Binding of 13C-enriched oleic acid to bovine serum albumin and to three large proteolytic fragments of albumin-two complementary fragments corresponding to the two halves of albumin and one fragment corresponding to the carboxyl-terminal domain-yielded unique.patterns of NMR resonances (chemical shifts and relative intensities) that were used to identify the locations of binding ofthe firstS mol ofoleic acid to the multidomain albumin molecule. The first 3 mol of oleic acid added to intact albumin generated three distinct NMR resonances as a result of simultaneous binding of oleic acid to three heterogeneous sites (primary sites). Two of these resonances were seen upon addition of 1 or 2 mol of oleic acid to fragments representing either the carboxyl-terminal half (residues 307-582) or the carboxyl-terminal domain (residues 377-582); the third resonance was seen upon addition of 1 mol of oleic acid to the fragment representing the amino-terminal half (residues 1-306). The resonance patterns for the fourth and fifth moles of oleic acid added to albumin (secondary sites) could not be duplicated by addition of more oleic acid to individual fragments. These resonance patterns were generated, however, when the two complementary fragments were mixed in equimolar proportions to form an albumin-like complex with a reconstituted middle domain. Thus, two primary fatty acid binding sites are assigned to the carboxyl-terminal domain, one primary site is assigned to the amino-terminal half, and the secondary sites are assigned to the middle domain. This distribution suggests albumin to be a less symmetrical binding molecule than theoretical models predict. This work also demonstrates the power of NMR for the study of microenvironments of individual fatty acid binding sites in specific domains.
13C and 31P NMR spectroscopy were used to monitor interactions of lyso 1-palmitoylphosphatidylcholine (LPPC) in the interfacial region of egg phosphatidylcholine (PC) bilayers and determine the effect of LPPC on the phospholipid bilayer structure. 13C NMR spectroscopy of small amounts (0.5-10 mol%) of 13C carbonyl-enriched LPPC cosonicated with egg PC to form small unilamellar vesicles (SUVs) revealed separate carbonyl signals for LPPC in the inner and outer leaflets of the vesicles. The ratio of LPPC in the outer leaflet to that in the inner leaflet was > or = 3/1. Exchange of LPPC between bilayer leaflets ("flip-flop") was too slow to be measured (t1/2 > 12 h). Albumin added to the external buffer of LPPC/PC vesicles was shown by 13C NMR to extract LPPC only from the outer leaflet. LPPC was a poor detergent in egg PC multilayers and SUVs. Stable SUVs were prepared by cosonicating egg PC with up to 30 mol% LPPC, and preformed SUVs incorporated up to 40 mol % of LPPC (added as an aqueous solution) without undergoing any morphological changes as evidenced by 31P NMR spectroscopy. The presence of oleic or palmitic acid did not have observable effects on properties of LPPC in SUVs, such as the localization of the LPPC carbonyl in the interface, and the transbilayer distribution and movement of LPPC. The apparent pKa of the fatty acid (FA) carboxyl at the membrane interface (7.7) measured by 13C NMR was not affected by LPPC, but the FA carboxyl carbon resonance showed linewidth changes near the apparent pKa that were dependent on the FA/LPPC ratio. These data suggest weak interactions in the interfacial region between FA and LPPC when both lipids are present at low levels in PC vesicles.
Diacylglycerols are minor constituents of membrane lipids, yet are essential in the activation and membrane association of protein kinase C. Solid-state 13C NMR experiments have been used to characterize the orientation of the glycerol backbone of dipalmitoylglycerol (DPG) and dipalmitoylphosphatidylcholine (DPPC) in egg phosphatidylcholine (PC) bilayers. The 13C NMR spectra of both DPG and DPPC specifically 13C-labeled at the sn-2 chain carbonyl exhibit a single narrow resonance (approximately 2 ppm) in liquid-crystalline egg PC bilayers. In contrast, specific 13C-labeling of both the sn-1 and sn-2 chain carbonyls results in an additional broad component (24-32 ppm) with an axially symmetric line shape. These data reveal that DPG has a distinct motionally-averaged structure in PC bilayers that is similar to that of DPPC and is not significantly affected by the absence of the large polar PC headgroup. The NMR line shapes are roughly consistent with the results of previous FTIR and NMR studies that indicate the sn-1 chain extends from the C1 carbon of the glycerol backbone into the hydrophobic interior of the bilayer, while the sn-2 chain first extends parallel to the bilayer surface and incorporates a bend at the ester linkage in order to keep the sn-1 and sn-2 chains parallel. However, the data suggest that the time-averaged orientation of the glycerol backbone is tilted from the bilayer normal, in contrast to the nearly parallel orientation observed in the crystal structures of phosphatidylcholines and phosphatidylethanolamines or the perpendicular orientation observed in the crystal structures of diacylglycerols.
A new approach to study phospholipase A2 mediated hydrolysis of phospholipid vesicles, using 13C NMR spectroscopy, is described. [13C]Carbonyl-enriched dipalmitoylphosphatidylcholine (DPPC) incorporated into nonhydrolyzable ether-linked phospholipid bilayers was hydrolyzed by phospholipase A2 (Crotalus adamanteus). The 13C-labeled carboxyl/carbonyl peaks from the products [lyso-1-palmitoylphosphatidylcholine (LPPC) and palmitic acid (PA)] were well separated from the substrate carbonyl peaks. The progress of the reaction was monitored from decreases in the DPPC carbonyl peak intensities and increases in the product peak intensities. DPPC peak intensity changes showed that only the sn-2 ester bond of DPPC on the outer monolayer of the vesicle was hydrolyzed. Most, but not all, of the DPPC in the outer monolayer was hydrolyzed after 18-24 h. There was no movement of phospholipid from the inner to the outer monolayer over the long time periods (18-24 h) examined. On the basis of chemical shift measurements of the product carbonyl peaks, it was determined that, at all times during the hydrolysis reaction, the LPPC was present only in the outer monolayer of the bilayer and the PA was bound to the bilayer and was approximately 50% ionized at pH approximately 7.2. Bovine serum albumin extracted most of the LPPC and PA from the product vesicles, as revealed by chemical shift changes after addition of the protein. The capability of 13C NMR spectroscopy to elucidate key structural features without the use of either shift reagents or separation procedures which may alter the reaction equilibrium makes it an attractive method to study this enzymatic process.
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