Deane D. McIntyre

Physical and structural characterization of yersiniophore, a siderophore produced by clinical isolates of Yersinia enterocolitica

Clinical isolates of Yersinia enterocolitca, which belong to mouse-lethal serotypes, produce the siderophore yersiniophore. Siderophore production was shown to be iron regulated and to reach maximum production in late log phase. Yersiniophore is a fluorescent siderophore with maximum excitation at 270 nm and a major emission peak at 428 nm. Absorption maxima were seen at 210 and 250 nm with a low broad peak from 280 to 320 nm. Purification of unchelated yersiniophore for structural analysis was made difficult by low yields (1–2 mg mg-1), and susceptibility to acid hydrolysis, oxidation and possibly polymerization. Yersinophore was therefore purified as an Al3+ chelate, which was found to be stable in solution for several weeks. To purify Al3+-yersiniophore, unchelated yersiniophore was first extracted from culture supernatants with dichloromethane, concentrated by rotary evaporation and adsorbed to a DEAE-sephacel column. Al3+-yersiniophore was eluted with 0.01 m AlCl3 and further purified by HPLC. The structure was established by a combination of elemental analysis, high resolution mass spectrometry and two-dimensional NMR experiments. Yersiniophore is a phenolate-thiazole siderophore with the formula C21H24N3O4S3Al and a molecular weight of 505.07404 when chelated to Al3+. The structure of yersiniophore was determined to be closely related to the structures of pyochelin, produced by Pseudomonas aeruginosa, and anguibactin, produced by Vibrio anguillarum.

Specific (CD2CD2SCHD2)-C-12-D-beta-C-12-D-gamma-C-13-H-epsilon Isotopomer Labeling of Methionine To Characterize Protein Dynamics by H-1 and C-13 NMR Relaxation Dispersion

Protein dynamics on the micro- to millisecond time scale is increasingly found to be critical for biological function, as demonstrated by numerous NMR relaxation dispersion studies. Methyl groups are excellent probes of protein interactions and dynamics because of their favorable NMR relaxation properties, which lead to sharp signals in the 1H and 13C NMR spectra. Out of the six different methyl-bearing amino acid residue types in proteins, methionine plays a special role because of its extensive side-chain flexibility and the high polarizability of the sulfur atom. Methionine is over-represented in many protein–protein recognition sites, making the methyl group of this residue type an important probe of the relationships among dynamics, interactions, and biological function. Here we present a straightforward method to label methionine residues with specific 13CHD2 methyl isotopomers against a deuterated background. The resulting protein samples yield NMR spectra with improved sensitivity due to the essentially 100% population of the desired 13CHD2 methyl isotopomer, which is ideal for 1H and 13C spin relaxation experiments to investigate protein dynamics in general and conformational exchange in particular. We demonstrate the approach by measuring 1H and 13C CPMG relaxation dispersion for the nine methionines in calcium-free calmodulin (apo-CaM). The results show that the C-terminal domain, but not the N-terminal domain, of apo-CaM undergoes fast exchange between the ground state and a high-energy state. Since target proteins are known to bind specifically to the C-terminal domain of apo-CaM, we speculate that the high-energy state might be involved in target binding through conformational selection.

Investigation of skeletal muscle denervation and reinnervation using magnetic resonance spectroscopy

OBJECTIVE: To determine changes in skeletal muscle metabolism after nerve transection and repair and to correlate metabolic changes with functional recovery. STUDY DESIGN AND SETTING: Male Wistar rats were divided into 6 experimental groups plus a control group. The posterior tibial nerve was transected and reapproximated. At varying times after surgery (1, 2, 4, 6, or 8 weeks) animals were sacrificed, the gastrocnemius muscle was harvested, and proton nuclear magnetic resonance (NMR) spectroscopy was performed. Functional recovery was measured using the sciatic function index. RESULTS: Animals undergoing nerve repair all showed functional recovery whereas the nonrepaired nerve group did not. Concentration of glucose and lactate increased after denervation and then returned toward normal. Choline concentration decreased and then returned toward normal. In animals not undergoing nerve repair, the metabolic abnormalities persisted and showed no sign of recovery over the 8-week observation period. CONCLUSIONS: 1H NMR spectroscopy is a potentially useful tool to study changes in skeletal muscle metabolism after motor nerve injury. SIGNIFICANCE: NMR spectroscopy is rapidly developing into a clinically useful tool. High-field magnets have improved resolution and data acquisition. Basic experiments, such as those described here, will help guide the use of NMR spectroscopy in clinical medicine and will also lead to a better understanding of basic mechanisms of nerve injury and repair.

Nitrogen-14 NMR relaxation, and reorientation behavior of dissolved dinitrogen

In recent years, nitrogen- 14 and - 15 NMR spectroscopy has become an important technique in organic chemistry and in biochemistry ( Z-4). A frequent occurrence in the NMR spectra of both nuclei is the presence of a signal at approximately -7 1.5ppm (referred to neat nitromethane; -66 ppm referred to aqueous nitrate). This resonance, which has been observed in water (4) and a range of organic solvents (.5-7), has been the subject of some confusion in the literature. In the case of nitrogen-14 NMR spectra, the signal for the quadrupolar nucleus has a remarkably narrow linewidth (about 25 Hz) compared to those recorded for a wide variety of other substances. This indicates a fairly high degree of electronic symmetry about the nitrogenatom and/or a very short correlation time ( 7,). In a recent report (8)) this signal has been assigned to dissolved dinitrogen on the basis that it could be removed by degassing´the solution; however, no reference was made to the fact that it had a remarkably narrow linewidth compared to other 14N NMR resonances. When detected in the 15NCIDNP spectra of the decomposition products of diazonium ions (5, 6) as well as azo compounds ( 7)) the signal has been assigned either to dinitrogen (6, 7) or to a terminal diazonium nitrogen (5). This work is in general agreement with our own observations over a period of years which indicate that the signal arises from dissolved dinitrogen. We have measured the 14N chemical shift of dissolved N2 in a number of solvents at 25°C and have also determined the T, and T2 relaxation times under a variety of conditions in solvents of different viscosity. This Note is concerned with a discussion of these 14N NMR observations and with the determination of the correlation time of dissolved dinitrogen which permits the determination of both the enthalpy and the entropy of activation via the Eyring equation. These results will be compared with earlier reported data regarding the chemical shift and relaxation of liquid nitrogen obtained under a variety of conditions ( 9-12). All spectra were obtained on a Bruker AM-400 wide-bore NMR spectrometer operating in the FT mode at a frequency of 28.9 MHz for 14N and 40.5 MHz for 15N, using a 10 mm broadband probe. Typical conditions for the acquisition of 14N spectra

14 N and 15N NMR characterization and the identification in sulphur–ammonia solution of the S7N– ion

The cyclic sulphur imide, S7NH, and the thermally unstable S7N–-ion have been characterized by 14N and 15N NMR spectroscopy; the existence of S7N–, in addition to S4N– and small amounts of S3N–, in sulphur–ammonia solutions is demonstrated by 14N NMR spectroscopy.

Anomalous shielding and hidden partner chemical exchange in the 1H NMR spectra of the bisurethane diazetidines, the 1,2-diaryl-3,5-dialkyl-6,7-dialkoxycarbonyl-4-oxo-6,7-diazabicyclo [3.2.0]hept-2-enes

The bicyclic diazetidines (3), (11), and (17), have trans-pyramidal nitrogens, the ester group on N(7) being sterically protected and very unreactive. Its alkyl group is close to the face of the aryl ring attached to C(1) and shows remarkable shielding effects in its 1H NMR spectrum, while the alkyl group of the N(6) ester shows a more normal resonance. In the methyl esters the OMe group of the N(7) ester is ca. 1 ppm upfield of the other ester OMe group and in each of the oxymethylene compounds one of the protons of the N(7) ester group is upfield of its (unshielded) diastereotopic partner by ca. 2 ppm.These shielded resonances are extremely broadened at room temperature, especially at high field. DNMR studies show that on cooling, the shielded absorption in each case broadens further and then sharpens into two peaks, an effect due to the interconversion of two major conformations with one or more of very low population, an example of ‘hidden partner exchange.’ The exchange processes responsible, which occur between protons in the N(7) ester group in very shielded (major conformations) and normal (minor) environments, must involve either N(7) inversion or N(7)–CO rotation.The di-methyl and -trichloroethyl compounds (3a) and (3c), were investigated in detail in CD2Cl2 and C7D8. In (3a) the N(7) ester OMe resonance of the minor partner was found at δ 3.65 (ca. 3%, 200 K, CD2Cl2), its identity being confirmed by low-temperature examination of the deuteriated isomers (20) and (21). In (3c) there are two minor conformations, ca. 2% each.Two major and at least one minor conformation were observed for the dibenzyl and dimethyl esters (3d) and (11); the latter also showed a very shielded Me in the ring C(5) ethyl group. The presence of a minor conformation was not conclusively detected in the diethyl ester (3b), but was inferred from the broadening of the upfield signals of its two major conformations. The acecylcone derived compound (17) showed three major conformations (46 : 36 : 18%), the second and third together (unresolved) being the most shielded in the N(7) group, though less so than in the other diazetidines.Rotation of the C(1) Ph ring has also been identified as a conformational process in all the diazetidines [except (17)] but is quite independent of the processes leading to anomalous shielding of the ester groups.