A detailed study on the structure, dynamics, and thermodynamic behavior of phosphatidylcholine/cholesterol (PC/CHOL) mixtures was undertaken using differential scanning calorimetry (DSC) and solid-state nuclear magnetic resonance (NMR) spectroscopy. DSC thermograms of mixtures of cholesterol (CHOL) with 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-phosphatidylcholine (DSPC), and 1,2-diarachidoyl-sn-phosphatidylcholine (DAPC) showed a broadening of the first-order gel-->liquid crystalline transition and a decrease in the transition enthalpy, indicating a gradual loss of cooperativity for high CHOL concentrations. DPPC and DSPC were labeled with 13C at the carbonyl group of the sn-2 chain and 2H was introduced into the middle of the sn-2 chain at the 6- and 12-position for DPPC and DSPC, respectively. The 13C and 2H NMR spectra of each labeled lipid were studied as a function of temperature and CHOL concentration. The residual quadrupole splitting in the 2H NMR spectra, delta nu Q perpendicular, was analyzed as a function of temperature and composition. For CHOL concentrations less than 30 mol %, a precipitous change in delta nu Q perpendicular occurs near the chain melting temperature of the phospholipid. Further increases in CHOL concentration broaden the transition and shift the midpoint to higher temperature, indicating the presence of a new phase at higher CHOL contents. Moreover, at a given temperature, delta nu Q perpendicular increases with increasing cholesterol content, which indicates a more ordered structure. The 13C NMR spectra in the gel state consisted of a superposition of two components which can be attributed to both gel-like and fluid phospholipid domains in the bilayer. This two-component spectrum can be simulated quantitatively with a two-parameter chemical exchange model, which permits the fraction of each form and the exchange rate to be determined as a function of temperature and composition. At high CHOL contents the line width of the fluid component broadens, suggesting an increase in the exchange rate between the domains. These results were interpreted in terms of a temperature composition diagram with one region L beta', two regions LGI and LGII, and one liquid crystalline region L alpha, with LG denoting "liquid-gel" type phases. Liquid-gel phases correspond to phases with increased order in the hydrocarbon chains (in comparison to that of the pure PC bilayer in the L alpha phase) combined with fast limit axial diffusion that averages the 13C NMR spectrum to a "fluidlike" line.(ABSTRACT TRUNCATED AT 400 WORDS)

Isotopically labeled tyrosines have been selectively incorporated into bacteriorhodopsin (bR). A comparison of the low-temperature bR570 to K Fourier transform infrared-difference spectra of these samples and normal bR provides information about the role of tyrosine in the primary phototransition. Several tyrosine contributions to the difference spectrum are found. These results and comparison with the spectra of model compounds suggest that a tyrosinate group protonates during the bR570 to K transition. This conclusion is strongly supported by the results of UV difference spectroscopy.Elucidation of the mechanism by which bacteriorhodopsin (bR), a light-driven proton pump in the purple membrane of Halobacterium halobium functions remains an important problem in biology (1). Two molecular events have been implicated in the bR primary phototransition. (i) An all-trans to 13-cis isomerization of the retinal chromophore has been deduced from resonance Raman measurements (2). (ii) Movement of a proton has been surmised from picosecond visible absorption data (3). It is not known, however, which bR groups are involved in proton transfer or how this event is coupled to retinal chromophore isomerization.Recently, our group and others (4-12) have begun to study the molecular alterations occurring during the bR photocycle with Fourier transform infrared (FTIR) spectroscopy. It has been demonstrated by this work that FTIR is sufficiently sensitive to detect changes occurring in single chemical groups in both the bR chromophore and the protein component. It is important for further progress that contributions to the FTIR-difference spectrum from specific amino acid residues be identified. This can be accomplished by selectively incorporating in bR isotopically labeled amino acids such as [E-15N]lysine (13). Such an approach was recently used by Englehard et al., who were able to identify the 1760 cm-1 carboxyl vibration (4, 7) as due to an aspartic acid residue (11).We report here the results of FTIR measurements on bR samples containing L-ring-deuterated tyrosine and L-[ring-4-13C]tyrosine (carbon label nearest hydroxyl group). We compare these difference spectra with FTIR measurements on model tyrosine compounds at high and low pH and p2H. Our results indicate that one or more tyrosines change during the bR570 to K phototransition and suggest that a tyrosinate group in bR570 becomes protonated by K. These conclusions are strongly supported by UV difference measurements, which also suggest the possible involvement of tryptophan at this stage of the photocycle. (14) and was purified by recrystallization. All isotope substitutions were verified by NMR spectroscopy. MATERIALS AND METHODSHalobacterium halobium R1 was grown in a synthetic medium like that of Gochnauer and Kushner (15) Purple membrane was isolated by the method of Oesterhelt and Stoeckenius (16). Specific activity measurements indicated that 50-80% of the tyrosine residues were labeled in various preparations. Amino acid analysis showed that <10% of t...

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.

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Phospholipid bilayers consisting of a 60:40 mixture of N-palmitoylsphingomyelin and dimyristoylphosphatidylcholine orient in a strong magnetic field. The orientation is easily observed in 31P- and 2H-nuclear magnetic resonance spectra where the intensity of the perpendicular edges of the powder lineshapes are enhanced. The lineshapes indicate that the long axis of the molecule is perpendicular to the magnetic field.