereas Pr3+ ions included in the inner volume of the vesicles did not affect the 13C resonances of melittin bound to the outer vesicle membrane. A wedge-like structure was derived for the membrane-bound melittin, the lysine side chains of which are freely accessible to the aqueous solvent.