An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated 15N, 13C-labeled proteins

Summary A biosynthetic strategy has recently been developed for the production of ¹⁵N, ¹³C, ²H-labeled proteins using ¹H₃C-pyruvate as the sole carbon source and D₂O as the solvent. The methyl groups of Ala, Val, Leu and Ile (2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a ¹⁵N, ¹³C, ²H, ¹H₃C-labeled SH2 domain at 3°C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30–40 kDa molecular weight range.     

An enhanced-sensitivity pure absorption gradient 4D 15N, 13C-edited NOESY experiment

Summary An enhanced-sensitivity gradient 4D ¹⁵N, ¹³C-edited NOESY experiment is presented. Gradients are employed to suppress artifacts, eliminate the intense H₂O signal and select for the coherence transfer pathway involving ¹⁵N magnetization. The latter use of the gradients results in a decrease in the number of phase cycle steps by a factor of two relative to the previously published 4D sequence (Kay et al., 1990) allowing for the recording of spectra with increased resolution per unit measuring time. Theoretical sensitivity enhancements of as much as a factor of can be expected over the previously published sequence, neglecting the effects of relaxation and pulse imperfections.     

Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy

We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D₂O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both ¹H and ¹⁵N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for ¹H–¹⁵N correlations in dipolar coupling based experiments for H₂O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based ¹H–¹⁵N correlation experiments yield a nearly constant SNR for samples prepared with ≤30% H₂O. Samples in which more H₂O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in ¹H T ₁ in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H₂O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H₂O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible ¹H,¹H interactions increases. At low levels of deuteration (≥60% H₂O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24 kHz employing perdeuterated samples of the chicken α-spectrin SH3 domain.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Binding of palladium(II) complexes to guanine, guanosine or guanosine 5-monophosphate in aqueous solution: potentiometric and NMR studies

The interaction of guanine, guanosine or 5^′-GMP (guanosine 5^′-monophosphate) with [Pd(en)(H₂O)₂](NO₃)₂ and [Pd(dapol)(H₂O)₂](NO₃)₂, where en is ethylenediamine and dapol is 2-hydroxy-1,3-propanediamine, were studied by UV-Vis, pH titration and ¹H NMR. The pH titration data show that both N1 and N7 can coordinate to [Pd(en)(H₂O)₂]²⁺ or [Pd(dapol)(H₂O)₂]²⁺. The pK_a of N1-H decreased to 3.7 upon coordination in guanosine and 5^′-GMP complexes, which is significantly lower than that of ∼9.3 in the free ligand. In strongly acidic solution where N1-H is still protonated, only N7 coordinates to the metal ion, but as the pH increases to pH ∼3, ¹H NMR shows that both N7-only and N1-only coordinated species exist. At pH 4-5, both N1-only and N1,N7-bridged coordination to Pd(II) complexes are found for guanosine and 5^′-GMP. The latter form cyclic tetrameric complexes, [Pd(diamine)(μ-N1,N7-Guo]₄⁴⁺ and [Pd(diamine)(μ-N1,N7-5^′-GMP)]₄H_x^(4−x)−, (x=2,1, or 0) with either [Pd(en)(H₂O)₂](NO₃)₂ or [Pd(dapol)(H₂O)₂](NO₃)₂. The pH titration data and ¹H NMR data agree well with the exception that the species distribution diagrams show the initial formation of the N1-only and N1,N7-bridged complexes to occur at somewhat higher pH than do the NMR data. This is due to a concentration difference in the two sets of data.     

1H(C) and 1H(N) total NOE correlations in a single 3D NMR experiment. 15N and 13C time-sharing in t1 and t2 dimensions for simultaneous data acquisition

Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as ¹⁵N and ¹³C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous ¹³C and ¹⁵N spin labeling coherence in both t ₁ and t ₂ dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between ¹H_N and ¹H_C resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, ¹⁵N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins.     

A novel approach for assessing protein synthesis in channel catfish, Ictalurus punctatus

A comprehensive understanding of animal growth requires adequate knowledge of protein synthesis (PS), which in fish, has traditionally been determined by the flooding dose method. However, this procedure is limited to short-term assessments and may not accurately describe fish growth over extended periods of time. Since deuterium oxide (²H₂O) has been used to non-invasively quantify PS in mammals over short- and long-term periods, we aimed at determining if ²H₂O could also be used to measure PS in channel catfish. Fish were stocked in a 40-L aquarium with ~ 4% ²H₂O and sampled at 4, 8 and 24 h (n = 6 at each time period) to determine ²H-labeling of body water (plasma), as well as protein-free and protein-bound ²H-labeled alanine. The labeling of body water reflected that of aquarium water and the labeling of protein-free alanine remained constant over 24 h and was ~ 3.8 times greater than that of body water. By measuring ²H-labeled alanine incorporation after 24 h of ²H₂O exposure we were able to calculate a rate of PS: 0.04 ± 0.01% h^− 1. These results demonstrate that PS in fish can be effectively measured using ²H₂O and, because this method yields integrative measures of PS, is relatively inexpensive and accounts for perturbations such as feeding, it is a novel and practical assessment option.     

New pulsed field gradient NMR experiments for the detection of bound water in proteins

Summary New H₂O-selective homonuclear and heteronuclear 2D NMR experiments have been designed for the observation of protein hydration (PHOGSY, Protein Hydration Observed by Gradient Spectroscop Y). These experiments utilize selective excitation of the H₂O resonance and pulsed field gradients for coherence selection and efficient H₂O suppression. The method allows for a rapid and sensitive detection of H₂O molecules in labelled and unlabelled proteins. In addition it opens a way to measure the residence time of water bound to proteins. Its application to uniformly ¹⁵N-labelled FKBP-12 (FK-506 binding protein) is demonstrated.     

Structure determination of proteins in <Superscript>2</Superscript>H<Subscript>2</Subscript>O solution aided by a deuterium-decoupled 3D HCA(N)CO experiment

We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone ¹Hα and ¹³Cα to those of ¹³C′ in the preceding residue. By applying ²H decoupling, the experiment was accomplished with high sensitivity comparable to that of HCA(CO)N. When combined with HCACO, HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone ¹³C′ and amide ¹⁵N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for ¹³C/¹⁵N-labeled GB1 dissolved in ²H₂O. The quality of the GB1 structure determined in ²H₂O was similar to that determined in H₂O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures in ²H₂O or H₂O at high pH values.     

Direct assessment of water exchange on a Gd(III) chelate bound to a protein

 The Gd(III) complex of 4-pentylbicyclo[2.2.2]octane-1-carboxyl-di-l-aspartyl-lysine-derived DTPA, [GdL(H₂O)]^2–, binds to serum albumin in vivo, through hydrophobic interaction. A variable temperature ¹⁷O NMR, EPR, and Nuclear Magnetic Relaxation Dispersion (NMRD) study resulted in a water exchange rate of k ²⁹⁸ _ex=4.2×10⁶ s^–1, and let us conclude that the GdL complex is identical to [Gd(DTPA)(H₂O)]^2– in respect to water exchange and electronic relaxation. The effect of albumin binding on the water exchange rate has been directly evaluated by ¹⁷O NMR. Contrary to expectations, the water exchange rate on GdL does not decrease considerably when bound to bovine serum albumin (BSA); the lowest limit can be given as k _{ex, GdL-BSA}=k _ex, GdL / 2. In the knowledge of the water exchange rate for the BSA-bound GdL complex, the analysis of its NMRD profile at 35  °C yielded a rotational correlation time of 1.0 ns, one order of magnitude shorter than that of the whole protein. This value is supported by the longitudinal ¹⁷O relaxation rates. This indicates a remarkable internal flexibility, probably due to the relatively large distance between the protein- and metal-binding moieties of the ligand. Received: 25 June 1998 / Accepted: 11 August 1998     

Structural modifications of oxidized insulin B-chain induced by glycerol addition

Summary ¹H-NMR studies of the bovine insulin S-sulfonated B-chain are reported in H₂O/D₂O (9/1) and in glycerol-d ₅ (5 M) using two-dimensional NMR spectroscopy. The first results show that the oxidized insulin B-chain secondary structure differs from that of native insulin by a loss of the α-helix between the two disulfide bridges and that the glycerol favours the structuring of the peptide.     

Yttrium containing head-on complexes of silico- and germanotungstate: Synthesis, structure and solution properties

Two dimeric head-on complexes of yttrium containing silico- and germanotungstate were isolated from the one-pot reaction of Y(NO₃)₃·6H₂O with the lacunary Na₁₀[MW₉O₃₄]·16H₂O (M = Si and Ge) building blocks in an acetate buffer at pH 4.5. Both polyanions were structurally characterized using various solid-state analytics, such as single-crystal X-ray diffraction, single-crystal X-ray analysis shows that both polyanions crystallize in the monoclinic crystal system (S.G. P2₁/c). FT-IR spectroscopy, or thermogravimetric analysis. The stability of the polyanion in aqueous solution was studied by multinuclear NMR spectroscopy (¹⁸³W, ⁸⁹Y, ²⁹Si, ¹³C, and ¹H). As expected, the ¹⁸³W NMR spectra display six peaks in the intensity ratio of 4:4:2:4:4:4 which indicates that both polyanions exist as dimeric entities in aqueous solution.     

A triple-resonance pulse scheme for selectively correlating amide1HN and15N nuclei with the1Hα proton of the preceding residue

Summary A 3D¹H–¹⁵N–¹³C triple resonance experiment is presented that contains exclusively cross peaks between the¹H^N and¹⁵N nuclei of one residue with the H of the preceding residue. The pulse sequence, designed to minimize the time coherence, is transverse on nuclei with short T₂ values. The experiment consists of coherence transfers via one-bond couplings from the H^N via N, CO, C to the H and back to the H^N for detection; it is called HN(COCA)HA. The experiment was tested on uniformly¹⁵N- and¹³C-enriched T4 lysozyme.     

New chiral macrocyclic lanthanide complexes derived from (1R,2R)-1,2-diaminocyclohexane and 2,6-diformylpyridine

The new enantiopure complexes [LnL](NO₃)₃ · nH₂O (Ln = Dy⁺³, Ho⁺³, Er⁺³, Lu⁺³) and [LnL]Cl₃ · nH₂O (Ln = Nd⁺³, Sm⁺³, Gd⁺³, Tb⁺³, Dy⁺³, Ho⁺³, Er⁺³, Tm⁺³, Lu⁺³) of the chiral macrocycle L derived from (1R,2R)-1,2-diaminocyclohexane and 2,6-diformylpyridine have been synthesised. The preference of macrocycle L for the heavier lanthanide(III) ions has been established on the basis of competition reaction. The complexes have been characterised by NMR spectroscopy and mass spectrometry. ¹H NMR signals of deuterated water solutions of the Ce⁺³, Nd⁺³ and Eu⁺³ complexes have been assigned on the basis of the COSY and HMQC spectra, and for the remaining lanthanide complexes the signals were assigned on the basis of linewidths analysis. The paramagnetic shifts of the series of lanthanide complexes [LnL](NO₃)₃ · nH₂O and [LnL]Cl₃ · nH₂O have been analysed using both crystal-field dependent and independent methods in order to separate contact and dipolar contributions and establish isostructurality along the series of lanthanide complexes in solution. The data obtained for nitrate derivatives in organic solvent indicate rather irregular deviations from the plots based on those methods, while the plots obtained for water solutions show the characteristic brake in the middle of the lanthanide series, that is interpreted as a result of change of the number of axially coordinated water molecules. The apparent inconsistencies of results obtained on the basis of crystal-field independent method are discussed.     

A new method for random mutagenesis by error-prone polymerase chain reaction using heavy water

Error-prone polymerase chain reactions (epPCRs) are often used to introduce mutations in random mutagenesis, which has been used as a tool in protein engineering. Here, we developed a new method of epPCR using heavy water as a solvent instead of normal water (H₂O). Rhodopsin cDNA of the Ayu fish (Plecoglossus altivelis) was used as a template and was amplified using five different conditions: (A) 100% H₂O with no Mn²⁺, (B) 100% H₂O/0.6 mM Mn²⁺, (C) 99% D₂O with no Mn²⁺, (D) 99% D₂O/0.6 mM Mn²⁺ and (E) 99% H₂¹⁸O with no Mn²⁺. The 13,960 (for each of the conditions A to D) and 33,504 (for condition E) base pairs were sequenced. A maximum error rate of 1.8 × 10⁻³ errors/bp was detected in condition D, without any particular hot-spot mutations. A high preference for AT → GC transitions was observed in condition D, whereas a high preference for transitions over transversions was observed in condition C. All of the mutations observed in condition E were transversions. When conditions A and C were applied to another template, the honeybee actin gene, the results were comparable to those for Ayu rhodopsin. Based on these results, the use of heavy water, instead of H₂O, as a solvent for epPCR can introduce random mutations without positional bias, template dependency or decreased yield. Our new epPCR method, and possibly combining the use of D₂O and H₂¹⁸O, may be a powerful random mutagenesis technique.     

Study on the interaction of a copper(II) complex containing the artificial sweetener aspartame with human serum albumin

A copper(II) complex containing aspartame (APM) as ligand, Cu(APM)₂Cl₂·2H₂O, was synthesized and characterized. In vitro binding interaction of this complex with human serum albumin (HSA) was studied at physiological pH. Binding studies of this complex with HSA are useful for understanding the Cu(APM)₂Cl₂·2H₂O–HSA interaction mechanism and providing guidance for the application and design of new and more efficient artificial sweeteners drive. The interaction was investigated by spectrophotometric, spectrofluorometric, competition experiment and circular dichroism. Hyperchromicity observed in UV absorption band of Cu(APM)₂Cl₂·2H₂O. A strong fluorescence quenching reaction of HSA to Cu(APM)₂Cl₂·2H₂O was observed and the binding constant (K_f) and corresponding numbers of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy change (?H) and entropy change (?S) were calculated to be ?458.67 kJ mol^?1 and ?1,339 J mol^?1 K^?1 respectively. According to the van’t Hoff equation, the reaction is predominantly enthalpically driven. In conformity with experimental results, we suggest that Cu(APM)₂Cl₂·2H₂O interacts with HSA. In comparison with previous study, it is found that the Cu(II) complex binds stronger than aspartame.     

Synthesis of Me2LSn(o-CH3-C2B10H10): Crystal structure of Sn ← O intramolecularly coordinated organotin compound containing 1-methyl-o-carborane

The synthesis and molecular structure of Me₂LSn(1-Me-1,2-C₂B₁₀H₁₀) (3), L is 2,6-(t-BuOCH₂)₂C₆H₃⁻, the precursor for the synthesis of other Sn ← O intramolecularly coordinated organotin(IV) compounds containing 1-methyl-o-carborane (1-Me-1,2-C₂B₁₀H₁₁) is reported. Compound 3 was characterized by the help of ¹H, ¹¹B, ¹³C, ¹¹⁹Sn NMR spectroscopy and X-ray diffraction technique. For the preparation of 3, a novel triorganotin(IV) compound Me₂LSnCl (2) has been prepared since the reaction of sterically more demanding Ph₂LSnCl (1) with 1-Me-1,2-C₂B₁₀H₁₀Li did not occur.     

Improved 3D gd-HCACO and gd-(H)CACO-TOCSY experiments for isotopically enriched proteins dissolved in H2O

Summary Pulsed field gradients were incorporated into the HCACO experiment for acquiring spectra on isotopically enriched protein samples dissolved in H₂O. Excellent water suppression and spectral quality were achieved using the modified pulse sequence (gd-HCACO), as demonstrated for a ¹³C-/¹⁵N-labeled sample of the SH2 domain from the hematopoietic cellular kinase dissolved in 90% H₂O/10% D₂O. Strong correlations for all residues were observed in the gd-HCACO spectrum, even for residues having -protons resonating exactly at the H₂O frequency. The HCACO-TOCSY experiment was modified to correlate intraresidue ¹³C (rather than ¹H), carbonyl (¹³C), and aliphatic side-chain protons [(H)CACO-TOCSY]. Pulsed field gradients were also incorporated into the (H)CACO-TOCSY experiment for water suppression.     

Efficient assignment of methyl resonances: Enhanced sensitivity by gradient selection in a DE-MQ–(H)CC<Superscript><Emphasis Type="Italic">m</Emphasis></Superscript>Ht <Superscript><Emphasis Type="Italic">m</Emphasis></Superscript>–TOCSY experiment

We present a gradient selected and doubly sensitivity-enhanced DE-MQ–(H)CC ^m H ^m –TOCSY experiment for the sequence-specific assignment of methyl resonances in ¹³C,¹⁵N labeled proteins. The proposed experiment provides improved sensitivity and artifact suppression relative to the phase-cycled experiments. One part of the ¹³Cchemical shift evolution takes place under heteronuclear multiple quantum coherence, whereas the other part occurs under ¹³C single quantum coherence in a semi-constant time fashion. The feasibility of the experiment was assessed using ¹⁵N,¹³C labeled Mus musculus coactosin (16 kDa), having a rotational correlation time of 14.5 ns at 15 °C in D₂O. A 16-h experiment on 600 MHz ¹H yielded good quality data and enabled the assignment of 70 out of 72 methyl groups in coactosin. As well as being an improved approach for methyl resonance assignment, this experiment can also be highly valuable for the rapid assignment of methyl resonances in SAR by NMR studies.     

Hydrolysis study of the bifunctional antitumour compound RAPTA-C, [Ru(eta6-p-cymene)Cl2(pta)

The hydrolysis of [Ru(η⁶-p-cymene)Cl₂(PTA)] (PTA = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decanephosphine; RAPTA-C) was studied using UV-visible (UV-vis) spectrophotometry and NMR spectroscopy. In analogy to in silico studies, [Ru(η⁶-p-cymene)Cl(H₂O)(PTA)]⁺ was found to be the most abundant hydrolysis product, although the dihydrolysed species [Ru(η⁶-p-cymene)(OH)(H₂O)(PTA)]⁺ and the dichloro compound are present. Rate constants for the different aquation and anation steps and the equilibrium constants were determined. Hydrolysis is suppressed at high chloride concentrations. These results have important implications on the mode of action of the RAPTA drug candidates.