Streptavidin Crystals Research Articles

A Metal-Chelating Lipid for 2D Protein Crystallization via Coordination of Surface Histidines

Two-dimensional protein crystallization on lipid monolayers is becoming a powerful technique for structure determination as well as materials applications. However, progress has been hindered by the requirement of a unique affinity lipid for each new protein of interest. Metal ion coordination by surface-accessible histidine side chains provides a convenient and general method for targeting of proteins to surfaces. Here we present the synthesis and characterization of a metal-chelating lipid which has been designed to target proteins to Langmuir monolayers and promote their two-dimensional crystallization based on histidine coordination. The lipid utilizes the metal chelator iminodiacetate (IDA) as the hydrophilic headgroup and contains unsaturated, oleyl tails to provide the fluidity necessary for two-dimensional protein crystallization. The lipid is shown to bind copper from the subphase strongly when incorporated in Langmuir monolayers. In addition, it is possible to form copper-containing monolayers by spreading the premetalated lipid on the subphase in the absence of copper. Fluorescence microscopy reveals the binding and crystallization of the protein streptavidin, promoted by the simultaneous coordination of two surface-accessible histidine side chains to the IDA−Cu lipid.

Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines.

A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented. This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface. This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals. Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket. The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

PROTEIN CRYSTALLIZATION Method may have broad applications

An ingenious new method of assembling two-dimensional arrays of protein molecules may lead to a general, simple technique for the usually arduous task of protein crystallization, as well as to a new breed of biomolecular imprints for materials design. Protein crystallization—which is vital to enable protein structure determination by electron or X-ray diffraction methods—is one of chemistry's most vexing problems. The technique was developed by groups led by California Institute of Technology chemical engineer Frances H. Arnold and by University of Washington bioengineers Viola Vogel and Patrick S. Stayton. In an article to be published in the May 14 issue of the Proceedings of the National Academy of Sciences , the two groups describe successful 2-D crystallization of the protein streptavidin on a lipid monolayer studded with copper. Streptavidin already has been studied extensively. Researchers not only have created 2-D crystals of streptavidin using biotinylated lipids [ Biophys. J. , 59 , 387 (1991)...

Two-Dimensional Crystallization of Streptavidin Studied by Quantitative Brewster Angle Microscopy

Brewster angle microscopy (BAM) is extended to probe protein adsorption and aggregation processes at interfaces quantitatively. Since no p-polarized light is reflected from the plain air−water inte...

Topochemistry for preparing ligands that dimerize receptors

Background: The cyclic, disulfide-containing peptide cyclo-Ac-[Cys-His-Pro-Gln-Gly-Pro-Pro-Cys]-NH 2, binds to streptavidin with high affinity. In streptavidin-peptide cocrystals of space group 1222, cyclic peptide monomers are bound on adjacent streptavidin tetramers related by a crystallographic two-fold symmetry axis. We set out to determine whether the disulfide bonds of the peptide, presented close to one another in the crystal, could undergo disulfide interchange to form a dimer. Results: When juxtaposed, the disulfides of neighboring peptides undergo disulfide interchange, breaking and forming covalent disulfide bonds, to produce a peptide dimer adopting the symmetry of the crystal. This is the first example of a chemical transformation mediated by a protein crystal lattice. The structure of the streptavidin-bound monomer, and that of the dimer that was eventually produced from it in the crystal, were both determined from the same single crystal studied at different times. The two-fold symmetric peptide dimer was independently synthesized and shown to form crystals of dimerized streptavidin. Conclusions: We have shown that formation of a covalently linked peptide dimer can be mediated by a protein crystal lattice. The dimer thus produced dimerizes its target, streptavidin, suggesting that solid-state (or topochemical) reactions of this kind may be broadly useful for the preparation of ligands that can dimerize other protein targets.

Visualization of β-sheets and some amino acid side chains in 400 kV electron images of streptavidin crystallized on phospholipid monolayers

Two-dimensional (2-D) protein crystallization on lipid monolayers is an emerging technique with potential application to structure elucidation of biological macromolecules (Uzgiris and Kornberg, 1983). Among more than a dozen proteins or macromolecular assemblies which have been crystallized by this technique, streptavidin 2-D crystals diffract electrons beyond 3 Å (Kubalek, et al., 1991), indicating that structural information from them is retrievable to near-atomic resolution. This 15 kD protein, synthesized by Streptomyces avidinii, is probably the single most widely used protein in modern biochemical assays. It forms nearly irreversible complexes (Kd ≈ 10-15 M) with the vitamin biotin and biotinylated molecules, which can be detected by a variety of methods.2-D streptavidin crystals were grown on biotinylated lipid monolayers by a method similar to that of Kubalek, et al., 1991. They were preserved in vitreous ice for electron cryomicroscopy. Electron images were recorded at 400 kV under flood beam illumination at a dose of 8 e-/Å2. Phases from four micrographs were merged.

The Mechanism of Protein Crystal Growth from Lipid Layers

Two-dimensional (2D) crystals of proteins on lipid monolayers can initiate the formation of large three-dimensional (3D) crystals suitable for X-ray diffraction studies. The role of the 2D crystals in this process has not been firmly established. While it is likely that the 2D crystals serve as nuclei for epitaxial crystal growth, other mechanisms, such as non-specific nucleation induced by the high local concentration of the protein at the surface of the lipid layer, cannot be excluded. Using streptavidin as a model system, we have now firmly established that 3D crystal growth from 2D crystals on lipid layers occurs by epitaxy. We show that 2D crystals of streptavidin (space groupC222) on biotinated lipid layers nucleate the growth of a 3D crystal form (space groupI4I22) that possesses a structural similarity with the 2D crystal, but have no effrect on the growth of 3D crystal forms (I222 andP21) that are unrelated to the 2D crystal. At lower pH, a new 3D crystal form (space groupP1), unrelated to the previously described 2D crystals, grew from lipid layers. This discovery initially raised concern about the validity of the epitaxial mechanism, but these concerns were alleviated with the subsequent discovery of a structurally related 2DP1 crystal that grew in similar solution conditions. Some parameters affecting epitaxial growth of both the P1 andI4I22 crystals were investigated, revealing several noteworthy features of the epitaxial growth. (1) 2D crystals are very effective nucleating agents; for instance, theP1 2D crystals can direct the growth ofP1 3D crystals even under conditions that favour the growth of other crystal forms. (2) The epitaxial 3D crystal grow very rapidly and at amazingly low protein concentrations;P1 3D crystals can be grown from solutions as low as 10μM streptavidin. (3) There is no obligate requirement for the deposition of pre-formed 2D crystals; lipid layers alone are equally effective at promoting epitaxial crystal growth.

Charging phenomena observed on biological specimens in a 400-kV electron cryo-microscope

There is an ever present need in electron microscopy of biological specimens to improve the quality of the recorded data to approach the atomic resolution capability of the modem instruments. The method adopted which has yielded the only significant advance in this direction is to directly phase theelectron diffraction amplitudes of 2-dimensional crystals using the Fourier transforms of high resolution images. Cryo methods have improved the preservation and stability of the specimen to the relatively high electron dose irradiation which is required for imaging compared to electron diffraction. In the cases, however, where the actual power of the structure factors was compared with those that were transferred by the microscope’s imaging system, a large attenuation was measured. Spot-scan imaging has been shown to improve the image quality by reducing the beam-induced movement. Charging of the specimen appears to cause similar deleterious effects on image resolution, but it requires different methods aimed at resolution improvement. Proteins are insulators and because of secondary electron loss they will become positively charged during irradiation. Defocused diffraction brightfield imagescan display the charged nature of an irradiated specimen as an apparent increase in mass. A comparison wasmade of the degree of charging and discharging between using 100 and 400 keV electrons on streptavidin crystals in glucose and catalase crystals in amorphous ice. We show that when the region of the irradiated sample includes part of the supporting carbon film, the charge build-up is prevented.

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.

Molecular recognition in biotin-streptavidin systems and analogues at the air-water interface

Specific interaction between biotin and the protein streptavidin in monolayers of synthetic lipids with biotin headgroups has been shown to lead to formation of highly ordered two-dimensional streptavidin crystals. The same behaviour is observed when using desthiobiotin as lipid headgroup which exhibits a significantly lower binding constant compared with biotin (5 × 10 13 M -1 compared with 10 15 M -1). This offers the possibility of detaching competetively the 2D crystalline streptavidin layer by addition of free biotin to the aqueous phase. Use of lipoic acid as lipid headgroup ( K a = 7 × 10 7 M −1) leads to formation of small snisotropic protein domains indicating a crystallization of streptavidin as already observed with the biotin and desthiobiotinlipids. Interaction of the protein with HABA-lipids ( K a = 10 8 M −1) under the same conditions does not lead to domain formation at the air-water interface, although the monolayer behaviour of the HABA lipid is significantly altered by active streptavidin in the subphase.

Molecular recognition processes at functionalized lipid surfaces: a neutron reflectivity study

The specific binding of proteins to functionalized monolayers on aqueous subphases has been characterized by neutron reflectivity measurements. As a model for the investigation of a recognition process on a molecular length scale, streptavidin and biotin were used because of the high specific affinity between them. Reflectivities from the aqueous surfaces covered with biotinylated monolayers before and after the adsorption of proteins were collected with a novel fixed wavelength liquid surface neutron reflectometer. In quantitative terms, binding was found to occur at a biotin surface concentration as low as 1 unit/1250 Å 2. The data are consistent with the formation of a monomolecular protein layer at the interface, directly underneath the lipid monolayer. The thickness of the protein film is 44 ± 2 Å, comparable in size to the smallest axis of the unit cell of three-dimensional streptavidin crystals (48 Å). It is found that the composition of the functionalized surface monolayer critically influences the two-dimensional aggregration behavior of the protein underneath.

Improved transfer of two-dimensional crystals from the air/water interface to specimen support grids for high-resolution analysis by electron microscopy

Electron crystallographic analysis of two-dimensional crystals grown on lipid layers at the air/water interface has been limited by loss or damage during transfer of the crystals to an electron microscope support grid. Two methods of transfer are described which are applicable on a small scale (10 μ1 of protein solution) and which give greatly improved results for streptavidin crystals on biotinylated lipid layers. In the first method, a hydrophobic grid surface was produced by coating a carbon support film with a thin layer of SiO 2, followed by alkylation with dimethyloctadecylchlorosilane. The transfer efficiency of protein crystals approached 50% coverage of the alkylated grid surface. The degree of order of crystals transferred to the alkylated grid surface and preserved in negative stain was significantly improved over that of crystals transferred directly to a carbon support film. In the second method, crystals at the air/water interface were transferred to a holey carbon support film. The efficiency of transfer across the holes was virtually 100%, as nearly every hole was completely covered with crystals. After preservation of the crystals in 1% glucose and cooling to liquid nitrogen temperature, electron diffraction was obtained that extended to 1/2.8 Å -1 resolution. This result demonstrates that two-dimensional crystals grown on lipid layers at the air/water interface can be sufficiently well-ordered, even after transfer to a support grid, to yield high-resolution structural information.

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.

Characterization and crystallization of core streptavidin.

We have characterized a streptavidin product that had been reduced to a minimal size that still retained full biotin-binding activity. This core streptavidin is proteolyzed at both ends at points that correspond closely with the termini of hen egg white avidin. Core streptavidin is more soluble than is the parent molecule. We have grown three different types of crystals of core streptavidin. The symmetry properties of these crystals prove that the molecule is a tetramer organized in tetrahedral (D2) point symmetry. The crystallographic response to the interaction of biotin with core streptavidin indicates that some conformational change accompanies ligand binding. We are attempting to determine the three-dimensional structure of streptavidin and its complex with selenobiotin from these crystals of core streptavidin.