J.K. Blasie

On the location of 1-anilino-8-naphthalene-sulfonate in lipid model systems: An X-ray diffraction study

Abstract The electron density distribution across the magnesium stearate bimolecular leaflet is calculated from the relative intensities of discrete X-ray diffraction patterns from magnesium stearate multilayers. The generalized Patterson function P′(χ) calculated for a system with 3 unit cells. The autocorrelation function P0(χ) is determined from P′(χ). The electron density distribution μ(χ) is calculated by a deconvolution of P0(χ) through a recursion process. Diffraction is recorded also from magnesium stearate multilayers with large numbers of unit cells. Phase angles are determined from μ(χ) and by swelling experiments. Electron density distributions ρ(χ) are calculated by Fourier syntheses. 1-Anilino-8-naphthalene-sulfonate (ANS) is incorporated into this system and the effects on its structure are discussed. X-ray diffraction data from lecithin/cardiolipin dispersions are presented. These diffracted intensities are a continuous function of the diffraction angle; they can be considered to arise from randomly oriented single bimolecular leaflets. The generalized Patterson function P′(χ) is calculated and it is shown that in this case P′(χ) is identical to the autocorrelation function P0(χ). μ(χ) is calculated by a deconvolution of P0(χ). ANS in different concentrations is incorporated in the lecithin/cardiolipin bimolecular leaflet and the resulting perturbation of its structure is discussed.

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.

Studies on the orientations of the mitochondrial redox carriers. I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase-lipid model membrane.

The liganded derivatives of mitochondrial cytochrome c oxidase have been prepared in hydrated oriented multilayers of membranous cytochrome c oxidase. The optical spectra of the liganded derivatives recorded at an angle of 45 degrees between the incident light beam and the normal to the planes of the membranes in the multilayers show dichroic ratios of almost 2 in the visible region and 1.2-1.4 in the Soret region. The dichroic ratios were found to be similar for both cytochromes a and a3. Electron paramagnetic resonance spectra of the azide, sulfide, and formate complexes of cytochrome c oxidase obtained as a function of the orientation of the applied magnetic field relative to the planes of the membranes in the multilayer confirm the optical data and demonstrate that both hemes of cytochrome c oxidase are oriented such that the angle between the heme normal and the membrane normal is approximately 90 degrees.

Temperature and frequency dependence of longitudinal proton relaxation times in sonicated lecithin dispersions

Abstract The temperature dependence of the longitudinal relaxation time, T 1 for various protons in sonicated dipalmitoyllecithin vesicles at 60 and 220 MHz are compared. In order to qualitatively explain the frequency dependence of T 1 values the anisotropy of motion of individual protons in the phospholipid molecule must be explicitly taken into account. The difficulties in interpreting NMR relaxation times in terms of specific motions in the phospholipid molecule are discussed.

Structural and dynamical studies of mixed chlorophyll/phosphatidylcholine bilayers via x-ray diffraction, absorption polarization spectroscopy and nuclear magnetic resonance.

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidylcholine fatty acids chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a 10(-9)-10(-10)s time scale in the ideally mixed two-component bilayer.

The determination of the separate Ca2+ pump protein and phospholipid profile structures within reconstituted sarcoplasmic reticulum membranes via X-ray and neutron diffraction.

We have previously compared the electron density profiles for several highly-functional reconstituted sarcoplasmic reticulum membranes with that for the isolated sarcoplasmic reticulum membrane (Herbette, L., Scarpa, A., Blasie, J.K., Wang, C.T., Saito, A. and Fleischer, S. (1981) Biophys. J. 36, 47-72). In this paper, we compare the separate calcium pump protein profile within these reconstituted sarcoplasmic reticulum membranes, as derived by X-ray and neutron diffraction methods, with that within isolated sarcoplasmic reticulum membranes. In addition, the time-average perturbation of the lipid bilayer by the incorporated calcium pump protein within these reconstituted sarcoplasmic reticulum membranes has been determined in some detail.

The location of redox centers in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex by resonance X-ray diffraction

Abstract The technique of resonance X-ray diffraction (Blasie, J.K. and Stamatoff, J. (1981) Annu. Rev. Biophys. Bioeng. 10, 451–452) utilizing synchrotron radiation was used to determine the locations of the cytochrome c heme iron atom and the photosynthetic reaction center iron atom within the profile of a reconstituted membrane. The accuracy of these determinations was better than ±2 Ȧ. The cytochrome c heme iron atom → reaction center iron atom vector was determined to have a magnitude of approx. 44 Ȧ projected onto the membrane profile and to span most of the lipid hydrocarbon core of the membrane profile. Since the reaction center iron atom interacts magnetically with the primary quinone electron acceptor Q I over a distance of less than 10 Ȧ, the primary light-induced electron-transfer reactions for this system generate the electric charge separation between oxidized cytochrome c + and Fe-Q − I across most (approx. 2 3 ) of the membrane profile including most or all of the lipid hydrocarbon core of the membrane.

Biological membrane structure as “seen” by X-ray and neutron diffraction techniques

ConclusionThe elastic X-ray/neutron diffraction techniques described in this review are currently capable of providing substantial information concerning the time-averaged structures and intermolecular ordering of molecular components within a dynamic membrane structure. In addition, the time resolution of the elastic X-ray diffraction technique, afforded by intense synchrotron and laser plasma X-ray sources, now permits this structural information to be obtained over a range of time scales from nanoseconds to milliseconds and upwards following an excitation of the membrane system. This time-averaged and time-resolved structural information may provide considerable insight into structure-function relationships in biological membranes and, especially when combined with structural information on the membrane proteins involved at atomic resolution, may provide this insight at the atomic level.

The location of redox centers in biological membranes determined by resonance X-ray diffraction. II. Analysis of the resonance diffraction data

In the preceding paper (Stamatoff, J., Eisenberger, P., Blasie, J.K., Pachence, J.M., Tavormina, A., Erecinska, M., Dutton P.L. and Brown, G. (1982) Biochim. Biophys. Acta 679, 177-187), we described the observation of resonance X-ray scattering effects from intrinsic metal atoms associated with redox centers in membrane proteins on the lamellar X-ray diffraction from oriented multilayers of reconstituted membranes. In this paper, we discuss the possible methods of analysis of such data and present the results of our model refinement analysis concerning (a) the location of the cytochrome c heme iron atom in the profile structure of a reconstituted membrane containing a photosynthetic reaction center-cytochrome c complex and (b) the location of the heme a and a3 iron atoms in the profile structure of a reconstituted membrane containing cytochrome oxidase. The former results are of special importance because they provide a test of the validity of the resonance diffraction data and the methods of analysis, since the location of cytochrome c in the reaction center-cytochrome c membrane profile is known independently of the resonance diffraction experiments.

The location of redox centers in biological membranes determined by resonance x-ray diffraction. I. Observation of the resonance effect.

We have developed resonance X-ray diffraction methods to locate for the first time intrinsic metal atoms associated with redox centers within biological membrane systems. The study of membranes containing dilute concentrations of resonant scatterers has been made possible by the development of synchrotron radiation sources of X-rays. The technique permits altering the scattering power of a particular atom relative to others by varying the incident X-ray energy. Thus, this method may be used to locate a metal atom within a complex integral protein without chemical modification of the membrane. We present resonance diffraction data taken with synchroton radiation for two different membrane systems: cytochrome oxidase incorporated into lipid vesicles and a photosynthetic reaction center-cytochrome c complex also reincorporated into lipid vesicles.