Two-dimensional crystallization of streptavidin: in pursuit of the molecular origins of structure, morphology, and thermodynamics

The streptavidin two-dimensional (2D) crystallization model has served as a paradigm for molecular self-assembly at interfaces. We have developed quantitative Brewster angle microscopy for the in situ measurement of spatially resolved relative protein surface densities. This allows investigation of both the thermodynamics and morphologies of 2D crystal growth. For crystal structure analysis, we employ TEM on grown crystals transferred to solid substrates. Comparison of results between commercially available streptavidin, recombinant streptavidin, and site-directed streptavidin mutants has provided insight into the protein protein and protein-lipid interactions that underlie 2D crystallization.     

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.     

Two-dimensional crystals of streptavidin on biotinylated lipid layers and their interactions with biotinylated macromolecules.

Streptavidin forms two-dimensional crystals when specifically bound to layers of biotinylated lipids at the air/water interface. The three-dimensional structure of streptavidin determined from the crystals by electron crystallography corresponds well with the structure determined by x-ray crystallography. Comparison of the electron and x-ray crystallographic structures reveals the occurrence of free biotin-binding sites on the surface of the two-dimensional crystals facing the aqueous solution. The free biotin-binding sites could be specifically labeled with biotinylated ferritin. The streptavidin/biotinylated lipid system may provide a general approach for the formation of two-dimensional crystals of biotinylated macromolecules.     

A crystallographic molecular lattice builder applied to model lipid bilayers

It is often desirable for noncrystallographers to generate graphical models of three-dimensional crystal structures based on published coordinates of the atoms that make up the crystallographic unit cells. This type of visualization is particularly important for small-molecule crystals, such as lipid crystals, where one may be interested in investigating interactions between the individual molecules in addition to their conformations. BILAYER BUILDER is a program that generates a portion of the entire crystal structure from the coordinates of the molecules in a single unit cell. It gives users of small desktop computers, such as the Apple Macintosh, the capability to generate and examine model crystal structures with a molecular graphics display program. BILAYER BUILDER stores the crystal coordinates in a Brookhaven Protein Data Bank file format for possible use in a variety of applications on many different computers. Initially, it was written for use with lipid crystals and bilayers but may be used for building an assortment of molecular crystals.     

Two-dimensional crystallization of streptavidin by nonspecific binding to a surface film: study with a scanning electron microscope.

A two-dimensional (2D) crystal of streptavidin has been obtained by a nonspecific binding method. The protein molecules were bound and formed a dense packing on the film of poly(1-benzyl-L-histidine) spread at the surface of protein solution. The surface film was moderately heated to stimulate crystallization of bound streptavidin. A potential of this method for obtaining 2D crystals of soluble proteins is demonstrated. The present 2D crystal structure of streptavidin resembles that previously obtained by specific binding to biotinylated lipid. We show in addition that the 2D array of protein with usual size approximately 50 A can be imaged using a high resolution scanning electron microscope (HR-SEM) and subject to structural analysis at low resolution. Various limitations in HR-SEM degrade considerably the image quality. However, the usability of a bulk plate as specimen support would make HR-SEM a convenient tool, when such a substrate must be considered in application of protein arrays, and if an intrinsic low resolution is acceptable.     

Helical crystallization on lipid nanotubes: streptavidin as a model protein

In this study, we use streptavidin (SA) as a model system to study helical protein array formation on lipid nanotubes, an alternative to 2D studies on lipid monolayers. We demonstrate that wild-type and a mutant form of SA form helical arrays on biotinylated lipid nanotubes. 3D maps from helical arrays of wild-type and mutant SA were reconstructed using two different approaches: Fourier-Bessel methods and an iterative single particle algorithm. The maps show that wild-type and mutant streptavidin molecules order differently. The molecular packing arrangements of SA on the surface of the lipid nanotubes differ from previously reported lattice packing of SA on biotinylated monolayers. Helical crystallization on lipid nanotubes presents an alternative platform to explore fundamentals of protein ordering, intermolecular protein interaction and phase behavior. We demonstrate that lipid nanotubes offer a robust and reproducible substrate for forming helical protein arrays which present a means for studying protein structure and structure-function relationships.     

Streptavidin crystals as nanostructured supports and image-calibration references for cryo-EM data collection

For cryo-EM structural studies, we seek to image membrane proteins as single particles embedded in proteoliposomes. One technical difficulty has been the low density of liposomes that can be trapped in the approximately 100nm ice layer that spans holes in the perforated carbon support film of EM grids. Inspired by the use of two-dimensional (2D) streptavidin crystals as an affinity surface for biotinylated DNA (Crucifix et al., 2004), we propose to use the crystals to tether liposomes doped with biotinylated lipids. The 2D crystal image also serves as a calibration of the image formation process, providing an absolute conversion from electrostatic potentials in the specimen to the EM image intensity, and serving as a quality control of acquired cryo-EM images. We were able to grow streptavidin crystals covering more than 90% of the holes in an EM grid, and which remained stable even under negative stain. The liposome density in the resulting cryo-EM sample was uniform and high due to the high-affinity binding of biotin to streptavidin. Using computational methods, the 2D crystal background can be removed from images without noticeable effect on image properties.     

Influence of bile salt molecular species on cholesterol crystallization from supersaturated model biles

Time-sequential enzymatic determination of cholesterol (CH) crystals harvested by ultrafiltration, and concomitant polarizing light microscopy observations corroborated the striking importance of the bile salts (BS) species in determining CH crystals formation rate from supersaturated model biles incubated in vitro. The more hydrophilic tauroursodeoxycholate, taurohyocholate, glycohyocholate, taurohyodeoxycholate, glycohyodeoxycholate and glyco-3α, hydroxy-6 oxo-5ß-cholanate inhibited CH precipitation through the formation of a stabilized liquid-crystalline phase. In contrast, in all hydrophobic systems (taurine (T) and glycine (G) conjugates of cholate (C), deoxycholate (DC) and chenodeoxycholate (CDC)), CH crystals precipitated with time. When crystallized CH concentrations were plotted vs. time, the figures showed a sigmoidal pattern, consistent with the transition from metastable systems to stable equilibrium states. Over the equilibration period, the nucleation kinetics (as inferred from enzymatic measurements) and all crystallization events (as microscopically observed) were both shifted in time, depending on the BS species: they were earliest in CDC systems, then in DC systems, and finally in C systems. In the latter, the delay was clearly due to the formation of a transient labile liquid-crystalline phase. G-conjugation also induced a significant delay in CH precipitation, compared to T-conjugation. At last, maximum crystallized CH concentrations at equilibrium were in the decreasing order: C > CDC > DC and T-conjugates > G-homologues. All data are discussed in connection with BS hydrophobicities, with predictions from the phase equilibria of aqueous biliary lipid systems and with new insights into CH crystal habits.     

Two-dimensional crystallization of avidin on biotinylated lipid monolayers.

Two-dimensional crystals of avidin were obtained on mixed lipid monolayers containing biotinylated lipids (N-biotinyl-dipalmitoyl-L-alpha-phosphatidyl ethanolamine and dioleoyl phosphatidyl choline) by specific interaction. Image analysis of electron micrographs of these crystals revealed p2 symmetry with the unit cell parameters a = 66 +/- 2 A, b = 68 +/- 1 A, and gamma = 121 +/- 4 degrees. The projection map showed, at a resolution of about 27 A, that the four subunits within one avidin molecule are separated into two parts. Comparison between avidin and streptavidin reveals that avidin molecule binds to the lipid monolayer in an orientation similar to that of streptavidin.     

Electron crystallography of macromolecular periodic arrays on phospholipid monolayers

Electron crystallography has the potential of yielding structural information equivalent to x-ray diffraction. The major difficulty has been preparing specimens with the required structural order and size for diffraction and imaging in the electron microscope. 2D crystallization on phospholipid monolayers is capable of fulfilling both of these requirements. Crystals can form as a result of specific interactions with a protein's ligand or an analog, suitably linked to a lipid tail; or on a surface of complementary head-group charge. With such choices, the availability of a suitable lipid is limited only by synthetic chemistry. Ultimately, it is the quality and regularity of the protein-protein interactions that determine the crystalline order, as it is with any protein crystal. In the case of streptavidin, the monolayer crystal diffracts beyond 2.5 Å. A 3 Å projection map reconstructed from electron diffraction amplitudes and phases from images shows density which can be interpreted as β-sheets and clusters of side chains. It remains to be shown that the monolayer crystals are flat and diffract as well at high tilt angle as untilted. Technological issues such as charging must be resolved. With parallel advances in data collection and processing, electron crystallography of monolayer macromolecular crystals will eventually take its place beside x-ray crystallography and NMR as a routine and efficient structural technique.     

Streptavidin-binding and -dimerizing ligands discovered by phage display, topochemistry, and structure-based design

Structural and mechanistic determinants of affinity of streptavidin-binding peptide ligands discovered by phage display are reviewed along with the use of streptavidin as a paradigm for structure-based design. A novel way of producing protein-dimerizing ligands in the streptavidin model system is discussed, in which crystal packing topochemically mediates or even catalyzes dimerization of adjacent bound ligands whose reactive ligating groups are presented toward one another in productive orientations in the crystal lattice. Finally, through crystallography on a set of streptavidin complexes with small molecule and peptide ligands at multiple pHs in two space groups, the mechanism by which ligands enhance intersubunit stabilization of the streptavidin tetramer is probed.     

Molecular dynamics study of polyethylene chain non-isothermal crystallisation: effects of chain length and branch structure

The molecular dynamics simulations in this work were aimed to provide a molecular insight into chain structure effects on non-isothermal crystallisation of polyethylene (PE) chains. The crystallisation behaviours were influenced by chain length and cooling rate in linear PE chain crystallisation: C100 and C150 were unable to fold into crystals. From C1000 to C3000, crystallisation abilities became stronger as chain length increased. From C5000 to C14000, chain length had no influence on crystallisation abilities. Final morphologies changed from rotator phase to single crystal domain, and to multi crystal domains as chain length increased. The formation of multi crystal domains with longer chain was easier than with the shorter chain in identical conditions. Branch content influenced not only the crystallisation kinetics but also final morphologies in non-isothermal crystallisation. The branches were defective in nucleation process, which was reflected in the crystal growth process. Crystallisation temperature, rate and crystallinity decreased, and the morphologies became disordered as branch content increased. Changes of final morphologies from single crystal domain to multi crystal domains were found under the influence of branch content and cooling rate. Trans-rich phenomenon was observed, and the trans-state population increment was prior to crystallinity increment. Crystallisation processes began at different crystallisation temperature when the trans-state populations reached a critical value which was independent of branch content.     

Foundations of chemical microscopy. 3. Derivatives of some chiral phenylalkylamines and phenylalkylamino acids with 5-nitrobarbituric acid

At one time, 5-nitrobarbituric acid (also known as dilituric acid) was extensively used as a chemical reagent for the qualitative identification of a variety of basic substances by light microscopy. This methodology was based on evaluation of observed crystal morphologies, because skilled observers could associate a unique crystal habit with the products formed with analytes when those were crystallized using standardized methods. As part of a study to understand the scientific foundations that permitted chemical microscopy to function as a useful analytical technique during its heyday, the products formed by dilituric acid with resolved and racemic phenylalkylamines and phenylalkylamino acids were characterized using a variety of physical analytical techniques. It was found that the different crystal morphologies associated with each of the crystalline adducts were derived from the ability of the systems to form differing structural types and/or hydrate crystal forms upon crystallization.     

Crystallographic data for Streptomyces avidinii streptavidin

Crystallization conditions are reported for Streptomyces avidinii streptavidin with and without bound biotin. X-ray examination of the free and bound crystal forms shows the streptavidin-biotin complex crystals to be most suitable for high resolution structure analysis. A complete x-ray data set to 2.6 A resolution was collected for the streptavidin-biotin crystals using a two-dimensional area detector. Reduction and analysis of the x-ray diffraction pattern show that the complex crystallizes in the tetragonal space group I4(1)22 (a = b = 98.4 A, c = 125.8 A), with half of the streptavidin tetramer in the crystallographic asymmetric unit.     

Crystal morphology study of N,N'-diacetylchitobiose by molecular dynamics simulation

Bisphenoidal shape of α-N,N′-diacetylchitobiose (α-(GlcNAc)₂) monohydrate crystals was obtained from aqueous solution. Crystal morphology was studied by computer simulation. Theoretical morphologies calculated by classic models (a BFDH model, a surface free-energy method, and an AE model) deviated significantly from that from the experimental crystal habit. Therefore, a solvent effect was considered by introducing an interface layer model based on molecular dynamics simulation in order to bridge this gap. The results of the simulation showed that the calculated habit is much closer to the experiment, meaning that the interface layer model describes the solvent effect very well. This model may also be applied for other oligosaccharide systems to study the relationship among crystal morphology, crystal structure, and solvation effects.     

Cryoelectron microscopy of a nucleating model bile in vitreous ice: formation of primordial vesicles

Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23-31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10-20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation.     

Rational design of lipid for membrane protein crystallization

The lipidic cubic phase has been used to grow crystals of membrane proteins for high-resolution structure determination. However, the original, so-called, in meso method does not work reliably at low temperatures, where proteins are generally more stable, because the hosting lipid turns solid. The need existed therefore for a lipid that forms the cubic phase and that supports crystal growth at low temperatures. We created a database of phase diagrams and used it to design such a lipid. X-ray diffraction showed that the new lipid exhibits designed phase behavior. Further, it produces diffraction quality membrane protein crystals by the in meso method at 6 degrees C. This demonstrates that lipidic materials, like their protein counterparts are amenable to rational design. The same approach as used in this study should find application in extending the range of membrane proteins crystallizable by the in meso method and in tailoring transport of cubic phases for controlled delivery and uptake.     

溶液流动对等电溶菌酶晶体生长的影响

本文对等温自由生长和强制性溶液生长的等电溶菌酶的晶体形态进行了研究,发现这些形态变化与溶液相的流动密切相关,指出生物晶体生长停止是由于生长晶体周围的溶质贫乏造成的;通过某些手段减薄或消除这一溶质贫乏区,就可以保证晶体的持续生长。本文的研究对改善大尺寸晶体的生长提供了一条途径。     

The intravacuolar organic matrix associated with calcium oxalate crystals in leaves of Vitis

In Vitis (grape) calcium oxalate crystals form in a needle-like morphology unique to plants, presenting an intriguing system of biological control over mineral formation. Crystals develop within an organic matrix which appears to provide control over the sites and forms of crystal deposition; however, little is known about the chemical nature of the matrix. A procedure has been developed to isolate crystals along with their associated intravacuolar matrix from leaves of grape, and studies have been initiated into the chemical composition of the matrix by characterizing elemental content, carbohydrates, and protein. The isolated matrix consisted of two structural phases, membrane chambers enclosing developing crystals, and a water-soluble phase surrounding the crystal chambers. Elemental analysis detected substantial calcium and potassium, as well as some iron in the water-soluble phase. Analysis of the water-soluble matrix by GC-MS showed that it contained an unusual polymer with novel glucuronic acid linkages. In addition, linkage analysis indicated 5-linked arabinans, arabinogalactan, and various mannosyl units typical of complex carbohydrates of N-linked glycoproteins. SDS—PAGE analysis of the water-soluble matrix and crystal chambers showed that each had distinct banding profiles in silver-stained gels, with prominent 60 and 70 kDa polypeptides in crystal chamber extracts. Demineralization studies provided direct evidence that the isolated matrix promotes crystal nucleation. The findings about the organic matrix associated with calcium oxalate crystals in grape are discussed in relation to crystal nucleation and growth and features shared with animal and microbial biomineralization systems.     

Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 1. Phase behavior and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings

We developed equilibrium phase diagrams corresponding to aqueous lipid compositions of upper small intestinal contents during lipid digestion and absorption in adult human beings. Ternary lipid systems were composed of a physiological mixture of bile salts (BS), mixed intestinal lipids (MIL), principally partially ionized fatty (oleic) acid (FA) plus racemic monooleylglycerol (MG), and cholesterol (Ch), all at fixed aqueous-electrolyte concentrations, pH, temperature, and pressure. The condensed phase diagram for typical physiological conditions (1 g/dL total lipids, FA:MG molar ratio of 5:1, pH 6.5, 0.15 M Na+ at 37 degrees C) was similar to that of a dilute model bile [BS/lecithin (PL)/Ch] system [Carey, M. C., & Small, D. M. (1978) J. Clin. Invest. 61, 998-1026]. We identified two one-phase zones composed of mixed micelles and lamellar liquid crystals, respectively, and two two-phase zones, one composed of Ch monohydrate crystals and Ch-saturated micelles and the other of physiologic relevance composed of Ch- and MIL-saturated mixed micelles and unilamellar vesicles. A single large three-phase zone in the system was composed of Ch-saturated micelles, Ch monohydrate crystals, and liquid crystals. Micellar phase boundaries for otherwise typical physiological conditions were expanded by increases in total lipid concentration (0.25-5 g/dL), pH (5.5-7.5), and FA:MG molar ratio (5-20:1), resulting in a reduction of the size of the physiological two-phase zone. Mean particle hydrodynamic radii (Rh), measured by quasielastic light scattering (QLS), demonstrated an abrupt increase from micellar (less than 40 A) to micelle plus vesicle sizes (400-700 A) as this two-phase zone was entered. With relative lipid compositions within this zone, unilamellar vesicles formed spontaneously following coprecipitation, and their sizes changed markedly as functions of time, reaching equilibrium values only after 4 days. Further, vesicle Rh values were influenced appreciably by MIL:mixed bile salt (MBS) ratio, pH, total lipid concentration, and FA:MG ratio, but not by Ch content. In comparison, micellar systems equilibrated rapidly, and their Rh values only slightly influenced by physical-chemical variables of physiological importance. In contrast to the BS-PL-Ch system [Mazer, N. A., & Carey, M. C. (1983) Biochemistry 22, 426-442], no divergence in micellar sizes occurred as the micellar phase boundary was approached. The ionization state of FA at simulated "intestinal" pH values (5.5-7.5) in the micellar and physiologic two-phase zones was principally that of 1:1 sodium hydrogen dioleate, an insoluble swelling "acid soap" compound. By phase separation and analysis, tie-lines for the constituent phase in the two-phase zone demonstrated that the mixed micelles were saturated with MIL and Ch and the coexisting vesicles were saturated with MBS, but not with Ch.(ABSTRACT TRUNCATED AT 400 WORDS)