L. Herbette

A direct analysis of lamellar x-ray diffraction from hydrated oriented multilayers of fully functional sarcoplasmic reticulum

The profile structure of functional sarcoplasmic reticulum (SR) membranes was investigated by X-ray diffraction methods to a resolution of 10 A. The lamellar diffraction data from hydrated oriented multilayers of SR vesicles showed monotonically increasing widths for higher order lamellar reflections, indicative of simple lattice disorder within the multilayer. A generalized Patterson function analysis, previously developed for treating lamellar diffraction from lattice-disordered multilayers, was used to identify the autocorrelation function of the unit cell electron density profile. Subsequent deconvolution of this autocorrelation function provided the most probable unit cell electron density profile of the SR vesicle membrane pair. The resulting single membrane profile possesses marked asymmetry, suggesting that a major portion of the Ca++ -ATPase resides on the exterior of the vesicle. The electron density profile also suggests that the Ca++-dependent ATPase penetrates into the lipid hydrocarbon core of the SR membrane. Under conditions suitable for X-ray analysis, SR vesicles prepared as partially dehydrated oriented multilayers are shown to conserve most of their ATP-induced Ca++ uptake functionality, as monitored spectrophotometrically with the Ca++ indicator arsenazo III. This has been verified both in resuspensions of SR after centrifugation and slow partial dehydration, and directly in SR multilayers in a partially dehydrated state (20-30 percent water). Therefore, the profile structure of the SR membrane that we have determined may closely resemble that found in vivo.

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.

Time-resolved x-ray diffraction studies of the sarcoplasmic reticulum membrane during active transport.

X-ray and neutron diffraction studies of oriented multilayers of a highly purified fraction of isolated sarcoplasmic reticulum (SR) have previously provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to approximately 10-A resolution. These studies used biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via phospholipid transfer proteins. Time-resolved x-ray diffraction studies of these oriented SR membrane multilayers have detected significant changes in the membrane profile structure associated with phosphorylation of the Ca2+-ATPase within a single turnover of the Ca2+-transport cycle. These studies used the flash photolysis of caged ATP to effectively synchronize the ensemble of Ca2+-ATPase molecules in the multilayer, synchrotron x-radiation to provide 100-500-ms data collection times, and double-beam spectrophotometry to monitor the Ca2+-transport process directly in the oriented SR membrane multilayer.

Comparison of the profile structures of isolated and reconstituted sarcoplasmic reticulum membranes.

The profile structures of functional reconstituted sarcoplasmic reticulum (RSR) membranes were investigated as a function of the lipid/protein (L/P) ratio via x-ray diffraction studies of hydrated oriented multilayers of these membranes to a resolution of 10-15 A, and neutron diffraction studies on these multilayers to lower resolutions. Our results at this stage of investigation indicate that reconstitution of SR with variable amounts of Ca2+ pump protein for L/P ratios greater than 88 results in closed membraneous vesicles in which the Ca2+ pump protein is distributed asymmetrically in the membrane profile; a majority of the protein density is contained primarily in the extravesicular half of the membrane profile whereas a relatively lesser portion of the protein spans the hydrocarbon core of the RSR membranes. These RSR membranes are functionally similar and resemble isolated light sarcoplasmic reticulum in both profile structure and function at a comparable L/P ratio. Reconstitution with greater amounts of Ca2+ pump protein (e. g. L/P approximately 50-60) resulted in substantially less functional membranes with a dramatically thicker profile structure.

Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio

The ATP-induced Ca2+ accumulation efficiency and rates of Ca2+ uptake of the reconstituted sarcoplasmic reticulum (RSR) model membrane system were measured over an extended range of lipid-to-protein (L/P) molar ratios and were compared to those of isolated light sarcoplasmic reticulum (LSR). Highly purified sarcoplasmic reticulum (SR), dissociated in the presence of deoxycholate, was reconstituted for several L/P ratios, according to the same procedure, forming closed membranes vesicles composed of greater than 95% Ca2+ pump protein and SR lipids which were capable of ATP-induced Ca2+ accumulation in the absence of oxalate or other Ca2+ precipitating agents. This suggests that dissociation of SR and reconstitution to form RSR does not significantly affect the ability of the Ca2+ pump protein incorporated into the SR lipid bilayer to establish Ca2+ gradients. Electron micrographs of fixed and stained dispersions of RSR revealed a structural organization of the membrane that was dependent upon the L/P molar ratio. RSR with L/P greater than 88 were composed of closed vesicles whose membranes stained asymmetrically, similar to that observed for LSR. Closed vesicles of RSR with L/P less than 88 were composed of membrane that stained symmetrically. In addition, reconstituted SR preparations with well-defined L/P molar ratios greater than 88 possess a functional behavior similar to that of LSR (in the absence of oxalate, energy efficiencies are 60-70% and apparent initial uptake rates are 80% that of isolated LSR controls); RSR preparations with L/P less than 88 are characterized by significantly depressed values of the energy efficiencies and apparent initial uptake rates especially at low L/P ratios. Thus, we are the first to report a reconstituted SR model membrane system capable of attaining rates of Ca2+ uptake comparable to isolated LSR controls at comparable L/P ratios in the absence of oxalate or other Ca2+ precipitating agents.

Large-scale structural changes in the sarcoplasmic reticulum ATPase appear essential for calcium transport.

Model refinement calculations utilizing the results from time-resolved x-ray diffraction studies indicate that specific, large-scale changes (i.e., structural changes over a large length scale or long range) occur throughout the cylindrically averaged profile structure of the sarcoplasmic reticulum ATPase upon its phosphorylation during calcium active transport. Several physical-chemical factors, all of which slow the kinetics of phosphoenzyme formation, induce specific, large-scale changes throughout the profile structure of the unphosphorylated enzyme that in general are opposite to those observed upon phosphorylation. These results suggest that such large-scale structural changes in the ATPase occurring upon its phosphorylation are required for its calcium transport function.

31P NMR Studies of Oriented Multilayers Formed from Isolated Sarcoplasmic Reticulum and Reconstituted Sarcoplasmic Reticulum: Evidence that “Boundary-Layer” Phospholipid is not Immobilized

Studies were made of the angular dependence of the /sup 31/P NMR signal from oriented sarcoplasmic reticulum membranes. Similar spectra were obtained from oriented reconstituted sarcoplasmic reticulum membranes with lipid-to-protein ratios ranging from 42.1 to 110.1 and from oriented bilayer membranes formed from sarcoplasmic reticulum phospholipids. The dependence of the /sup 31/P NMR spectra on the alignment of the membranes with respect to the magnetic field was used to draw two conclusions about the motion of the phospholipid molecules that contribute to the observed spectra. First, the phosphate group and the two adjacent methylene groups are able to rotate rapidly (i.e., faster than 10/sup -5/ s) around the normal to the plane of the membrane. Second, calibration experiments showed that all (100 +/- 7%) of the phospholipid molecules in the membrane can be accounted for in the observed spectra. Thus, essentially all the phospholipid molecules in the sarcoplasmic reticulum and the reconstituted sarcoplasmic reticulum membranes have the same motion in the polar headgroup region as found in model bilayer membranes. (JMT)

STATIC AND TIME-RESOLVED STRUCTURAL STUDIES OF THE Ca2+-ATPase OF ISOLATED SARCOPLASMIC RETICULUM

Publisher Summary The X-ray and neutron diffraction studies of oriented multi-layers of isolated light sarcoplasmic reticulum (SR) have provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to ∼ 10A resolution. These studies utilized biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via exchange proteins. The nature of the time-resolved X-ray diffraction studies of the SR membrane can be summarized briefly as follows: the Ca2+- transport process is initiated essentially synchronously throughout the ensemble of Ca2+-ATPase molecules within an oriented multilayer of isolated SR membranes via the flash-photolysis of caged-adenosine triphosphate (ATP). The time-scale of the effective synchronization of the ensemble depends initially upon the duration of the UV light flash required to produce a sufficient quantity of ATP in the multilayer to sustain several turnovers of the Ca2+-ATPase molecules and is ultimately limited to the millisecond time-scale due to the kinetics of the dark-reactions of the photolysis process.