Abstract:
Solid-state nuclear magnetic resonance (NMR) spectroscopy finds growing application to inorganic and organic materials, biological samples, polymers, proteins, and cellular membranes. However, this technique is often neither included in laboratory curricula nor typically covered in undergraduate courses. On the other hand, spectroscopy and molecular structure taught in second-semester undergraduate physical chemistry courses meet the minimal background prerequisites for interpreting data obtained in many solid-state NMR experiments. A solid-state 2H NMR experiment is described in which the student obtains and interprets the spectrum of a powder sample of hexamethylbenzene-d18 using a solution NMR spectrometer as found in many undergraduate institutions. A quadrupolar-echo pulse sequence is applied to the sample to obtain the 2H NMR spectrum. The spectrum of a randomly oriented powder sample consists of two spectral branches with broad shoulders. The quadrupolar frequencies corresponding to the nuclear spin transitions are interpreted in terms of molecular mobility in the solid state, that is, 3-fold rotation of the methyl groups and 6-fold rotation of the hexamethylbenzene ring. In this way, students discover that solid substances may have significant molecular motions. This undergraduate investigation employing solid-state 2H NMR provides an informative exposure to state-of-the-art research techniques by using facilities that are already in place at many undergraduate institutions. Furthermore, it demonstrates a real-life manifestation of quantum mechanics as explained in physical chemistry courses, as well as fundamentals of molecular motions such as rotation of aromatic ring compounds and methyl groups in the solid state. © 2012 The American Chemical Society and Division of Chemical Education, Inc.
PMID: 18393486;PMCID: PMC2722912;Abstract:
Planar supported lipid bilayers that are stable under ambient atmospheric and ultra-high-vacuum conditions were prepared by cross-linking polymerization of bis-sorbylphosphatidylcholine (bis-SorbPC). X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed to investigate bilayers that were cross-linked using either redox-initiated radical polymerization or ultraviolet photopolymerization. The redox method yields a more structurally intact bilayer; however, the UV method is more compatible with incorporation of transmembrane proteins. UV polymerization was therefore used to prepare cross-linked bilayers with incorporated bovine rhodopsin, a light-activated, G-protein-coupled receptor (GPCR). A previous study (Subramaniam, V.; Alves, I. D.; Salgado, G. F. J.; Lau, P. W.; Wysocki, R. J.; Salamon, Z.; Tollin, G.; Hruby, V. J.; Brown, M. F.; Saavedra, S. S. J. Am. Chem. Soc. 2005, 127, 5320-5321) showed that rhodopsin retains photoactivity after incorporation into UV-polymerized bis-SorbPC, but did not address how the protein is associated with the bilayer. In this study, we show that rhodopsin is retained in supported bilayers of poly(bis-SorbPC) under ultra-high-vacuum conditions, on the basis of the increase in the XPS nitrogen concentration and the presence of characteristic amino acid peaks in the ToF-SIMS data. Angle-resolved XPS data show that the protein is inserted into the bilayer, rather than adsorbed on the bilayer surface. This is the first study to demonstrate the use of ultra-high-vacuum techniques for structural studies of supported proteolipid bilayers. © 2008 American Chemical Society.
Changes in lipid composition have recently been shown to exert appreciable influences on the activities of membrane-bound proteins and peptides. We tested the hypothesis that the conformational states of rhodopsin linked to visual signal transduction are related to biophysical properties of the membrane lipid bilayer. For bovine rhodopsin, the meta I-meta II conformational transition was studied in egg phosphatidylcholine (PC) recombinants versus the native rod outer segment (ROS) membranes by means of flash photolysis. Formation of metarhodopsin II was observed by the change in absorbance at 478 nm after a single actinic flash was delivered to the sample. The meta I/meta II ratio was investigated as a function of both temperature and pH. The data clearly demonstrated thermodynamic reversibility of the transition for both the egg PC recombinants and the native ROS membranes. A significant shift of the apparent pK(a) for the acid-base equilibrium to lower values was evident in the egg PC recombinant, with little meta II produced under physiological conditions. Calculations of the membrane surface pH using a Poisson-Boltzmann model suggested the free energies of the meta I and meta II states were significantly affected by electrostatic properties of the bilayer lipids. In the ROS membranes, phosphatidylserine (PS) is needed for full formation of meta II, in combination with phosphatidylethanolamine (PE) and polyunsaturated docosahexaenoic acid (DHA; 22:6omega3) chains. We propose that the PS surface potential leads to an accumulation of hydronium ions, H(3)O(+), in the electrical double layer, which drive the reaction together with the large negative spontaneous curvature (H(0)) conferred by PE plus DHA chains. The elastic stress/strain of the bilayer arises from an interplay of the approximately zero H(0) from PS and the negative H(0) due to the PE headgroups and polyunsaturated chains. The lipid influences are further explained in terms of matching of the bilayer spontaneous curvature to the curvature at the lipid/rhodopsin interface, as formulated by the Helfrich bending energy. These new findings guide current ideas as to how bilayer properties govern the conformational energetics of integral membrane proteins. Moreover, they yield knowledge of how membrane lipid-protein interactions involving acidic phospholipids such as PS and neutral polyunsaturated DHA chains are implicated in key biological functions such as vision.
PMID: 9718305;Abstract:
The orientation of prosthetic groups in membrane proteins is of considerable importance in understanding their functional role in energy conversion, signal transduction, and ion transport. In this work, the orientation of the retinylidene chromophore of bacteriorhodopsin (bR) was investigated using 2H NMR spectroscopy. Bacteriorhodopsin was regenerated with all-trans-retinal stereospecifically deuterated in one of the geminal methyl groups on C1 of the cyclohexene ring. A highly oriented sample, which is needed to obtain individual bond orientations from 2H NMR, was prepared by forming hydrated lamellar films of purple membranes on glass slides. A Monte Carlo method was developed to accurately simulate the 2H NMR line shape due to the distribution of bond angles and the orientational disorder of the membranes. The number of free parameters in the line shape simulation was reduced by independent measurements of the intrinsic line width (1.6 kHz from T(2e experiments) and the effective quadrupolar coupling constant (38.8- 39.8 kHz from analysis of the line shape of a powder-type sample). The angle between the C1-(1R)-1-CD3 bond and the purple membrane normal was determined with high accuracy from the simultaneous analysis of a series of 2H NMR spectra recorded at different inclinations of the uniaxially oriented sample in the magnetic field at 20 and -50 °C. The value of 68.7 ± 2.0°in dark-adapted bR was used, together with the previously determined angle of the C5-CD3 bond, to calculate the possible orientations of the cyclohexene ring in the membrane. The solutions obtained from 2H NMR were then combined with additional constraints from linear dichroism and electron cryomicroscopy to obtain the allowed orientations of retinal in the noncentrosymmetric membrane structure. The combined data indicate that the methyl groups on the polyene chain point toward the cytoplasmic side of the membrane and the N-H bond of the Schiff base to the extracellular side, i.e., toward the side of proton release in the pump pathway.
PMID: 3741833;Abstract:
31P NMR studies of rod outer segment (ROS) and sarcoplasmic reticulum (SR) membranes have been performed under conditions where broad and narrow spectral components can be clearly resolved. Control studies of an anhydrous, solid powder of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), as well as aqueous binary mixtures of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), demonstrate clearly that broad spectral components can be detected. For the codispersions of DSPC and DOPC in the mixed-phase region at 22°C, the 31P NMR spectra consist of a superposition of a broad component and a narrow, axially symmetric component, due to coexisting solid and liquid-crystalline domains, which are in slow exchange on the 31P NMR time scale. The 31P NMR spectra of the native ROS and SR membranes, however, consist of only a narrow component, to within experimental error, indicating that most or all of the phospholipids are in the liquid-crystalline (Lα) phase at 22°C. The above conclusions are in agreement with many, but not all, previous studies [see, e.g., Yeagle, P. L. (1982) Biophys. J. 37, 227-239]. It is estimated that at most 10% of the phospholipids in the ROS and SR membranes could give rise to broad 31P NMR spectral components, similar to those seen for anhydrous or solid-phase lipids, corresponding to ∼7 phospholipids/rhodopsin molecule and ∼11 phospholipids/ Ca2+-ATPase molecule, respectively. © 1986 American Chemical Society.
