Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Measurement of 13Cα-13CO cross-relaxation rates in 15N-/13C-labelled proteins

Summary Internal motions play an important role in the biological function of proteins and NMR relaxation studies may characterize them over a wide range of frequencies. An experimental pulse scheme is proposed for the measurement of the ¹³CO-¹³C cross-relaxation rate. For sensitivity reasons, this measurement is performed in an indirect manner through several coherence transfer steps, which should thus be calibrated independently. Contributions of other relaxation pathways can be eliminated by the determination of the initial slope of the buildup curve. The cross-relaxation rates have been determined on a ¹⁵N-/¹³C-labelled 116-residue protein and the significant variations along the sequence have been interpreted as evidence of an increased amount of fast local motion.     

Quantitative J correlation methods for the accurate measurement of 13C′-13Cαdipolar couplings in proteins

Methods are described for the precise and accurate measurement of one-bond dipolar (13)C'-(13)C(alpha) couplings in weakly aligned proteins. The experiments are based on the principle of quantitative J correlation, where (1)J(C'C(alpha)) (or (1)J(C'C(alpha)) + 1D(C'C(alpha)) is measured from the relative intensity of two interleaved 3D TROSY-HN(CO)CA or 3DTROSY-HNCO spectra recorded with dephasing intervals of zero (reference spectrum) and approximately 3/(2(1)J(C'C(alpha)) (attenuated spectrum). In analogy to other quantitative J correlation techniques, the random error in the measured (1)J(C'C(alpha)) value is inversely proportional to the signal-to-noise ratio in the reference spectrum. It is shown that for weakly aligned proteins, with the magnitude of the alignment tensor of D(a)(NH) < or = 10-15 Hz, the systematic errors are typically negligible. The methods are demonstrated for the third IgG-binding domain of protein G (GB3) and a-synuclein in complex with a detergent micelle, where errors in (1)D(C'C(alpha)) of less than 0.1 Hz and ca. 0.2 Hz,respectively, are estimated. Remarkably, the dipolar couplings determined for GB3 are in even better agreement with the recently refined 1.1-angstroms X-ray structure than the input (13)C'-(13)C(alpha) couplings used for the refinement.     

Intraresidue 1H-15N-13C′ and 1Hα-13Cα-13C′ dipole-CSA relaxation interference as a source of constraints for structural refinement of metal-binding sites in zinc-finger proteins

¹H(i)-¹⁵N(i)-¹³C(i) dipole-chemical shift anisotropy (CSA) relaxation interference was quantified for the ¹³C,¹⁵N labeled zinc-finger protein qCRP2(LIM2). The cross-correlation rates obtained for residues located in the metal coordination sites of qCRP2(LIM2) show a high degree of correlation with the peptide plane torsion angles and taken from the solution structure. ¹H(i)-¹⁵N(i)-¹³C(i) as well as ¹³C(i)-¹H(i)-¹³C(i) dipole-CSA cross-correlation rates were subsequently used to improve the geometry of the metal binding site. The optimized dihedral angles of the two zinc-binding sites in qCRP2(LIM2) are in better agreement with values obtained from crystal structures of other zinc-finger proteins and thus establish the utility of this approach to improve the metal-binding site geometry of zinc-finger proteins studied by NMR spectroscopy in solution.     

The role of protein–solvent hydrogen bond dynamics in the structural relaxation of a protein in glycerol versus water

We used MD simulations to investigate the dependence of the dynamics of a soluble protein, RNase A, on temperature and solvent environment. Consistent with neutron scattering data, the simulations predict that the protein undergoes a dynamical transition in both glycerol and aqueous solutions that is absent in the dry protein. The temperature of the transition is higher, while the rate of increase with temperature of the amplitudes of motion on the 100 ps timescale is lower, in glycerol versus water. Analysis of the dynamics of hydrogen bonds revealed that the protein dynamical transition is connected to the relaxation of the protein-solvent hydrogen bond network, which, in turn, is associated with solvent translational diffusion. Thus, it appears that the role of solvent dynamics in affecting the protein dynamical transition is qualitatively similar in water and glycerol.     

13C–13C NOESY spectra of a 480 kDa protein: solution NMR of ferritin

Molecular size has limited solution NMR analyses of proteins. We report ¹³C–¹³C NOESY experiments on a 480 kDa protein, the multi-subunit ferritin nanocage with gated pores. By exploiting ¹³C-resonance-specific chemical shifts and spin diffusion effects, we identified 75% of the amino acids, with intraresidue C–C connectivities between nuclei separated by 1–4 bonds. These results show the potential of ¹³C–¹³C NOESY for solution studies of molecular assemblies >100 kDa.     

CD measurement of aqueous protein solution at high temperature up to 180 °C —Thermodynamic analysis of thermophilic protein by pressure-proof cell compartment

Summary During investigation of thermal transitions of peptides and proteins, the transition temperature is occasionally too high to trace the whole transition profile. In order to solve this problem and perform conformational analysis at high temperature, we have recently developed a pressure-proof cell compartment for circular dichroism measurements. Here we demonstrate how well this system works to collect CD spectra, at high temperature up to 180°c in aqueous solution. Ribosome recycling factor (RRF), which consists of two domains; three stranded α-helix bundle domain (Domain I) and β/α/β domain (Domain II), was used as an example. We constructed models of isolated Domain I substituting Domain II with tripeptide (Gly-Gly-Gly) and compared these models from mesophilic and thermophilic bacteria. The melting profiles of these models revealed that thermal stability is enhanced by the increased enthalpy provided by hydrogen bonds and ionic pairing.     

Determination of 13Cα relaxation times in uniformly 13C/15N-enriched proteins

Summary Relaxation times of ¹³C carbons of uniformly ¹³C/¹⁵N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T₁, T₂ and the heteronuclear NOE of ¹³C in uniformly ¹³C/¹⁵N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T₁ and the heteronuclear NOE were similar to those of the corresponding ¹⁵N experiments published previously. The determination of T₂ for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T₂ evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T₂ evolution period were simulated in order to optimize the bandwidth for which reliable T₂ relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual ¹H–¹³C cross peaks, in a series of (¹H, ¹³C)-ct-HSQC spectra with a modified CPMG sequence as well as a T_1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.     

Modulation of Bax mitochondrial insertion and induced cell death in yeast by mammalian protein kinase Cα

Protein kinase Cα (PKCα) is a classical PKC isoform whose involvement in cell death is not completely understood. Bax, a major member of the Bcl-2 family, is required for apoptotic cell death and regulation of Bax translocation and insertion into the outer mitochondrial membrane is crucial for regulation of the apoptotic process. Here we show that PKCα increases the translocation and insertion of Bax c-myc (an active form of Bax) into the outer membrane of yeast mitochondria. This is associated with an increase in cytochrome c (cyt c) release, reactive oxygen species production (ROS), mitochondrial network fragmentation and cell death. This cell death process is regulated, since it correlates with an increase in autophagy but not with plasma membrane permeabilization. The observed increase in Bax c-myc translocation and insertion by PKCα is not due to Bax c-myc phosphorylation, and the higher cell death observed is independent of the PKCα kinase activity. PKCα may therefore have functions other than its kinase activity that aid in Bax c-myc translocation and insertion into mitochondria. Together, these results give a mechanistic insight on apoptosis regulation by PKCα through regulation of Bax insertion into mitochondria.     

Study of the influence of temperature on the dynamics of the catalytic cleft in 1,3-1,4-β-glucanase by molecular dynamics simulations

The dependence of some molecular motions in the enzyme 1,3-1,4-β-glucanase from Bacillus licheniformis on temperature changes and the role of the calcium ion in them were explored. For this purpose, two molecular dynamics simulated trajectories along 4 ns at low (300 K) and high (325 K) temperatures were generated by the GROMOS96 package. Several structural and thermodynamic parameters were calculated, including entropy values, solvation energies, and essential dynamics (ED). In addition, thermoinactivation experiments to study the influence of the calcium ion and some residues on the activity were conducted. The results showed the release of the calcium ion, which, in turn, significantly affected the movements of loops 1, 2, and 3, as shown by essential dynamics. These movements differ at low and high temperatures and affect dramatically the activity of the enzyme, as observed by thermoinactivation studies. The first two authors contributed equally to this work     

α-1 acid glycoprotein inhibits insulin responses by glucose oxidation,protein synthesis and protein breakdown in mouse C2C12 myotubes

Increased plasma α-1 acid glycoprotein (AGP) is correlated with reduced growth rates in neonatal swine. The specific physiological mechanisms contributing to this relationship are unknown. This study was performed to determine if AGP can modify muscle metabolism by examining glucose oxidation and protein synthesis in the C2C12 muscle cell line. Cells were used for experiments 4 days post-fusion as myotubes. Myotubes were exposed to AGP for 24 h, with the last 4 h used to monitor ¹⁴C-glucose oxidation or to measure protein synthesis by incorporation of ³H-tyrosine. Treatment of C2C12 myotubes with mouse AGP (100 µg/ml) reduced glucose oxidation (P<0.01, n=3 trials), whereas bovine insulin (1 µM) stimulated glucose oxidation (P<0.05, n=3 trials). Treatment with AGP in combination with insulin reduced ¹⁴C-glucose oxidation (P<0.05, n=3 trials), similar to the effect of AGP alone. Glucose transport, as measured by ³H-deoxyglucose uptake, was increased by 38% with 1 µM insulin (P<0.05, n=3 trials), whereas AGP alone increased glucose uptake by 36% (P<0.05, n=3 trials). The combination of insulin and AGP in the medium resulted in an 88% increase in glucose uptake (P<0.01, n=3 trials). Protein synthesis was measured by ³H-tyrosine incorporation into C2C12 myotubes. Insulin stimulated a 18% increase in ³H-tyrosine incorporation (P<0.05, n=6 trials). The incorporation of ³H-tyrosine into myotubes was reduced by 20% with AGP incubation (P<0.01, n=6 trials), like the 20% decrease in ³H-tyrosine incorporation in response to the combination of AGP and insulin (P<0.01, n=6 trials). Protein breakdown, as measured by the release of ³H-tyrosine from C2C12 myotubes, was reduced 27% by insulin (P<0.01, n=6 trials). Treatment with AGP had no effect on protein breakdown (P>0.05, n=6 trials), whereas incubation with both AGP and insulin reduced ³H-tyrosine release by 15% (P<0.01, n=6 trials). First, these data indicate that the acute phase protein AGP can interact with the skeletal muscle to reduce glucose oxidation, but this is not the result of an effect on glucose transport. Second, AGP can specifically reduce protein synthesis. Lastly, AGP can inhibit insulin-stimulated glucose oxidation, protein synthesis and breakdown.     

α-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases

α-Tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with α-tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2 × 10⁶ cells. A saturating dose of TS (40 μmol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 μmol/l), and much more than Trolox (40 μmol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of α-tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that α-tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.     

Structure and dynamics of two β-peptides in solution from molecular dynamics simulations validated against experiment

We have studied two different beta-peptides in methanol using explicit solvent molecular dynamics simulations and the GROMOS 53A6 force field: a heptapeptide (peptide 1) expected to form a left-handed 3(14)-helix, and a hexapeptide (peptide 2) expected to form a beta-hairpin in solution. Our analysis has focused on identifying and analyzing the stability of the dominant secondary structure conformations adopted by the peptides, as well as on comparing the experimental NOE distance upper bounds and 3J-coupling values with their counterparts calculated on the basis of the simulated ensembles. Moreover, we have critically compared the present results with the analogous results obtained with the GROMOS 45A3 (peptide 1) and 43A1 (peptide 2) force fields. We conclude that within the limits of conformational sampling employed here, the GROMOS 53A6 force field satisfactorily reproduces experimental findings regarding the behavior of short beta-peptides, with accuracy that is comparable to but not exceeding that of the previous versions of the force field. GCE legend Conformational clustering analysis of the simulated ensemble of a ss-hexapeptide with two different simulation setups (a and b). The central members of all of the clusters populating more than 5% of all of the structures are shown, together with the most dominant hydrogen bonds and the corresponding percentages of cluster members containing them.     

Syntheses of 1-O-benzyl-α-glucoside and 1-O-benzyl-α-maltoside by transglycosylation of α- amylase from soluble starch in aqueous solution

Glycosides, 1-O-benzyl--glucoside (BG) and 1- O-benzyl--maltoside (BM), were synthesized from soluble starch and benzyl alcohol by transglycosylation with an -amylase in a water system. BG was mostly obtained in a reaction mixture of pH 5.0, while BM was synthesized in pH 8.0. The synthesized glycosides had -configuration linkage between sugar and benzyl alcohol. The BG was rapidly hydrolyzed to benzyl alcohol and glucose by -glucosidase. The BM was hydrolyzed to BG and glucose below pH 5.0 by the -amylase used for its synthesis but it was not hydrolyzed above pH 8.0.     

Selectively 13C-enriched DNA: Evidence from 13C1′ relaxation rate measurements of an internal dynamics sequence effect in the lac operator

Summary In order to study some internal dynamic processes of the lac operator sequence, the ¹³C-labeled duplex 5d(C₀G₁C₂T₃C₄A₅C₆A₇A₈T₉T₁₀) · d(A₁₀A₉T₈T₇G₆T₅G₄A₃G₂C₁G₀)3 was used. The spreading of both the H1 and C1 resonances brought about an excellent dispersion of the ¹H1-¹³C1 correlations. The spinlattice relaxation parameters R(C_z), R(C_x,y) and R(H_zC_z) were measured for each residue of the two complementary strands, except for the 3-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387–7401; and (1990) J. Am. Chem. Soc., 112, 4989–4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1-H1 fragments exhibited both slow (_s = 1.5) and fast (_f = 20 ps) restricted libration motions (S _inf2 ^sups =0.74 to 1.0 and S _inf2 ^supf =0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5-terminal residues showed large internal motions (S²=0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters S _inf2 ^supf and S _inf2 ^sups along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (S _inf2 ^supf =0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(C_z) and R(HzC_z) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(C_x,y) for the purine residues in the (53) G₄A₃ and A₁₀A₉T₈T₇ sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213–216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.     

Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of ΔC p of protein unfolding

Cold and heat denaturation of the double mutant Arg 3→Glu/Leu 66→Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D ¹H NMR spectroscopy in the temperature range from −12°C in supercooled water up to +70°C. The fraction of unfolded protein, f _u, was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be −11°C and 39°C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, ΔC _p of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.     

Glutamine prevents myostatin hyperexpression and protein hypercatabolism induced in C2C12 myotubes by tumor necrosis factor-α

Depletion of skeletal muscle protein mainly results from enhanced protein breakdown, caused by activation of proteolytic systems such as the Ca2+-dependent and the ATP-ubiquitin-dependent ones. In the last few years, enhanced expression and bioactivity of myostatin have been reported in several pathologies characterized by marked skeletal muscle depletion. More recently, high myostatin levels have been associated with glucocorticoid-induced hypercatabolism. The search for therapeutical strategies aimed at preventing/correcting protein hypercatabolism has been directed to inhibit humoral mediators known for their pro-catabolic action, such as TNFα. The present study has been aimed to investigate the involvement of TNFα in the regulation of both myostatin expression and intracellular protein catabolism, and the possibility to interfere with such modulations by means of amino acid supplementation. For this purpose, C2C12 myotubes exposed to TNFα in the presence or in the absence of amino acid (glutamine or leucine) supplementation have been used. Myotube treatment with TNFα leads to both hyperexpression of the muscle-specific ubiquitin ligase atrogin-1, and enhanced activity of the Ca²⁺-dependent proteolytic system. These changes are associated with increased myostatin expression. Glutamine supplementation effectively prevents TNFα-induced muscle protein loss and restores normal myostatin levels. The results shown in the present study indicate a direct involvement of TNFα in the onset of myotube protein loss and in the perturbation of myostatin-dependent signaling. In addition, the protective effect exerted by glutamine suggests that amino acid supplementation could represent a possible strategy to improve muscle mass.