The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane.

One of the key pieces of information from pressure denaturation experiments is the standard volume change for unfolding (Delta V(o)). The pressure dependence of the volume change, the standard compressibility change (Delta K(o)T), is typically assumed to be zero in the analysis of these experiments. We show here that this assumption can be incorrect and that the neglect of compressibility differences can skew the interpretation of experimental results. Analysis of experimental, variable-pressure NMR data for bovine pancreatic ribonuclease A in 2H2O at pH 2.0 and 295 K yielded the following statistically significant, non-zero values: Delta K(o) T = 0.015 +/- 0.002 mL mol-1 bar-1, Delta V(o) = -21 +/- 2 mL mol-1, and Delta G(o) = 2.8 +/- 0.3 kcal mol-1. The experimental protein stability is in good agreement with one (Delta G(o) = 2.5 kcal mol-1) determined independently for the same protein by calorimetry at atmospheric pressure under equivalent conditions [Makhatadze, G. I., Clore, G. M., and Gronenborn, A. M. (1995) Nat. Struct. Biol. 2, 852-855]. The positive value for Delta K(o)T indicates that the denatured form of ribonuclease A is more compressible than the native form; this is explained in terms of an interplay between the intrinsic compressibility of the protein and solvation effects. When the same data were fitted to a model that assumes a zero compressibility change, the Delta G(o) value of 4. 0 +/- 0.1 kcal mol-1 returned by the model no longer agreed with the independent measurement, and the Delta V(o) returned by the model was a very different -59 +/- 1 mL mol-1. By contrast, it was not possible to carry out a similar thermodynamic analysis of fluorescence spectroscopic data for the denaturation of staphylococcal nuclease to yield well-defined values of Delta G(o), Delta V(o), and Delta K(o)T. This limitation was shown by evaluation of synthetic data to be intrinsic to spectroscopic data whose analysis requires fitting of the plateaus at either side of the transition. Because NMR data do not have this requirement, they can be analyzed more rigorously.

ABSTRACT Formation of methanethiol from methionine is widely believed to play a significant role in development of cheddar cheese flavor. However, the catabolism of methionine by cheese-related microorganisms has not been well characterized. Two independent methionine catabolic pathways are believed to be present in lactococci, one initiated by a lyase and the other initiated by an aminotransferase. To differentiate between these two pathways and to determine the possible distribution between the pathways, 13 C nuclear magnetic resonance (NMR) performed with uniformly enriched [ 13 C]methionine was utilized. The catabolism of methionine by whole cells and cell extracts of five strains of Lactococcus lactis was examined. Only the aminotransferase-initiated pathway was observed. The intermediate and major end products were determined to be 4-methylthio-2-oxobutyric acid and 2-hydroxyl-4-methylthiobutyric acid, respectively. Production of methanethiol was not observed in any of the 13 C NMR studies. Gas chromatography was utilized to determine if the products of methionine catabolism in the aminotransferase pathway were precursors of methanethiol. The results suggest that the direct precursor of methanethiol is 4-methylthiol-2-oxobutyric acid. These results support the conclusion that an aminotransferase initiates the catabolism of methionine to methanethiol in lactococci.

Theory,experimental aspects, and use in structure calculation of cross-correlated relaxation rates measured on zero- and double-quantum coherences in liquid state NMR are presented. The relative size of the interaction depends on the projection angle between the two tensorial interactions. The tensorial interaction can be either a dipolar interaction or a chemical shift anisotropy relaxation mechanism (CSA). Effects of additional sources of relaxation on the cross-correlated relaxation rates are analyzed. Also, an easy-to-use formalism is given to manipulate different cross-correlated relaxation interactions. The application addresses measurement of the backbone angle ψ in a protein by measuring dipole(15N–1H)–dipole(13Cα–1Hα) and CSA(15N)–dipole(13Cα–1Hα) cross-correlated relaxation rates. It is shown that ambiguities due to the 3 cos2θ-1 dependence of one cross-correlated relaxation rate can be overcome by measuring additional cross-correlated relaxation rates. The use of cross-correlated relaxation rates is demonstrated in structure calculations.

Pursuant to the original description by Kay, Ikura, Tschudin, and Bax, a growing variety of double- and triple-resonance, three-dimensional (3D) and four-dimensional (4D) nuclear magnetic resonance (NMR) experiments have been developed. Although fully equipped commercial NMR spectrometer consoles produced after 1990 have been capable of performing these demanding experiments, earlier consoles lack the necessary decoupling capabilities and requisite number of transmitter channels. Thus, although the original two-dimensional (2D) triple-resonance experiments have been able to be carried out some of with a standard 1985 vintage Bruker Instruments AM-400 NMR spectrometer and BSV-3 X-nucleus decoupler, it was needed to make extensive modifications in order to perform multinuclear 3D and 4D experiments. Kay et al. described the adaptation of a Bruker AM console for early 3D and 4D triple-resonance experiments. With more extensive modifications, most of the current multinuclear 3D and 4D experiments have been able to be performed with older model Bruker Instruments AM-500 and AM-600 consoles and BSV-3 X-nucleus decouplers.

Sequence-specific assignments were determined for the diamagnetic proton resonances from recombinant Anabaena 7120 heterocyst ferredoxin (M(r) = 11,000) produced in Escherichia coli. Several samples selectively labeled with nitrogen-15 were prepared for use in two-dimensional heteronuclear multiple quantum coherence (HMQC) [Müller, L. (1979) J. Am. Chem. Soc. 101, 4481-4484] experiments. A sample uniformly labeled with nitrogen-15 was also prepared and used in two three-dimensional experiments: NOESY-HMQC and TOCSY-HMQC [Zuiderweg, E. R. P., & Fesik, S. W. (1989) Biochemistry 28, 2387-2391; Marion, D., Ikura, M., Tsuchudin, R., & Bax, A. (1989) J. Magn. Reson. 85, 393-399]. The sequential assignment strategy relied on the detection of 15N-edited interresidue 1H alpha i/1HNi+1 NOE connectivities. Starting points and checks were provided by HMQC spectra of the selectively labeled samples. A sample doubly labeled with carbon-13 and nitrogen-15 was also prepared and used in three triple-resonance experiments: HNCA, HNCO, and HN(CO)CA [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667; Kay, L. E., Ikura, M., Tschudin, R., & Bax, A. (1990) J. Magn. Reson. 89, 496-514]. The HNCA and HN(CO)CA spectra, which were used to confirm assignments from NOE connectivities, provided independent sequential assignments from spin couplings. Resonances from 18 residues were not seen in the diamagnetic region of the NMR spectrum. Several of these residues are very close to the [2Fe-2S] cluster, and their absence is explained by paramagnetic broadening and/or shifting.(ABSTRACT TRUNCATED AT 250 WORDS)

Publisher Summary The objectives of this chapter are 2-fold. First, it presents basic unifying features of pulse sequences so that the underlying mechanics of even complicated sequences become more transparent. Second, a step-by-step guide to present the practical implementation and processing of multidimensional experiments is illustrated. Much progress has resulted from generalization of heteronuclear twodimensional (2D) NMR experiments with 13 C- and 15 N-labeled biomolecules to higher dimensions. The ultimate goal of NMR investigations of biomolecules is to obtain structural and dynamic information. To this end, many specialized experimental techniques similar to those described above have been developed, which allow the measurement of parameters that provide distance and dihedral angle constraints. In conjunction with the methods described in this chapter, computer-automated resonance assignments and spectral analysis techniques should facilitate efficient studies of larger biomolecules and are expected to accelerate the pace of NMR contributions to structural biochemistry.

Backbone 1H, 13C, and 15N NMR assignments were obtained for the complex of chicken muscle adenylate kinase (AK) with its bisubstrate analog, MgAP5A [magnesium P1,P5-bis(5'-adenosyl)-pentaphosphate]. The assignments were used to elucidate the secondary structures and the enzyme-MgAP5A interactions. The work involves two unusual features: the molecular weight of AK (21.6 kDa) is one of the largest, on a monomeric basis, for which nearly complete assignment has been reported to date, and the assignment was performed at pH 7.1 instead of the acidic pH used for most other proteins. The results are summarized as follows. Firstly, unambiguous sequential assignments of backbone resonances have been achieved effectively by the combined use of two sequential assignment methods: NOE-directed assignments and the recently developed 1J-coupling-directed assignments. The starting points of the assignments were provided by several specifically labeled enzyme samples. Over 90% of the backbone 1H, 13C, and 15N resonances have been assigned. Secondly, spin system information was obtained from the HCCH-TOCSY and HCCH-COSY experiments as well as from 2D homonuclear NMR data. Overall, the side-chain resonances of ca. 40% of the residues, including most of the those displaying NOEs with the adenosine moieties of MgAP5A, have been assigned. Thirdly, secondary structural elements in the AK-MgAP5A complex were identified by extensive analyses of 1H-15N 2D HMQC-NOESY and 3D NOESY-HMQC spectra. Overall, the enzyme consists of ca. 60% alpha-helices and a five-stranded parallel beta-sheet. The results are compared with the secondary structure of the free AK from porcine muscle in crystals [Dreusicke, D., Karplus, P. A., & Schulz, G. E. (1988) J. Mol. Biol. 199, 359-371]. Lastly, most of the intermolecular NOEs between AK and the adenosine moieties of MgAP5A have been identified: Thr39, Leu43, Gly64, Leu66, Val67, Val72, and Gln101 are in proximity to the adenosine moiety of the adenosine 5'-monophosphate site, whereas Thr23 is in proximity to that of the adenosine 5'-triphosphate site. These data are discussed in relation to previous results from site-directed mutagenesis, NMR, and X-ray studies and in relation to the mechanism of catalysis.

The backbone H-1 and N-15 resonances of unligated staphylococcal nuclease H124L (recombinant protein produced in Escherichia coli whose sequence is identical to the nuclease produced by the V8 strain of Staphylococcus aureus) have been assigned by three-dimensional (3D) H-1-N-15 NOESY-HMQC NMR spectroscopy at 14.1 tesla. The protein sample used in this study was labeled uniformly with N-15 to a level greater than 95% by growing the E. coli host on a medium containing [99% N-15]ammonium sulfate as the sole nitrogen source. The assignments include 82% of the backbone H-1(N) and H-1(alpha) resonances as well as the N-15 resonances of non-proline residues. Secondary structural elements (alpha-helices, beta-sheets, reverse turns, and loops) were determined by analysis of patterns of NOE connectivities present in the 3D spectrum.

This chapter discusses the methodological aspects of experiments that employ heteronuclear coherence transfer for measurement or assignment of chemical shifts, coupling constants, or relaxation times. It will not only describe the heteronuclear 2D- or 3D-shift correlation experiments, but also includes selective and non-selective 1D techniques designed in particular for the measurement of heteronuclear couplings and chemical shifts of low-γ nuclei, spin-echo experiments intended for multiplicity editing, and experiments with “passive” selection of the satellite lines. The chapter also provides an overview on the application of these techniques in inorganic, organoelement, and organometallic chemistry. It discusses that from a methodical point of view, interesting aspects in future evolutions of nX, mY correlation experiments can be foreseen in an increased use of 3D experiments with 1H detection for further improvements in sensitivity, resolving power, and the incorporation of pulsed field gradients that are now a consolidated strategy for 1H -detected inverse experiments. The latter aspect would be particularly interesting, as the possibility to obtain very “clean” spectra with minimum artefacts should enable to extend the application of indirect detection metal NMR studies to lower the concentration ranges and improve indirect detection through very small coupling constants, thus further increasing the chemical applicability of these methods.

The flavodoxin isolated from Anabaena 7120 grown under iron-limiting conditions has been studied in its oxidized form. Flavodoxin 95%+ enriched in VN was obtained by growing the cyanobacterium with 98% VN nitrate as the sole nitrogen source. A one-dimensional H NMR (500 MHz) spectrum has been collected, as well as a double-quantum-filtered COSY spectrum in D2O. One-dimensional VN NMR (50.68 MHz) spectra have been obtained by observing nitrogen directly and by using the INEPT pulse sequence. Results of a two-dimensional H-detected VN experiment allowed the correlation of VN and H resonances of VN-/sup I/H groups. The four nitrogen resonance of the FMN cofactor have been assigned: N(1), 188.0 ppm; N(3), 162.5 ppm; N(5) 335.0 ppm; and N(10), 163.5 ppm. The resonance assigned to N(3) is coupled to a proton at 10.9 ppm. Shifts in the positions of these VN resonances, compared to corresponding ones in free FMN, suggest that these positions are strongly hydrogen-bonded in the oxidized state as has been observed with lower molecular weight flavodoxins.

The acid-base titration of bleomycin-A2 in D2O solution at 35 +/- 5 degrees has been monitored by 13C n.m.r. spectroscopy at 67.89 MHz. The following pKDa values were obtained: 3.68 +/- 0.05 (secondary amine), 5.29 +/- 0.03 (imidazole), and 8.23 +/- 0.19 (primary amine), where KDa is the dissociation constant in D2O solution. The equilibrium isotope effects (pKDa--pKa in H2O) are: 0.70 +/- 0.06 (secondary amine), 0.28 +/- 0.04 (imidazole), and 0.85 +/- 0.19 (primary amine). Titration of the imidazole group of Bleo-A2 occurs at Npi, i.e. only Ntau is protonated in basic solution. Significant protonation shifts are almost completely limited to carbons of the N-terminal tetrapeptide, suggesting that the C-terminal tripeptide extends into the solvent and interacts to a minimal extent with the rest of the molecule. Long range protonation shifts associated with titration of the imidazole and secondary amine groups indicate that protonation of one or both of these sites is probably accompanied by significant conformational changes. The observed protonation shifts generally fail to correlate with Zn(II) complexation shifts reported by Dabrowiak et al. (1973, Biochemistry 17., 4090) indicating that ligation sites cannot unambiguously be determined from these complexation shifts. The complexation shifts previously attributed to coordination of the imidazole and carbamoyl groups probably result from conformational changes.