SEROLOGICAL STUDIES ON THE FORMATION OF PROTEIN PARASPORAL INCLUSIONS IN BACILLUS THURINGIENSIS

A strain of Bacillus thuringiensis has been isolated, and methods have been developed for separation of the crystalline, parasporal inclusions in a pure form. Normal sporulation with concomitant crystal formation takes place when cells are incubated under suitable conditions in a nutrient free medium. Serological techniques have been used to study the origin and development of the crystals. Rabbit antisera have been prepared to a vegetative cell extract, suspensions of crystals, and a solution of crystal protein (obtained by alkali treatment of crystals). Tests have been carried out mainly by the Ouchterlony gel plate technique. Crystal protein solutions were found to be more active than suspensions of intact crystals both in reaction with, and in neutralization of, the crystal antibodies. Antisera to the vegetative cell extract gave no reaction with crystal protein. Ultrasonic extracts of cells taken before or during crystal formation gave no reaction with the crystal antibodies. Tests with alkali extracts of disrupted cells showed that the crystal antigen is absent in vegetative cells but arises during sporulation. The appearance of the antigen can be correlated with the formation and growth of the crystals as followed by examination of disrupted cell preparations under the electron microscope. It can be concluded that the crystalline protein inclusions do not arise from precursors in the same antigenic state.     

S-layer protein from Thermus thermophilus HB8 assembles into porin-like structures

The cells of the extreme thermophile Thermus thermophilus are surounded by a regular layer (S-layer) built up by a protein with an apparent molecular mass of 100 kDa (P100). From purified membrane fractions, three different class of two-dimensional crystals can be obtained by following alternative extractive procedures. One of these crystals, with p6 symmetry, clearly represents the native S-layer detected by freeze etching on whole cells, while the other two, showing p2 and p3 symmetries respectively, closely resemble aggregates of bacterial porins. We demonstrate here by limited protreolysis and Western blotting the surprising fact that the protein component of the three crystals is the P100 protein. Our biochemical data also show how this protein forms Ca²⁺-stabilized trimers in each crystal, which support the structural analysis that points to p3 units as the common structural block in all of them, and again resembles the situation found in bacterial porins.     

The role of lipids and salts in two-dimensional crystallization of the glycine-betaine transporter BetP from Corynebacterium glutamicum

The osmoregulated and chill-sensitive glycine–betaine transporter (BetP) from Corynebacterium glutamicum was reconstituted into lipids to form two-dimensional (2D) crystals. The sensitivity of BetP partly bases on its interaction with lipids. Here we demonstrate that lipids and salts influence crystal morphology and crystallinity of a C-terminally truncated BetP. The salt type and concentration during crystallization determined whether crystals grew in the form of planar-tubes, sheets or vesicles, while the lipid type influenced crystal packing and order. Three different lipid preparations for 2D crystallization were compared. Only the use of lipids extracted from C. glutamicum cells led to the formation of large, well-ordered crystalline areas. To understand the lipid-derived influence on crystallinity, lipid extracts from different stages of the crystallization process were analyzed by quantitative multiple-precursor ion scanning mass spectroscopy (MS). Results show that BetP has a preference for fatty acid moieties 16:0–18:1, and that a phosphatidyl glycerol (PG) 16:0–18:1 rich preparation prevents formation of pseudo crystals.     

Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation

Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, "in cubo", has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field.     

X-ray diffraction from membrane protein nanocrystals

Membrane proteins constitute >30% of the proteins in an average cell, and yet the number of currently known structures of unique membrane proteins is <300. To develop new concepts for membrane protein structure determination, we have explored the serial nanocrystallography method, in which fully hydrated protein nanocrystals are delivered to an x-ray beam within a liquid jet at room temperature. As a model system, we have collected x-ray powder diffraction data from the integral membrane protein Photosystem I, which consists of 36 subunits and 381 cofactors. Data were collected from crystals ranging in size from 100 nm to 2 μm. The results demonstrate that there are membrane protein crystals that contain <100 unit cells (200 total molecules) and that 3D crystals of membrane proteins, which contain <200 molecules, may be suitable for structural investigation. Serial nanocrystallography overcomes the problem of x-ray damage, which is currently one of the major limitations for x-ray structure determination of small crystals. By combining serial nanocrystallography with x-ray free-electron laser sources in the future, it may be possible to produce molecular-resolution electron-density maps using membrane protein crystals that contain only a few hundred or thousand unit cells.     

7 Å projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7 Å resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Automated screening of 2D crystallization trials using transmission electron microscopy: A high-throughput tool-chain for sample preparation and microscopic analysis

We have built and extensively tested a tool-chain to prepare and screen two-dimensional crystals of membrane proteins by transmission electron microscopy (TEM) at room temperature. This automated process is an extension of a new procedure described recently that allows membrane protein 2D crystallization in parallel (Iacovache et al., 2010). The system includes a gantry robot that transfers and prepares the crystalline solutions on grids suitable for TEM analysis and an entirely automated microscope that can analyze 96 grids at once without human interference. The operation of the system at the user level is solely controlled within the MATLAB environment: the commands to perform sample handling (loading/unloading in the microscope), microscope steering (magnification, focus, image acquisition, etc.) as well as automatic crystal detection have been implemented. Different types of thin samples can efficiently be screened provided that the particular detection algorithm is adapted to the specific task. Hence, operating time can be shared between multiple users. This is a major step towards the integration of transmission electron microscopy into a high throughput work-flow.     

Binding of a synthetic targeting peptide to a mitochondrial channel protein

Membrane crystals of the mitochondrial outer membrane channel VDAC (porin) fromNeurospora crassa were incubated with a 20-amino-acid synthetic peptide corresponding to the N-terminal targeting region of subunit IV of cytochrome oxidase. The peptide caused disordering and contraction of the crystal lattice of the membrane arrays. Also, new stain-excluding features were observed on the peptide-treated arrays which most likely correspond to sites at which the peptide accumulates. The stain exclusion zones associated with binding of the targeting peptide (and with binding of apocytochromec in an earlier study) have been localized on a two-dimensional density map of frozen-hydrated, crystalline VDAC previously obtained by cryo-electron microscopy. The results indicate that both the peptide and cytochromec bind to protein arms which extend laterally between the channel lumens. The finding that imported polypeptides bind to a specific region of the VDAC protein implicates this channel in the process by which precursor proteins are recognized at and translocated across the mitochondrial outer membrane.     

Surface topography and molecular stoichiometry of the mitochondrial channel, VDAC, in crystalline arrays.

The mitochondrial outer membrane contains a protein, called VDAC, that forms large aqueous pores. In Neurospora crassa outer membranes, VDAC forms two-dimensional crystalline arrays whose size and frequency can be greatly augmented by lipase treatment of these membranes (C. Mannella, Science 224, 165, 1984). Fourier filtration and surface reconstruction of freeze-dried/shadowed (45 degrees) arrays produced detailed images of two populations of crystals, whose lattices are mirror images of each other. Most likely, this technique has revealed both surfaces of the same two-dimensional crystal with lattice parameters: a = 12.3 +/- 0.1 nm, b = 11.2 +/- 0.1 nm, and theta = 109 +/- 1 degree. Three-dimensional reconstructions of the surface reliefs on both sides of the crystal show them to be very similar. The majority of the protein forming the channel appears to be at or below the level of the membrane. To address the issue of the number of 30-kDa polypeptides that form a VDAC channel, measurements of mass per unit area were carried out by analyzing scanning transmission electron micrographs of unstained, freeze-dried arrays. The crystal form used for mass analysis contained the same motif of six stain-accumulating centers per unit cell, with p2 symmetry as in the oblique configuration, but it had a different orientation relative to the lattice lines. These data yielded a surface density of 1.9 +/- 0.2 kDa/nm2, indicating that there is a one-to-one ratio between VDAC polypeptides and the channels visualized in filtered electron micrographs, and that VDAC membrane crystals contain 68% protein and 32% lipid by mass.     

Novel crystalline sheets of Na,K-ATPase induced by phospholipase A2

Treatment of purified preparations of Na,K-ATPase by phospholipase A2 has led to the formation of two-dimensional crystals of the protein. Control tests with another phospholipase and two detergents have shown that crystallization occurs as the result of hydrolysis and/or solubilization of the phospholipids in the enzyme vesicles. Experimentation with various buffer systems has indicated that reduction in the amount of phospholipids alone is sufficient for inducing the formation of crystalline sheets. Inclusion of crystal inducing ions in the buffer facilitates the crystallization process, resulting in more extensive arrays. The new crystalline sheets are exclusively dimeric with average unit cell dimensions: a = 15.8 +/- 0.4 nm, b = 4.9 +/- 0.2 nm, and gamma = 64 +/- 3 degrees. Examination of the micrographs shows that the initial intermolecular interaction leading to the formation of sheets is between the alpha subunits. Results from this study suggest that removal and/or modification of phospholipids by phospholipases could prove successful in crystallizing those membrane proteins in which excess lipid is the main barrier to the formation of two-dimensional arrays.     

Two-Dimensional Crystallization of Brush Border Myosin I

Brush border myosin-I (BBMI) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells, where it links the core bundle of actin filaments to the plasma membrane. An association of BBMI with anionic phospholipids has been shown to be mediated by a carboxy-terminal domain which is rich in basic amino acids. We have exploited this natural affinity of BBMI for negatively charged lipids to form two-dimensional (2D) crystals of this protein which are suitable for structural analysis by electron crystallographic techniques. The 2D crystals which we have obtained belong to one of two space groups, p22₁2₁or p2. We present here projection maps calculated from images of negatively stained crystals for each of these crystal types to a resolution of 20 Å and show that the asymmetric unit is the same in both crystal types.     

Crystallization and micro FT-IR spectroscopy investigation of cytochrome bc1 complex

A simple method to obtain large red crystals of cytochrome bc1 complex from beef heart mitochondria has been developed. These crystals are very stable. Their shapes are retained for a long time in tip-sealed Pasteur pipets placed in a refrigerator. The structure of crystalline cytochrome bc1 complex by micro FT-IR spectroscopy has been investigated. Based on the IR spectra of cytochrome c, the empirical assignments of the major infrared frequencies of cytochrome bc1 complex are given. Infrared frequencies and relative intensities of variable orientation and section of crystal are significantly different. These imply that infrared spectral characterization of the membrane protein crystallization is associated with the variable symmetries and orientations of the structure. Experimental results show that phospholipid exists in the crystal of cytochrome bc1 complex. The membrane protein is probably spanned on the mitochondrial membrane and buried in phospholipid bilayer in an asymmetric manner.     

Co‐ordinated functions of Mms proteins define the surface structure of cubo‐octahedral magnetite crystals in magnetotactic bacteria

Magnetotactic bacteria synthesize magnetosomes comprised of membrane‐enveloped single crystalline magnetite (Fe₃O₄). The size and morphology of the nano‐sized magnetite crystals (< 100 nm) are highly regulated and bacterial species dependent. However, the control mechanisms of magnetite crystal morphology remain largely unknown. The group of proteins, called Mms (Mms5, Mms6, Mms7, and Mms13), was previously isolated from the surface of cubo‐octahedral magnetite crystals in Magnetospirillum magneticum strain AMB‐1. Analysis of an mms6 gene deletion mutant suggested that the Mms6 protein plays a major role in the regulation of magnetite crystal size and morphology. In this study, we constructed various mms gene deletion mutants and characterized the magnetite crystals formed by the mutant strains. Comparative analysis showed that all mms genes were involved in the promotion of crystal growth in different manners. The phenotypic characterization of magnetites also suggested that these proteins are involved in controlling the geometries of the crystal surface structures. Thus, the co‐ordinated functions of Mms proteins regulate the morphology of the cubo‐octahedral magnetite crystals in magnetotactic bacteria.     

Cell and calcium oxalate crystal growth is coordinated to achieve high-capacity calcium regulation in plants

Summary Crystal idioblasts are cells which are specialized for accumulation of Ca²⁺ as a physiologically inactive, crystalline salt of oxalic acid. Using microautoradiographic, immunological, and ultrastructural techniques, the process of raphide crystal growth, and how crystal growth is coordinated with cell growth, was studied in idioblasts ofPistia stratiotes. Incorporation of⁴⁵Ca²⁺ directly demonstrated that, relative to surrounding mesophyll cells, crystal idioblasts act as high-capacity Ca²⁺ sinks, accumulating large amounts of Ca²⁺ within the vacuole as crystals. The pattern of addition of Ca²⁺ during crystal growth indicates a highly regulated process with bidirectional crystal growth. In very young idioblasts,⁴⁵Ca²⁺ is incorporated along the entire length of the needle-shaped raphide crystals, but as they mature incorporation only occurs at crystal tips in a bidirectional mode. At full maturity, the idioblast stops Ca²⁺ uptake, although the cells are still alive, demonstrating an ability to strictly regulate Ca transport processes at the plasma membrane. In situ hybridization for ribosomal RNA shows young idioblasts are extremely active cells, are more active than older idioblasts, and have higher general activity than surrounding mesophyll cells. Polarizing and scanning electron microscopy demonstrate that the crystal morphology changes as crystals develop and includes morphological polarity and an apparent nucleation point from which crystals grow bidirectionally. These results indicate a carefully regulated process of biomineralization in the vacuole. Finally, we show that the cytoskeleton is important in controlling the idioblast cell shape, but the regulation of crystal growth and morphology is under a different control mechanism.Abbreviation SEM scanning electron microscopy     

X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes

An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt-generated two-phase system. Monodispersity and homogeneity of protein-detergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis. By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper. The tetragonal form (space group P4(2); unit cell dimensions, a = b = 155 A, c = 172 A) is suitable for X-ray analysis. Low temperature induces a change of the space group to P4(2)22, with a single trimer in the asymmetric unit. This crystal form diffracts to a resolution beyond 2.9 A. The hexagonal crystal form (space group P6(3)22; unit cell dimensions, a = b = 93 A, c = 220 A) is limited in resolution to 4.5 A, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays.     

Electron crystallographic analysis of two-dimensional streptavidin crystals coordinated to metal-chelated lipid monolayers.

Coordination of individual histidine residues located on a protein surface to metal-chelated lipid monolayers is a potentially general method for crystallizing proteins in two dimensions. It was shown recently by Brewster angle microscopy (BAM) that the model protein streptavidin binds via its surface histidines to Cu-DOIDA lipid monolayers, and aggregates into regularly shaped domains that have the appearance of crystals. We have used electron microscopy to confirm that the domains are indeed crystalline with lattice parameters similar to those of the same protein crystallized beneath biotinylated lipid monolayers. Although BAM demonstrates that the two-dimensional protein crystals grown via metal chelation are distinct from the biotin-bound crystals in both microscopic shape and thermodynamic behavior, the two crystal types show similar density projections and the same plane group symmetry.     

Crystallization and micro FT-IR spectroscopy investigation of cytochrome bc_1 complex

A simple method to obtain large red crystals of cytochrome bc1 complex from beef heart mitochondria has been developed. These crystals are very stable. Their shapes are retained for a long time in tip-sealed Pasteur pipets placed in a refrigerator. The structure of crystalline cytochrome bc1 complex by micro FT-IR spectroscopy has been investigated. Based on the IR spectra of cytochrome c, the empirical assignments of the major infrared frequencies of cytochrome bc1 complex are given. Infrared frequencies and relative intensities of variable orientation and section of crystal are significantly different. These imply that infrared spectral characterization of the membrane protein crystallization is associated with the variable symmetries and orientations of the structure. Experimental results show that phospholipid exists in the crystal of cytochrome bc1 complex. The membrane protein is probably spanned on the mitochondrial membrane and buried in phospholipid bilayer in an asymmetric manner.     

Protein crystals in Adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489-492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors.     

On the formation of the pattern of crystal idioblasts — in Canavalia ensiformis DC

Summary Light and electron-microscope observations were made of the crystal idioblasts in the leaves of Canavalia. The crystal-containing cells occur as pairs in which the crystals, nuclei, and the majority of the chloroplasts are symmetrically arranged with regard to the common wall. The chloroplasts are found in the cytoplasm along this wall.The crystals originate in a vacuole. The space in which the young crystal develops is delimited by a membrane. One to several additional membranes surround the crystal inside the vacuole. Numerous vesicles are distributed between these vacuolar membranes. Dense groups of tubules or fibrils are oriented toward a portion of the crystal surface, suggesting that the material forming the crystal might be transported to the surface by these structures.The cytoplasm of the young idioblasts contains many mitochondria and dictyosomes with associated vesicles. Concentrations of what is assumed to be protein are present in the cytoplasm. These protein accumulations are not seen in neighboring cells, suggesting that protein synthesis is especially high in the idioblasts.In older crystal cells, layers of wall material are deposited on the wall between the two crystals of the pair and towards the cell wall adjacent to the mesophyll. Not only does the original wall become thickened but a new wall develops at the border of the crystal vacuole. Eventually this wall material becomes continuous and the crystal becomes, on two sides, directly connected with the wall.