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.     

Time-resolved x-ray diffraction study of the troponin-associated reflexions from the frog muscle.

The vertebrate skeletal muscle gives rise to a series of x-ray reflexions indexed as orders (n) of 77 nm, the even orders being meridional whereas the odd orders being near-meridional. The diffraction intensities associated with these reflexions originate from the axial period of 39 nm attributable to the repeat of troponin-tropomyosin on the thin filament. In the present study, the x-ray intensities of the furthest inner reflexions, A2 (n = 2) reflexion at an axial spacing of 1/39 nm-1 and A4 (n = 4) reflexion at 1/19 nm, of this series were measured with a time resolved manner. Upon activation of the frog striated muscle, the two reflexions underwent biphasic time courses of the intensity changes. With A2 reflexion, a rapid intensity increase by 16%, being completed by the time when tension rises to 5%, was followed by a slow intensity decrease down to 50%, which was associated with the tension rise. In both phases, lateral widths remained unchanged. A4 reflexion also behaves in the same way, although the first phase (the intensity increase) was not clear due to unsatisfactory statistics. We interpret phase one as being caused by conformational change of the troponin-tropomyosin complex upon binding of Ca2+ to troponin, whereas phase two being due to direct contribution of the mass of the myosin heads bound to the thin filament, although possible contribution of conformational changes of the regulatory proteins to phase two is not excluded. The results indicated that the calcium activation of the thin filament leads the onset of the actomyosin interaction.     

Structural transition of bacteriorhodopsin is preceded by deprotonation of Schiff base: microsecond time-resolved x-ray diffraction study of purple membrane

The structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of 7 angstroms by a time-resolved x-ray diffraction experiment utilizing synchrotron x rays from an undulator of SPring-8. The x-ray diffraction measurement system, used in coupling with a pulsed YAG laser, enabled us to record a diffraction pattern from purple membrane film at a time-resolution of 6 micros over the time domain of 5 micros to 500 ms. In the time domain, the functionally most important M-intermediate appears. A series of time-resolved x-ray diffraction data after photo-excitation showed clear intensity changes caused by the conformational changes of helix G in the M-intermediate. The population of the reaction intermediate was prominently observed at approximately 5 ms after a photo-stimulus. In contrast, absorption measurement indicated the deprotonation of the Schiff base predominantly occurred at approximately 300 micros after a photo-stimulus. These results showed that the conformational changes characterizing structurally the M-intermediate predominantly occur at a later stage of the deprotonation of the Schiff base. Thus, the M-intermediate can be divided into two metastable stages with different physical characteristics.     

High sensitivity electron diffraction analysis. A study of divalent cation binding to purple membrane.

A sensitive high-resolution electron diffraction assay for change in structure is described and harnessed to analyze the binding of divalent cations to the purple membrane (PM) of Halobacterium halobium. Low-dose electron diffraction patterns are subject to a matched filter algorithm (Spencer, S. A., and A. A. Kossiakoff. 1980. J. Appl. Crystallogr. 13:563-571). to extract accurate values of reflection intensities. This, coupled with a scheme to account for twinning and specimen tilt in the microscope, yields results that are sensitive enough to rapidly quantitate any structure change in PM brought about by site-directed mutagenesis to the level of less than two carbon atoms. Removal of tightly bound divalent cations (mainly Ca2+ and Mg2+) from PM causes a color change to blue and is accompanied by a severely altered photocycle of the protein bacteriohodopsin (bR), a light-driven proton pump. We characterize the structural changes that occur upon association of 3:1 divalent cation to PM, versus membranes rendered purple by addition of excess Na+. High resolution, low dose electron diffraction data obtained from glucose-embedded samples of Pb2+ and Na+ reconstituted PM preparations at room temperature identify several sites with total occupancy of 2.01 +/- 0.05 Pb2+ equivalents. The color transition as a function of ion concentration for Ca2+ or Mg2+ and Pb2+ are strictly comparable. A (Pb2(+)-Na+) PM Fourier difference map in projection was synthesized at 5 A using the averaged data from several nominally untilted patches corrected for twinning and specimen tilt. We find six major sites located on helices 7, 5, 4, 3, 2 (nomenclature of Engelman et al. 1980. Proc. Natl. Acad. Sci. USA. 77:2023-2027) in close association with bR. These partially occupied sites (0.55-0.24 Pb2+ equivalents) represent preferential sites of binding for divalent cations and complements our earlier result by x-ray diffraction (Katre et al. 1986. Biophys. J. 50:277-284).     

Low-angle x-ray diffraction of the erythrocyte membrane

Using low-angle X-ray diffraction technique it has been shown that preparing procedures can induce the phase separation in red blood cell membranes in the course of which proteins are removed from lipid bilayers. The latter form rather extensive regions and the diffraction pattern of asymmetric type changes to a symmetric one.     

Kinetics of the lamellar and hexagonal phase transitions in phosphatidylethanolamine. Time-resolved x-ray diffraction study using a microwave-induced temperature jump.

The kinetics of the thermotropic lamellar gel (L beta')/lamellar liquid crystal (L alpha) and L alpha/inverted hexagonal (HII) phase transitions in fully hydrated dihexadecylphosphatidylethanolamine (DHPE) have been studied. Measurements were made by using time-resolved x-ray diffraction (TRXRD) to monitor progress of the transitions. In these studies microwave energy at 2.5 GHz was used to increase the sample temperature rapidly and uniformly through the phase transition regions. The L beta'/L alpha and L alpha/HII transitions of DHPE were examined under active microwave heating and passive cooling. The transitions were found to be repeatable and reversible, and to have an upper bound on the time required to complete the transition of less than 3 s. Regardless of the direction of the transition, both phase transitions appeared to be two-state with no accumulation of intermediates to within the sensitivity limits of the TRXRD method. The rate and amplitude of the temperature jump can be controlled by regulating microwave radiation input power. A temperature jump rate of 29 degrees C/s was obtained at a final microwave power setting of 120 W. Comparisons between previously reported fluid flow (Caffrey, M. 1985. Biochemistry. 24:4826-4844) and microwave heating studies suggest that the determination of limiting transit times will require faster heating.     

An extended x-ray absorption fine structure study of the high-affinity cation-binding site in the purple membrane.

The structure of the high-affinity cation-binding site of bacteriorhodopsin was studied using extended x-ray absorption fine structure techniques. The results obtained for Mn2+ in aqueous solution and for the complex BR-Mn2+ (1:1 molar ratio) show great similarities, suggesting that Mn2+, when bound to this site, is coordinated with six atoms of oxygen, forming an octahedral disposition. The interatomic distance between the atoms of oxygen and the Mn2+ was found to be 2.17 A for the complex BR-Mn2+, similar to Mn2+ in solution (2.15 A). In addition, the absence of any other peak at greater distances in the Fourier-transformed spectrum indicates that neither phosphorus nor sulphur atoms are present in the second coordination shell. This suggests that this binding site is located in the protein, discarding the proximity of lipid polar headgroups.     

A dynamic x-ray diffraction study of anesthesia action. Thickening of the myelin membrane by n-pentane.

The structural changes induced in the myelin sheath by n-pentane nerve impulse blockage were studied by small-angle X-ray diffraction using a linear position-sensitive detector. The results show that the thickness of the myelin period lattice increases from 170 to 180 A during n-pentane treatment.     

The location of retinal in the purple membrane profile by neutron diffraction.

The trans-membrane location of retinal in the purple membrane of Halobacterium halobium, has been determined by low-angle neutron scattering studies on aqueous dispersions of the membranes. The membrane was bleached and regenerated with deuterated and with hydrogen-containing retinal. The modified retinal was obtained by extraction from bacteria grown in a totally deuterated medium. The determination of the retinal position is based on the differences in neutron scattering between a purple membrane sample with normal, protonated retinal and another sample with deuterated retinal. A distinct scattering density difference between the two preparations was observed. A direct structure determination was used with the retinal localized from a Fourier difference density profile. We conclude that the β-ionone ring portion of the retinal is situated centrally in the membrane.     

Time-resolved x-ray study of effect of sinusoidal length change on tetanized frog muscle.

Time-resolved x-ray diffraction studies were done on frog skeletal muscles with synchrotron radiation by applying sinusoidal length changes of frequency 10 Hz and amplitude approximately 1% to isometrically contracting muscles at approximately 17 degrees C. Distinct periodic intensity changes were observed in the 14.3-nm myosin meridional reflection and the equatorial 1,0 and 1,1 reflections. Response of the 14.3-nm reflection to the sinusoidal length change was nonlinear, as evidenced by a large second harmonic in its oscillatory intensity change, whereas the response of the equatorial 1,1 reflection was closely linear, as evidenced by almost sinusoidal intensity change. Intensity change of the 1,0 reflection was nearly antiphase to that of the 1,1 reflection. Integral widths of the 14.3-nm meridional reflection measured along the meridian and of the equatorial 1,1 reflection remained almost constant during tension development, while that of the 1,0 reflection tended to decrease. The widths of the 14.3-nm meridional reflection perpendicular to the meridian and of the equatorial 1,0 reflection appeared to undergo oscillatory changes in response to the sinusoidal length changes.     

Analysis of high-resolution electron diffraction patterns from purple membrane labelled with heavy-atoms

Progress in the structure determination of bacteriorhodopsin, the protein component of purple membrane from Halobacterium halobium has been limited by the lack of three-dimensional phase information between 6 and 3 A resolution. By analogy with X-ray methods, it is possible that heavy-atom labelling of the membrane crystal may provide heavy-atom derivatives that can be used for phasing by the multiple isomorphous replacement method. This paper describes the screening of heavy-atom compounds as potential derivatives, and the evaluation of the data collected from these heavy-atom-labelled membranes. Improvements in the methods for collecting electron diffraction data and analysing and merging the data are presented. Diffraction patterns of purple membrane samples were taken at -120 degrees C to minimize radiation damage. About 30 heavy-atom compounds were tested for use as potential derivatives. The diffraction patterns from labelled membranes were analysed by examining 6.5 A difference Fourier maps. Two heavy-atom compounds were selected for three-dimensional data collection at 3 A resolution. In addition, a full set of native data at -120 degrees C was collected to 2.7 A resolution. The intensity merging, heavy-atom derivative evaluation, heavy-atom refinement and the calculation of phases are presented. Phases are compared to those determined by electron microscope imaging, and limitations of the method are discussed. It is concluded that, with the present accuracy of data collection and the present magnitude of delta F/F available for the derivatives, the phasing power is too small. The phases that are obtained are not sufficiently accurate to provide a reliably interpretable map. It may be possible, however, to use the heavy-atom derivative data in difference Fourier calculations in which the presence of a peak would confirm the phases calculated from a model or obtained by electron microscope imaging.     

Structure of the outer mitochondrial membrane analysis of x-ray diffraction from the plant membrane

X-ray diffraction from centrifugally oriented specimens of plant outer mitochondrial membranes suggests that these membranes contain prominent in-plane subunits. The short lamellar repeat which these specimens display (as low as 5.1 nm) points to a predominantly internal localization of the protein components of these membranes. The simplest model for the putative in-plane subunit consistent with autocorrelation analysis of the normal-incidence diffraction data consists of two concentric rings of electron density with diameters of (approx.) 2 and 4 nm. These rings could represent the planar projections of concentric cylindrical shells, aligned normal to the membrane surface.     

Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X-ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH-values. The observed light-induced intensity changes of in-plane X-ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X-ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3-3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at -180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.     

Neocarzinostatin: an antitumor protein a preliminary x-ray diffraction study.

The antitumor protein, neocarzinostatin, has been crystallized and examined by X-ray diffraction. Crystals of this globular protein are of space group P2₁2₁2₁ with cell parameters a = 27.4Å, b = 33.9Åand c = 102.0Å. There is one molecule of approximately 27Ådiameter per asymmetric unit. Crystals soaked in a K₂HgI₄ solution give diffraction patterns significantly different from native crystal diffraction patterns.     

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. II. Comparison of diffraction patterns of photosynthetic units from various purple bacteria

Comparative X-ray diffraction studies, in conjunction with infrared absorption spectroscopy, were performed on chromatophores isolated from various purple photosynthetic bacteria in order to achieve a better understanding of the molecular structure of the photosynthetic unit. Purple non-sulfur bacteria used were Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas palustris. Chromatophores of Chromatium vinosum, as a typical example of purple sulfur bacteria, were also investigated. The results were as follows. Distinct equatorial X-ray diffraction patterns were obtained from chromatophores of all the bacteria examined. They showed diffuse, continuous diffraction patterns having several maxima, and the patterns are evidently distinguished from those of either crystalline or amorphous material. The pattern indicates that the photosynthetic unit in the chromatophore has a highly organized molecular structure in the plane of the membrane. Bacteria whose major photosynthetic pigment is bacteriochlorophyll alpha can be categorized in three groups from the viewpoint of near infrared absorption spectra. X-ray diffraction patterns are also grouped accordingly, although the differences are minimal and the patterns display common features. In other words, the bacteriochlorophyll forms, which are bacteriochlorophyll-protein complexes exhibiting different near-infrared absorption spectra, show different X-ray patterns: the molecular structure of photosynthetic units is closely related to the state of pigment in each complex, although the "X-ray" molecular structure is mainly concerned with the arrangement of constituent protein molecules at the present resolution, whereas the "spectroscopic" structure reflects the local environment of pigment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved changes in equatorial x-ray diffraction and stiffness during rise of tetanic tension in intact length-clamped single muscle fibers.

We report the first time-resolved x-ray diffraction studies on tetanized intact single muscle fibers of the frog. The 10, 11, 20, 21, 30, and Z equatorial reflections were clearly resolved in the relaxed fiber. The preparation readily withstood 100 1-s duration (0.4-s beam exposure) tetani at 4 degrees C (less than 4% decline of force and no deterioration in the 10, 11 equatorial intensity ratio at rest or during activation). Equatorial intensity changes (10 and 11) and fiber stiffness led tension (t1/2 lead 20 ms at 4 degrees C) during the tetanus rise and lagged during the isometric phase of relaxation. These findings support the existence of a low force cross-bridge state during the rise of tetanic tension and isometric relaxation that is not evident at the tetanus plateau. In "fixed end" tetani lattice expansion occurred with a time course similar to stiffness during the tetanus rise. During relaxation, lattice spacing increased slightly, while the sarcomere length remained isometric, but underwent large changes after the "shoulder" of tension. Under length clamp control, lattice expansion during the tetanus rise was reduced or abolished, and compression (2%) of the lattice was observed. A lattice compression is predicted by certain cross-bridge models of force generation (Schoenberg, M. 1980. Biophys. J. 30:51-68; Schoenberg, M. 1980. Biophys. J. 30:69-78).     

Flash spectroscopy of purple membrane.

Flash spectroscopy data were obtained for purple membrane fragments at pH 5, 7, and 9 for seven temperatures from 5 degrees to 35 degrees C, at the magic angle for actinic versus measuring beam polarizations, at fifteen wavelengths from 380 to 700 nm, and for about five decades of time from 1 microsecond to completion of the photocycle. Signal-to-noise ratios are as high as 500. Systematic errors involving beam geometries, light scattering, absorption flattening, photoselection, temperature fluctuations, partial dark adaptation of the sample, unwanted actinic effects, and cooperativity were eliminated, compensated for, or are shown to be irrelevant for the conclusions. Using nonlinear least squares techniques, all data at one temperature and one pH were fitted to sums of exponential decays, which is the form required if the system obeys conventional first-order kinetics. The rate constants obtained have well behaved Arrhenius plots. Analysis of the residual errors of the fitting shows that seven exponentials are required to fit the data to the accuracy of the noise level.     

X-ray diffraction and flash photolysis studies of M intermediate of lattice-contracted purple membrane

The effects of cross-linking and lattice contraction of purple membrane (PM) on the photodynamics of bacteriorhodopsin (bR) and on the tertiary structure were studied by flash photolysis and X-ray diffraction. To get a contracted lattice form of PM, native PM, and/or PM cross-linked by glutaraldehyde were treated with deoxycholate or Triton X-100. Part of the Triton-treated cross-linked PM was further incubated with Bio-Beads SM-2 to remove Triton X-100. In the modified PM, several long-lived components of the M intermediate appeared, the features of which were related to the environment of bR. Also, X-ray diffraction studies using synchrotron radiation were performed on the modified PM under intense light irradiation (lambda greater than 500 nm) in which 40-80% of bR was photoconverted to the M state. In the Triton-treated cross-linked PM dispersed in 0.25% Triton X-100, the unit cell of membrane crystalline lattice was enlarged from 58.8 to 59.8 A and the crystalline order decreased with irradiation. The analysis of X-ray diffraction patterns suggests that light-induced conformational changes of bR correlated with the Triton content of the environment and an increase of substitution disorder was caused by these changes, but the average location of bR was unchanged. However, the other modified PM showed no significant changes of diffraction, upon light irradiation.