Larry R. Brown

Heteronuclear isotropic mixing in liquids

Abstract We report on a new experimental NMR technique for heteronuclear polarization transfer in the rotating frame which utilizes a WALTZ-16 decoupling sequence applied synchronously to both nuclear species to effect magnetization transfer among J -coupled spins. The technique increases the tolerance to Hartmann-Hahn mismatch between coupled spins and provides easily phased one-bond correlation spectra. Long-range connectivities can also be elucidated with longer mixing times.

Comparison of membrane organization in mitochondria from yeast and rat liver by nuclear magnetic resonance spectroscopy

SummaryProton magnetic resonance (PMR) and carbon-13 magnetic resonance (CMR) spectra of intact, unsonicated yeast and rat liver motochondria show differences which may be correlated with the composition of the membranes. High resolution PMR and CMR signals in intact yeast mitochondria have been assigned to regions of fluid lipid-lipid interaction on the basis of spectra of extracted lipid and protein, and the temperature dependence of NMR signals from the intact membrane. PMR spectra suggest that about 20% of total yeast phospholipid is in regions where both intramolecular fatty acid chain mobility and lateral diffusion of entire phospholipid molecules are possible. No such regions appear to exist in rat liver mitochondria. For both yeast and rat liver mitochondria, comparison of PMR and CMR spectra suggests that about 50% of phospholipid appears to be in regions where intramolecular fatty acid chain motion is considerable, but lateral diffusion is restricted. The remaining phospholipid appears to have little inter- or intramolecular mobility. Since NMR observation of lipid extracts from membranes indicates that phospholipid-sterol interactions do not account for the spectra of intact mitochondria, these effects are interpreted in terms of extensive lipid-protein interactions.

Proton magnetic resonance spectroscopy of promitochondrial membranes from yeast grown under different regimes of lipid supplementation

SummaryPromitochondrial membranes, prepared fromSaccharomyces cerevisiae grown anaerobically under different conditions of lipid supplementation, have been examined by PMR spectroscopy. Promitochondria from cells cultured anaerobically in media containing both unsaturated fatty acid and sterol supplements, or containing unsaturated fatty acid alone, yield high resolution spectra similar to those which are characteristic of aerobic mitochondria. By contrast, promitochondrial membranes from cells grown only with sterol supplementation in order to deplete unsaturated fatty acid and total phospholipid content of the organelles, yielded PMR spectra which were very substantially broadened. These spectra are similar to those obtained with rat liver mitochondria.PMR spectra of promitochondria from each cell type dispersed in trifluoroacetic acid, or of extracted lipids or residual proteins similarly dispersed, were different only in detail. It appears, therefore, that in the native state membranes of unsaturated fatty acid-depleted promitochondria are structurally different from promitochondria of the other two cell types. The difference may be a consequence of altered lipid-to-protein ratios, and thus of changes in the extent of lipid domain formation in the membranes of these organelles.

Proton magnetic resonance spectroscopic studies using paramagnetics of primary and tertiary structure of proteins

On binding a paramagnetic broadening probe at the end of a random coil polypeptide chain, one obtains sequential broadening of the -CH resonances along the chain and, hence, can determine the sequence of the peptide. The method has been applied to tripeptides (0.25 mg of material being used), tetrapeptides and a hexapeptide under conditions such that gadolinium ions bind at the C-terminus or cupric ions at the N-terminus. Dimethylation of the lysine residues of lysozyrne and observation of the chemical shifts of the six dimethyl proton resonances as a function of pH allows the determination of their pK. From studies of selective broadening of the methyl resonances on addition of gadolinium ions and the known structure of the lysozyme—ion complex, it is possible to obtain the pK values of the lysine residues of lysozyme. Approaches to the determination of the structure in solution of proteins using paramagnetic broadening and shifting probes is discussed. Problems concerning the site of ion binding and the assignment of p.m.r. resonances arise when the crystal structure is unknown. The amount by which a proton magnetic resonance (p.m.r.) line is broadened by a paramagnetic probe with a long electronic relaxation time —e.g. gadolinium(III)—is dependent on the inverse sixth power of the distance between the nucleus giving rise to the resonance and the paramagnetic probe. Furthermore, if the paramagnetic probe is an ion which binds rapidly and reversibly at a specific site, then the width of the resonance line will progressively increase as the concentration of paramagnetic ion is increased until such time as the site is saturated. after which no further broadening will occur. On the binding of gadolinium ions at low concentration to lysozyme. there is preferential broadening of p.m.r. resonances that arise from protons which are closest to the binding 2 The binding site has been shown by x-ray studies3 to be located between glutamic acid 35 and aspartic acid 52. By the use of difference spectroscopy46, in this case the subtraction of a paramagnetically broadened spectrum from the spectrum of native lysozyme, it has been possible to observe the resonances from the side chains of valine 83 J. H. BRADBURY ET AL. 109 and alanine 11012. In addition, distance information has been obtained from these studies with broadening probes and also from the use of lanthanide ions with short electronic relaxation times (europium(III). praseodymium(HI)). which cause shifts of resonances2 . In this paper three applications are given of the use of paramagnetic ions in peptide and protein chemistry. 1. DETERMINATION OF PRIMARY STRUCTURE (SEQUENCE) OF PEPTIDES If a peptide is present as a random coil in aqueous solution (which can be achieved by use of a denaturant such as urea or guanidine hydrochloride). then the average distance from one end of the molecule to successive cx-CH groups along the peptide chain increases as one proceeds along the chain. Thus, addition of a small amount of a suitable paramagnetic ion which is bound at one end causes most broadening of the ct-CH resonance of the nearest amino acid residue, less of the second residue and very little of the third residue8. This effect is shown in Figure 1. for addition of gadolinium