Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization.

Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 ß-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.

The variable twist of actin and its modulation by actin-binding proteins.

Previous studies demonstrated that actin filaments have variable twist in which the intersubunit angles vary by approximately +/- 10 degrees within a filament. In this work we show that this variability was unchanged when different methods were used to prepare filaments for electron microscopy. We also show that actin-binding proteins can modulate the variability in twist. Three preparations of actin filaments were photographed in the electron microscope: negatively stained filaments, replicas of rapidly frozen, etched filaments, and frozen hydrated filaments. In addition, micrographs of actin + tropomyosin + troponin (thin filaments), of actin + myosin S1 (decorated filaments), and of filaments frayed from the acrosomal process of Limulus sperm (Limulus filaments) were obtained. We used two independent methods to measure variable twist based on Fourier transforms of single filaments. The first involved measuring layer line intensity versus filament length and the second involved measuring layer line position. We measured a variability in the intersubunit angle of actin filaments of approximately 12 degrees independent of the method of preparation or of measurement. Thin filaments have 15 degrees of variability, but the increase over pure actin is not statistically significant. Decorated filaments and Limulus filaments, however, have significantly less variability (approximately 2 and 1 degree, respectively), indicating a torsional stiffening relative to actin. The results from actin alone using different preparative methods are evidence that variable twist is a property of actin in solution. The results from actin filaments in the presence of actin-binding proteins suggest that the angular variability can be modulated, depending on the biological function.

Comparison of H+-ATPase and Ca2+-ATPase suggests that a large conformational change initiates P-type ion pump reaction cycles.

BACKGROUND: Structures have recently been solved at 8 A resolution for both Ca2+-ATPase from rabbit sarcoplasmic reticulum and H+-ATPase from Neurospora crassa. These cation pumps are two distantly related members of the family of P-type ATPases, which are thought to use similar mechanisms to generate ATP-dependent ion gradients across a variety of cellular membranes. We have undertaken a detailed comparison of the two structures in order to describe their similarities and differences as they bear on their mechanism of active transport. RESULTS: Our first important finding was that the arrangement of 10 transmembrane helices was remarkably similar in the two molecules. This structural homology strongly supports the notion that these pumps use the same basic mechanism to transport their respective ions. Despite this similarity in the membrane-spanning region, the cytoplasmic regions of the two molecules were very different, both in their disposition relative to the membrane and in the juxtaposition of their various subdomains. CONCLUSIONS: On the basis of the crystallization conditions, we propose that these two crystal structures represent different intermediates in the transport cycle, distinguished by whether cations are bound to their transport sites. Furthermore, we propose that the corresponding conformational change (E2 to E1 ) has two components: the first is an inclination of the main cytoplasmic mass by 20 degrees relative to the membrane-spanning domain; the second is a rearrangement of the domains comprising the cytoplasmic part of the molecules. Accordingly, we present a rough model for this important conformational change, which relays the effects of cation binding within the membrane-spanning domain to the nucleotide-binding site, thus initiating the transport cycle.

Keeping calcium in its place: Ca(2+)-ATPase and phospholamban.

Electron microscopy is gradually revealing more and more about the structure of the calcium pump from the sarcoplasmic reticulum, Ca(2+)-ATPase. The most recent result reveals the ATP-binding site, and two different avenues are being pursued towards achieving a higher resolution structure. Although no such structures are currently available for phospholamban, various spectroscopies and site-directed mutagenesis have been combined to produce a compelling structural model for its regulation of Ca(2+)-ATPase.

Structure of CaATPase: electron microscopy of frozen-hydrated crystals at 6 A resolution in projection.

Thin, three-dimensional crystals of CaATPase have been studied at high resolution by electron crystallography. These crystals were grown by adding purified CaATPase to appropriate concentrations of lipid, detergent and calcium. A thin film of crystals was then rapidly frozen and maintained in the frozen-hydrated state during electron microscopy. The resulting electron diffraction patterns extend to 4.1 A resolution and images contain phase data to 6 A resolution. By combining Fourier amplitudes from electron diffraction patterns with phases from images, a density map has been calculated in projection. Comparison of this map from unstained crystals with a previously determined map from negatively stained crystals reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and of the packing of molecules in the crystal, we have proposed a specific arrangement for the ten alpha-helices that have been suggested as spanning the bilayer.

Averaging data derived from images of helical structures with different symmetries.

There are many examples of macromolecules that form helical tubes or crystals, which are useful for structure determination by electron microscopy and image processing. Helical crystals can be thought of as two-dimensional crystals that have been rolled into a cylinder such that two lattice points are superimposed. In many real cases, helical crystals of a particular macromolecule derive from an identical two-dimensional lattice but have different lattice points superimposed, thus producing different helical symmetries which cannot be simply averaged in Fourier-space. When confronted with this situation, one can select images corresponding to one of the observed symmetries at the expense of reducing the number of images that can be used for data collection and averaging, or one can calculate separate density maps from each symmetry, then align and average them together in real-space. Here, we present a third alternative, which is based on averaging of the Fourier-Bessel coefficients, gn,l(r), and which allows the inclusion of data from all symmetry groups derived from a common two-dimensional lattice. The method is straightforward and simple in practice and is shown, through a specific example with real data, to give results comparable to real-space averaging.

Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy.

Thapsigargin (TG) is a potent inhibitor of Ca(2+)-ATPase from sarcoplasmic and endoplasmic reticula. Previous enzymatic studies have concluded that Ca(2+)-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nM and that this complex closely resembles the E(2) enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca(2+)-ATPase molecules in the E(2) conformation. In particular, we have compared 3D reconstructions of Ca(2+)-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca(2+)-ATPase in the reconstructions is very similar, demonstrating that the TG/Ca(2+)-ATPase complex does indeed physically resemble the E(2) conformation, in contrast to massive domain movements that appear to be induced by Ca(2+) binding. Difference maps reveal a consistent difference on the lumenal side of the membrane, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca(2+)-ATPase into our density maps reveals that this binding site is composed of the loops between transmembrane segments M3/M4 and M7/M8. Indirect effects are proposed to explain the effects of the S3 stalk segment on thapsigargin affinity as well as thapsigargin-induced changes in ATP affinity. Indeed, a second difference density was observed at the decavanadate-binding site within the three cytoplasmic domains, which we believe reflects an altered affinity as a result of the long-range conformational coupling that drives the reaction cycle of this family of ATP-dependent ion pumps.

Three-dimensional crystals of Ca2+-ATPase from sarcoplasmic reticulum: merging electron diffraction tilt series and imaging the (h, k, 0) projection.

Electron crystallography offers an increasingly viable alternative to X-ray crystallography for structure determination, especially for membrane proteins. The methodology has been developed and successfully applied to 2D crystals; however, well-ordered thin, 3D crystals are often produced during crystallization trials and generally discarded due to complexities in structure analysis. To cope with these complexities, we have developed a general method for determining unit cell geometry and for merging electron diffraction data from tilt series. We have applied this method to thin, monoclinic crystals of Ca2+-ATPase from sarcoplasmic reticulum, thus characterizing the unit cell and generating a 3D set of electron diffraction amplitudes to 8 A resolution with tilt angles up to 30 degrees. The indexing of data from the tilt series has been verified by an analysis of Laue zones near the (h, k, 0) projection and the unit cell geometry is consistent with low-angle X-ray scattering from these crystals. Based on this unit cell geometry, we have systematically tilted crystals to record images of the (h, k, 0) projection. After averaging the corresponding phases to 8 A resolution, an (h, k, 0) projection map has been calculated by combining image phases with electron diffraction amplitudes. This map contains discrete densities that most likely correspond to Ca2+-ATPase dimers, unlike previous maps of untilted crystals in which molecules from successive layers are not aligned. Comparison with a projection structure from tubular crystals reveals differences that are likely due to the conformational change accompanying calcium binding to Ca2+-ATPase.

Structure, transmembrane topology and helix packing of P-type ion pumps.

Electron microscopy has recently provided improved structures for P-type ion pumps. In the case of Ca(2+)-ATPase, the use of unstained specimens revealed the structure of the transmembrane domain. The composition of this domain has been controversial due to the variety of methods used to study the number and exact locations of transmembrane crossings within the sequence. After reviewing the results from several members of the family, we found a consensus for 10 transmembrane segments, and also that 10 helices fitted well into the structure of Ca(2+)-ATPase. Thus, we present the most detailed model for transmembrane structure so far, in the hope of stimulating more precise experimental strategies.

Co-reconstitution and co-crystallization of phospholamban and Ca(2+)-ATPase.

Significant advances have recently been made in understanding the regulation of Ca(2+)-ATPase by phospholamban and in modeling their structures. However, these insights would be furthered by determining the 3-D structure of both proteins within the membrane, thus revealing the structural basis for their interaction. To this end, we have developed methods for reconstituting purified Ca(2+)-ATPase with recombinant phospholamban. After reconstitution at high lipid-to-protein ratios, we have verified their functional association by measuring calcium transport and ATPase activity. Furthermore, we have grown co-crystals after reconstitution at low lipid-to-protein ratios. The structure of Ca(2+)-ATPase has recently been solved by cryoelectron microscopy at 8-A resolution, thus revealing transmembrane alpha-helices. Using a variety of constraints, we have associated these helices with the predicted transmembrane sequences to produce a detailed model for the packing of transmembrane helices. Structure determination of the co-crystals is currently underway, which we hope will eventually reveal the interaction of phospholamban with Ca(2+)-ATPase at a similar level of detail.

Development of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo diagnosis.

In this work, the development and applications of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo disease diagnosis including oral carcinoma are described. The optical diagnosis system was based on an OPO laser for multi-wavelength excitation and time-resolved detection. The pulsed Nd-YAG-pumped OPO laser system (6 ns, 20 Hz) is compact and has a rapid, broad, and uniform tuning range. Time-gated detection of intensified charge-coupled device (ICCD) making use of external triggering was used to effectively eliminate the laser scattering and contribute to the highly sensitive in vivo measurements. Artificial tissue-simulating phantoms consisting of polystyrene microspheres and tissue fluorophores were tested to optimize the gating parameters. 51-ns gate width and 39-ns gate delays were determined to be the optimal parameters for sensitive detection. In vivo measurements with the optical diagnosis system were applied to esophagus, stomach, and small intestine using an endoscope in canine animal studies. The rapid tuning capability of the optical diagnosis system contributed greatly to the optimization of wavelength for the observation of porphyrin in the small intestine. When the small intestine was thoroughly washed with water, the emission band which corresponds to porphyrin disappeared. Based on this observation, it was concluded that the detected signal was yielded by porphyrin-containing bile secretion. Also, multispectral analyses using multiple excitations from 415 to 480 nm at 5 nm intervals confirmed the porphyrin detection in the small intestine. The optical diagnosis system was also applied to the detection of human xenograft of oral carcinoma in mice using 5-aminolevulinic acid (5-ALA) which is a photodynamic therapy (PDT) drug. Significant differences in protoporphyrin IX fluorescence intensity between normal and tumor tissue could be obtained 2 hours after the injection of 5-ALA into mice due to the preferential accumulation of 5-ALA in tumors. Results reported herein demonstrate potential capabilities of the LIF-OPO system for in vivo disease diagnosis.

Neurobehavioral and immunological effects of prenatal cocaine exposure in rat.

Time-pregnant Sprague-Dawley rats were injected subcutaneously with 20 mg/kg of cocaine HCl or 0.9% saline daily from gestation days 15 through 21. Maternal plasma levels of approximately 720 ng/ml of cocaine did not alter maternal weight gain during treatment, duration of pregnancy, any of the litter variables or several indices of maternal behavior. Offsprings' body weight from birth to 30 days of age and physical maturation were not generally affected by prenatal cocaine exposure. While the development of surface righting, cliff avoidance, and the startle response was accelerated in cocaine-exposed offspring, acquisition of a preference for a social odor was unaltered. Prenatal cocaine also attenuated the locomotor response of the offspring to d-amphetamine and cocaine at PND 15; at PND 30 both of these catecholaminergic agonists increased activity in prenatal saline and prenatal cocaine offspring. However, the difference in plasma levels of cocaine at PND 30 suggests a possible down-regulation of adrenergic receptors following prenatal cocaine exposure. Decreased thymus/body weight ratios and splenomegaly were observed in prenatal cocaine animals at 55 days of age. Although complete neutralization of herpes simplex virus-type 1 was not observed, sera from prenatal cocaine offspring showed an increased rate of appearance of cytopathic effect, while sera from animals given cocaine postnatally showed a reduction in the rate at which viral infectivity was expressed in culture. These results indicate that prenatal cocaine exposure can alter neurobehavioral ontogeny and humoral immune responsitivity in the offspring.

Demonstration of an integrated capillary electrophoresis-laser-induced fluorescence fiber-optic sensor.

A unique integrated separation-based fiber-optic sensor for remote analysis, that incorporates capillary electrophoresis (CE) directly at the fiber sensing terminus is described for the first time. Based on laser-induced fluorescence detection, the sensor offers the potential for high sensitivity. Although the broad-band nature of fluorescence spectra limits selectivity, the high separation power of CE provides a unique dimension of selectivity, while permitting a design of diminutive size. Previously reported fluorescence-based sensors that utilize a chemical reagent phase to impart selectivity tend to be inflexible (not readily adaptable to the detection of different species) and "one-measurement-only" sensors. Conversely, the CE-based fiber-optic sensor described here is both versatile and reusable. The analysis speed and the potential for remote control are further attributes which make the system amenable to remote sensing. A "single-fiber" optical detection arrangement and a "single-reservoir" CE system with the fiber-optic probing the outlet of the separation capillary are employed. A preliminary evaluation of the separation characteristics of this CE-based sensor is presented. Highlights include an observed separation efficiency of up to 3000 theoretical plates (8 cm separation capillary) and migration time reproducibility of less than 10% for frontal mode CE separations. The potential utility of the sensor for remote analysis is demonstrated with separations involving the CE analysis of charged fluorescent dyes, CE analysis of metal complexes based on in situ complexation and micellar electrokinetic capillary chromatographic analysis of neutral fluorescent compounds.

Laser-induced fluorescence studies of polycyclic aromatic hydrocarbons (PAH) vapors at high temperatures.

In this work, we present the fluorescence spectra of anthracene and pyrene vapors at different elevated temperatures (from 150 to 650 degrees C) excited with the 337 nm line of a nitrogen laser. We describe the high temperature effects on the resulting spectral properties including spectral intensity, spectral bandwidth and spectral shift. We found that the PAH fluorescence spectral bandwidths become very broad as the temperature increases. The broadening is mainly due to thermal vibrational sequence congestion. We also have found that the fluorescence intensity of pyrene vapor increases with increasing temperature, which results from the increase of the pyrene vapor absorption cross section at 337 nm.

Synthesis and characterization of SERS gene probe for BRCA-1 (breast cancer).

A protocol for binding cresyl fast violet (CFV), a SERS-active dye (label) containing an aromatic amino group with a modified oligomer having a carboxy derivatized thymidine moiety using carbodiimide coupling has been achieved for the first time. Covalent coupling between CFV and the oligomer has been confirmed by mass spectral analysis of the labeled oligomer. The fluorescence, SERS and absorption characteristics of the labeled product have been evaluated. The chosen oligomer contains a BRCA-1 (breast cancer) sequence, and hence has the potential for being used as a gene probe to identify BRCA-1 gene. It has high potential for being used in polymerase chain reaction (PCR) amplification, as has been performed with labeled oligonucleotide for the HIV sequence.

Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing.

Structural studies of CaATPase from sarcoplasmic reticulum have so far been restricted to low resolution due to the poor order of two-dimensional crystal forms. However, we report that three-dimensional microcrystals of detergent-solubilized CaATPase diffract to 7.2 A in x-ray powder patterns and may therefore provide an opportunity to study CaATPase structure at higher resolutions. In the present study, we have characterized the symmetry and molecular packing of negatively stained crystals by electron microscopy (em). By altering the detergent-to-lipid ratio, different sized crystals were produced, which adhere to an em grid in different orientations. Thus, we obtained micrographs of three different projections and from these determined unit cell dimensions to be 151 X 51 X 158 A and the three-dimensional space group to be C2 with an angle beta very close to 90 degrees; x-ray powder patterns of hydrated, unstained crystals yielded dimensions of 166 X 58 X 164 A. Micrographs from each of two principal projections were averaged to produce two-dimensional density maps. Based on these maps and on the previously determined low-resolution structure of CaATPase, a packing diagram for these three-dimensional crystals is presented and major intermolecular contacts are proposed.

Growth conditions control the size and order of actin bundles in vitro.

The bonding rules for actin filament bundles do not lead to a particular packing symmetry, but allow for either regular or disordered filament packing. Indeed, both hexagonal and disordered types of packing are observed in vivo. To investigate factors which control bundle order, as well as size, we examined the effect of protein concentration on the growth of actin-fascin bundles in vitro. We found that bundles require 4-8 d to achieve both maximum size and order. The largest and best ordered bundles were grown at low fascin and high actin concentrations (an initial fascin/actin ratio of 1:200). In contrast, a much larger number of poorly ordered bundles were formed at ratios of 1:25 and 1:50, and most surprisingly, no bundles were formed at 1:300 or 1:400. Based on these observations we propose a two-stage mechanism for bundle growth. The first stage is dominated by nucleation, which requires relatively high concentrations of fascin and which is therefore accompanied by rapid growth. Below some concentration threshold, nucleation ceases and bundles enter the second stage of slow growth, which continues until the supply of fascin is exhausted. By analogy with crystallization, we hypothesize that slower growth produces better order. We are able to use this mechanism to explain our observations as well as previous observations of bundle growth both in vitro and in vivo.

Structural modelling of P-type ion pumps.

Over forty sequences of P-type ion pumps have been determined. They fall into five families showing between 20% and 50% identity in sequence. The conserved residues are concentrated in several regions which are found in all the pumps. All the defined functional sites are associated with conserved segments and provide a basis for subdivision into domains, to which tentative secondary and tertiary structures can be assigned. The domains have been assembled into a structure consisting of a conserved core with variable loops and deletions on the surface, which accommodates site mutants, affinity labels and known epitopes. This model has been correlated with the results of an electron crystallographic study of the Ca++ pump. Two types of crystal have been examined in negative stain and in amorphous ice; thin plates which diffract to 4A and long helical tubes which diffract to 15A. The plates have given a 6A projection map, some of which can be interpreted by difference from the map of the negatively stained crystal, as transmembrane helices. Three dimensional interpretation will require a tilt series. In the meantime, analysis of the tubes has given a 14A 3D map which clearly defines the cytoplasmic domains and their relation to the transmembrane region (Stokes and Toyoshima in preparation). Although it is not yet possible to assign specific functions to the cytoplasmic lobes, the structure at this resolution is consistent with the model.

The mechanics of calcium transport.

With the recent atomic models for the sarcoplasmic reticulum Ca(2+)-ATPase in the Ca(2+)-bound state, the Ca(2+)-free, thapsigargin-inhibited state, and the Ca(2+)-free, vanadate-inhibited state, we are that much closer to understanding and animating the Ca(2+)-transport cycle. These "snapshots" of the Ca(2+)-transport cycle reveal an impressive breadth and complexity of conformational change. The cytoplasmic domains undergo rigid-body movements that couple the energy of ATP to the transport of Ca2+ across the membrane. Large-scale rearrangements in the transmembrane domain suggest that the Ca(2+)-binding sites may alternately cease to exist and reform during the transport cycle. Of the three cytoplasmic domains, the actuator (A) domain undergoes the largest movement, namely a 110 degrees rotation normal to the membrane. This domain is linked to transmembrane segments M1-M3, which undergo large rearrangements in the membrane domain. Together, these movements are a main event in Ca2+ transport, yet their significance is poorly understood. Nonetheless, inhibition or modulation of Ca(2+)-ATPase activity appears to target these conformational changes. Thapsigargin is a high-affinity inhibitor that binds to the M3 helix near Phe256, and phospholamban is a modulator of Ca(2+)-ATPase activity that has been cross-linked to M2 and M4. The purpose of this review is to postulate roles for the A domain and M1-M3 in Ca2+ transport and inhibition.