Application of liquid chromatography-mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusions.

A new method involving zinc sulphate deproteinization was developed to study short chain fatty acids (SCFA) production in the colon and subsequent occurrence of SCFA in blood. SCFA were baseline separated in a 30 min cycle using ion-exclusion chromatography and detected by mass spectrometry. Concentrations could be measured down to 10 microM and isotopomeric distributions could be assessed, enabling the conduction of tracer studies to study changes in SCFA synthesis. The applicability of the method was tested in an extensively characterized pig model yielding portal SCFA concentrations ranging from 70 microM (butyric acid) to 150 microM (propionic acid) to 440 microM (acetic acid) prior to butyrate tracer infusion, reaching butyric acid isotopic steady state within 2 h.

A refined model of the chlorosomal antennae of the green bacterium Chlorobium tepidum from proton chemical shift constraints obtained with high-field 2-D and 3-D MAS NMR dipolar correlation spectroscopy.

Heteronuclear 2-D and 3-D magic-angle spinning NMR dipolar correlation spectroscopy was applied to determine solid-state (1)H shifts for aggregated bacteriochlorophyll c (BChl c) in uniformly (13)C-enriched light harvesting chlorosomes of the green photosynthetic bacterium Chlorobium tepidum. A complete assignment of 29 different observable resonances of the 61 protons of the aggregated BChl c in the intact chlorosomes is obtained. Aggregation shifts relative to monomeric BChl c in solution are detected for protons attached to rings I, II, and III/V and to their side chains. The 2(1)-H(3), 3(2)-H(3), and 3(1)-H resonances are shifted upfield by -2.2, -1, and -3.3 ppm, respectively, relative to monomeric BChl c in solution. Although the resonances are inhomogeneously broadened and reveal considerable global structural heterogeneity, the 5-CH and the 7-Me responses are doubled, which provides evidence for the existence of at least two relatively well-defined structurally different arrangements. Ab initio quantum chemical modeling studies were performed to refine a model for the self-assembled BChl c with two different types of BChl stacks. The BChl in the stacks can adopt either anti- or syn-configuration of the coordinative bond, where anti and syn designate the relative orientation of the Mg-OH bond relative to the direction of the 17-17(1) bond. The analogy between aggregation shifts for BChl c in the chlorosome and for self-assembled chlorophyll a/H(2)O is explored, and a bilayer model for the tubular supra-structure of sheets of BChl c is proposed, from a homology modeling approach.

Assignment of the nonexchanging protons of the alpha-spectrin SH3 domain by two- and three-dimensional 1H-13C solid-state magic-angle spinning NMR and comparison of solution and solid-state proton chemical shifts.

The assignment of nonexchanging protons of a small microcrystalline protein, the alpha-spectrin SH3 domain (7.2 kDa, 62 residues), was achieved by means of three-dimensional (3D) heteronuclear (1H-13C-13C) magic-angle spinning (MAS) NMR dipolar correlation spectroscopy. With the favorable combination of a high B(0)-field, a moderately high spinning frequency, and frequency-switched Lee-Goldburg irradiation applied during 1H evolution, a proton linewidth < or =0.5 ppm at 17.6 Tesla was achieved for the particular protein preparation used. A comparison of the solid-state 1H chemical shifts with the shifts found in solution shows a remarkable similarity, which reflects the identical protein structures in solution and in the solid. Significant differences between the MAS solid- and liquid-state 1H chemical shifts are only observed for residues that are located at the surface of the protein and that exhibit contacts between different SH3 molecules. In two cases, aromatic residues of neighboring SH3 molecules induce pronounced upfield ring-current shifts for protons in the contact area.

Backbone and side-chain 13C and 15N signal assignments of the alpha-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 Tesla.

The backbone and side-chain 13C and 15N signals of a solid 62-residue (u-13C,15N)-labelled protein containing the alpha-spectrin SH3 domain were assigned by two-dimensional (2D) magic angle spinning (MAS) 15N-13C and 13C-13C dipolar correlation spectroscopy at 17.6 T. The side-chain signal sets of the individual amino acids were identified by 2D 13C-13C proton-driven spin diffusion and dipolar recoupling experiments. Correlations to the respective backbone nitrogen signals were established by 2D NCACX (CX=any carbon atom) experiments, which contain a proton-nitrogen and a nitrogen-carbon cross-polarisation step followed by a carbon-carbon homonuclear transfer unit. Interresidue correlations leading to sequence-specific assignments were obtained from 2D NCOCX experiments. The assignment is nearly complete for the SH3 domain residues 7-61, while the signals of the N- and C-terminal residues 1-6 and 62, respectively, outside the domain boundaries are not detected in our MAS spectra. The resolution observed in these spectra raises expectations that receptor-bound protein ligands and slightly larger proteins (up to 20 kDa) can be readily assigned in the near future by using three-dimensional versions of the applied or analogous techniques.

Sample optimization and identification of signal patterns of amino acid side chains in 2D RFDR spectra of the alpha-spectrin SH3 domain.

Future structural investigations of proteins by solid-state CPMAS NMR will rely on uniformly labeled protein samples showing spectra with an excellent resolution. NMR samples of the solid alpha-spectrin SH3 domain were generated in four different ways, and their (13)C CPMAS spectra were compared. The spectrum of a [u-(13)C, (15)N]-labeled sample generated by precipitation shows very narrow (13)C signals and resolved scalar carbon-carbon couplings. Linewidths of 16-19 Hz were found for the three alanine C(beta )signals of a selectively labeled [70% 3-(13)C]alanine-enriched SH3 sample. The signal pattern of the isoleucine, of all prolines, valines, alanines, and serines, and of three of the four threonines were identified in 2D (13)C-(13)C RFDR spectra of the [u-(13)C, (15)N]-labeled SH3 sample. A comparison of the (13)C chemical shifts of the found signal patterns with the (13)C assignment obtained in solution shows an intriguing match.

Characterization of pheophytin ground states in Rhodobacter sphaeroides R26 photosynthetic reaction centers from multispin pheophytin enrichment and 2-D 13C MAS NMR dipolar correlation spectroscopy.

The electronic ground states of pheophytin cofactors potentially involved in symmetry breaking between the A and B branch for electron transport in the bacterial photosynthetic reaction center have been investigated through a characterization of the electron densities at individual atomic positions of pheophytin a from 13C chemical shift data. A new experimental approach involving multispin 13C labeling and 2-D NMR is presented. Bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26 were reconstituted with uniformly 13C biosynthetically labeled (plant) Pheo a in the two pheophytin binding sites. From the multispin labeled samples 1-D and 2-D solid-state 13C magic angle spinning NMR spectra could be obtained and used to characterize the pheophytin a ground state in the Rb. sphaeroides R26 RCs, i.e., without a necessity for time-consuming selective labeling strategies involving organic synthesis. From the 2-D solid state 13C-13C correlation spectra collected with spinning speeds of 8 and 10 kHz, with mixing times of 1 and 0.8 ms, many 13C resonances of the [U-13C]Pheo a molecules reconstituted in the RCs could be assigned in a single set of experiments. Parts of the pheophytins interacting with the protein, at the level of 13C shifts modified by binding, could be identified. Small reconstitution shifts are detected for the 17(2) side chain of ring IV. In contrast, there is no evidence for electrostatic differences between the two Pheo a, for instance, due to a possibly strong selective electrostatic interaction with Glu L104 on the active branch. The protonation states appear the same, and the NMR suggests a strong overall similarity between the ground states of the two Pheo a, which is of interest in view of the asymmetry of the electron transfer.