Streptavidin Crystals Research Articles

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation.

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community.

Genetically fused charged peptides induce rapid crystallization of proteins.

We utilized electrostatic interaction to induce rapid crystallization of streptavidin. Simply mixing streptavidins possessing either a positively or negatively charged peptide at their C-terminus generated diffraction-quality crystals in a few hours. We modified the streptavidin crystals with fluorescent molecules using biotin, demonstrating the concept of protein crystals as functional biomaterials.

3.1 Å structure of yeast RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion solved using streptavidin affinity grids

Despite significant advances in all aspects of single particle cryo-electron microscopy (cryo-EM), specimen preparation still remains a challenge. During sample preparation, macromolecules interact with the air-water interface, which often leads to detrimental effects such as denaturation or adoption of preferred orientations, ultimately hindering structure determination. Randomly biotinylating the protein of interest (for example, at its primary amines) and then tethering it to a cryo-EM grid coated with two-dimensional crystals of streptavidin (acting as an affinity surface) can prevent the protein from interacting with the air-water interface. Recently, this approach was successfully used to solve a high-resolution structure of a test sample, a bacterial ribosome. However, whether this method can be used for samples where interaction with the air-water interface has been shown to be problematic remains to be determined. Here we report a 3.1 Å structure of an RNA polymerase II elongation complex stalled at a cyclobutane pyrimidine dimer lesion (Pol II EC(CPD)) solved using streptavidin grids. Our previous attempt to solve this structure using conventional sample preparation methods resulted in a poor quality cryo-EM map due to Pol II EC(CPD)’s adopting a strong preferred orientation. Imaging the same sample on streptavidin grids improved the angular distribution of its view, resulting in a high-resolution structure. This work shows that streptavidin affinity grids can be used to address known challenges posed by the interaction with the air-water interface.

Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality.

Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5Å or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5Å, while the resolution of the map reached 4.0Å for the same number of particles, when the estimated resolution of streptavidin crystal was 4Å or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6Å, a marked improvement over the value of 3.9Å obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.

Tuning membrane protein mobility by confinement into nanodomains.

High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.

A Novel Platform for Tailoring Membrane Protein Mobility

High speed atomic force microscopy (HS-AFM) [1] allows for visualizing the dynamics and conformational changes of single soluble proteins [2]. For studying purified and reconstituted membrane proteins, planar lipid membranes have to be generated. This is usually done by spreading proteoliposomes on flat solid supports such as mica. However, these surfaces cause unwanted and vastly uncontrollable interactions that influence mobility, conformation as well as functionality of the membrane proteins. Hence, there is a need for flat, non-interacting supports.Here we show how streptavidin crystals grown on mica-supported lipid bilayers can be exploited as suspension for membranes containing biotinylated lipids. Such periodically suspended membranes (PSMs) are flat and mechanically stable, and are thus well suited for studying membrane proteins by HS-AFM and other surface specific techniques. Reconstituted proteins therein are provided with free space on the support side, while the crowded environment sets limitations to their lateral movement.Furthermore, our platform allows for tuning the lateral mobility of membrane proteins by glutaraldehyde-crosslinking of streptavidin to any value between unrestricted membrane diffusion and immobility, as exemplified for SecYEG (protein translocation channel) and GlpF [3,4] (aquaglyceroporin). Such tailoring to the scanning rate of the microscope enables conformational or docking studies of spatially confined proteins as we demonstrate by imaging GlpF at sub-molecular resolution and catching the motor protein SecA in the act of binding to SecYEG.[1] Ando, et al., Proceedings of the National Academy of Sciences 98.22 (2001): 12468-12472.[2] Preiner, et al., Nature communications 5 (2014).[3] Preiner, et al., Nano letters 15.1 (2014): 759-763.[4] Horner, et al., Science Advances 1.2 (2015): e1400083.

Flow-induced 2D protein crystallization: characterization of the coupled interfacial and bulk flows.

Two-dimensional crystallization of the protein streptavidin, crystallizing below a biotinylated lipid film spread on a quiescent air-water interface is a well studied phenomenon. More recently, 2D crystallization induced by a shearing interfacial flow has been observed at film surface pressures significantly lower than those required in a quiescent system. Here, we quantify the interfacial and bulk flow associated with 2D protein crystallization through numerical modeling of the flow along with a Newtonian surface model. Experiments were conducted over a wide range of conditions resulting in a state diagram delineating the flow strength required to induce crystals for various surface pressures. Through measurements of the velocity profile at the air-water interface, we found that even in the cases where crystals are formed, the macroscopic flow at the interface is well described by the Newtonian model. However, the results show that even in the absence of any protein in the system, the viscous response of the biotinylated lipid film is complicated and strongly dependent on the strength of the flow. This observation suggests that the insoluble lipid film plays a key role in flow-induced 2D protein crystallization.

Dip-pen nanolithography-assisted protein crystallization.

We demonstrate the use of dip-pen nanolithography (DPN) to crystallize proteins on surface-localized functionalized lipid layer arrays. DOPC lipid layers, containing small amounts of biotin-DOPE lipid molecules, were printed on glass substrates and evaluated in vapor diffusion and batch crystallization screening setups, where streptavidin was used as a model protein for crystallization. Independently of the crystallization system used and the geometry of the lipid layers, nucleation of streptavidin crystals occurred specifically on the DPN-printed biotinylated structures. Protein crystallization on lipid array patches is also demonstrated in a microfluidic chip, which opens the way toward high-throughput screening to find suitable nucleation and crystal growth conditions. The results demonstrate the use of DPN in directing and inducing protein crystallization on specific surface locations.

Substrate Effects on Structural Studies using Cryo-Em

In electron microscopy, glow discharge has been widely used to render the carbon film hydrophilic for better adhesion of aqueous solution. However, the target macromolecules (e.g. proteins) may also interact with the charge on the carbon film, which may introduce preferred orientation and/or some other artifacts. A promising alternative is to use two-dimensional (2D) crystal as a substrate. This method has been successfully employed to study DNA molecules, proteoliposomes, and multiprotein complexes. Here we will use liposomes (i.e. lipid vesicles) as a model system to study the effect of substrate on targeted structures. Four types of substrates will be studied: untreated carbon film, glow-discharged carbon film, holey carbon film, and 2D streptavidin crystal. Liposomes, doped with a few copies of biotinylated lipid and osmotically swollen to ensure a spherical shape, will be allowed to attach to the substrate before blotting and rapid freezing of the specimen. Then cryo-electron microscopy of tilted specimens and cryo-electron tomography will be employed, and the resulted images and tomographs will be compared with spherical liposome models. The deformation of the liposomes will be used to quantify the effect of the substrate on the targeted structure.

Minimizing electrostatic charging of an aperture used to produce in-focus phase contrast in the TEM

Microfabricated devices designed to provide phase contrast in the transmission electron microscope must be free of phase distortions caused by unexpected electrostatic effects. We find that such phase distortions occur even when a device is heated to 300 °C during use in order to avoid the formation of polymerized, carbonaceous contamination. Remaining factors that could cause unwanted phase distortions include patchy variations in the work function of a clean metal surface, radiation-induced formation of a localized oxide layer, and creation of a contact potential between an irradiated area and the surround due to radiation-induced structural changes. We show that coating a microfabricated device with evaporated carbon apparently eliminates the problem of patchy variation in the work function. Furthermore, we show that a carbon-coated titanium device is superior to a carbon-coated gold device, with respect to radiation-induced electrostatic effects. A carbon-coated, hybrid double-sideband/single-sideband aperture is used to record in-focus, cryo-EM images of monolayer crystals of streptavidin. Images showing no systematic phase error due to charging are achievable under conditions of low-dose data collection. The contrast in such in-focus images is sufficient that one can readily see individual streptavidin tetramer molecules. Nevertheless, these carbon-coated devices perform well for only a limited length of time, and the cause of failure is not yet understood.

Electron microscopy of biotinylated protein complexes bound to streptavidin monolayer crystals

Chemical biotinylation of protein complexes followed by binding to two-dimensional (monolayer) crystals of streptavidin is shown to be an effective way to prepare cryo-EM specimens from samples at low protein concentration. Three different multiprotein complexes are used to demonstrate the generality of this method. In addition, native thermosomes, purified from Sulfolobus solfataricus P2, are used to demonstrate that a uniform distribution of Euler angles is produced, even though this particle is known to adopt a preferred orientation when other methods of cryo-EM specimen preparation are used.

Cryo EM Study of Protein Complexes Bound to Streptavidin Crystal Layers

Conventional cryo-EM specimens use either continuous or holey, glow-discharge treated carbon films to prepare samples. As a result, particles are either immobilized by binding to an unnatural (i.e. carbon-film) surface, or they collide multiple times (due to rapid diffusion) with the freshly blotted air-water interface. In either case there is a risk that considerable structural heterogeneity may occur, or even that particles may undergo severe denaturation.Binding biotinylated particles to streptavidin monolayer crystals is a more structure-friendly way to immobilize particles. We have developed a protocol for preparing biotinylated protein complexes and tethering them to the surface of streptavidin monolayer-crystals. Three different multi-protein complexes have been used to demonstrate the generality of this method. The biotinylated protein complexes bind to the streptavidin crystal layer with high affinity, whereas the unbiotinylated control samples do not do so. In addition, the same technique can be used to produce a uniform distribution of Euler angles. The thermosomes, known to adopt a preferred orientation with other methods of cryo-EM specimen preparation, have been used as a test sample. Indeed, the results confirm that a uniform distribution of Euler angles is obtained for the biotinylated thermosome. We thus conclude that biotinylation, together with binding to 2-D crystals of streptavidin, is a better method to prepare protein complexes for cryo-EM study.

Manipulating Energy Landscapes To Tune Ordering in Biotemplated Nanoparticle Arrays

Two-dimensional non-close-packed crystals of the protein streptavidin, grown on phospholipid membranes, can serve as nanoscale templates capable of directing the formation of ordered nanoparticle arrays through site-specific electrostatic adsorption. Here we examine the effects of both interparticle and nanoparticle/lipid membrane electrostatic interactions on the degree of structural order exhibited by the templated nanoparticle array. Interparticle electrostatic repulsion is shown to have only marginal influence on nanoparticle ordering. In contrast, the degree of order exhibited by the templated array can be tuned by controlling the charge on the lipid membrane. Analysis of the local and global structure of arrays generated with negatively charged gold nanoparticles (∼6 nm) indicate improved long-range order when the lipid membrane supporting the protein crystal is derived from cationic lipid molecules as opposed to zwitterionic phospholipids. Furthermore, as nanoparticle size is reduced (∼3 nm), the presence of a charged lipid membrane is found to be essential, as smaller particles do not adhere to streptavidin crystals grown on zwitterionic membranes. These findings demonstrate that the composition of the lipid support can influence the efficacy of directed-assembly processes which utilize protein templates and are important results toward enhancing control over bottom-up nanofabrication applications.

Biotemplating of Metallic Nanoparticle Arrays Through Site-Specific Electrostatic Adsorption on Streptavidin Crystals

The protein streptavidin exhibits unique properties advantageous for "bottom-up" nanofabrication applications. It self-assembles into various 2-D crystalline lattices onto which nanoparticles can be attached through both electrostatic and ligand-receptor mechanisms. We examine the electrostatic adsorption of gold nanoparticles onto non-close-packed streptavidin crystals and show that site-specific attachment preferentially occurs in between protein molecules. The resulting nanoparticle arrangement consequently displays a long-range structural coherence with the underlying protein lattice, although with a reduced degree of order relative to that of the biological template. Monte Carlo simulations reveal that this remittent ordering is due to (1) the random offset between the nanoparticles and their respective adsorption sites and (2) nonspecific binding to the surface of the protein molecules. Overall, our results indicate that streptavidin crystals are capable of templating ordered nanoparticle arrays.

Effects of surface ligand density on lipid-monolayer-mediated 2D assembly of proteins

The two-dimensional (2D) assembly of the protein streptavidin on a biotin-bearing lipid monolayer was studied as a function of the surface density of biotin, a protein-binding ligand, by means of in situ X-ray scattering and optical Brewster angle microscopy measurements at the liquid–vapor interface. Although this model system has been studied extensively, the relationship between the surface biotin density and the adsorption, 2D phase behavior, and binding state of streptavidin has yet to be determined quantitatively. The observed equilibrium phase behavior provides direct structural evidence that the 2D crystallization of the lipid-bound streptavidin occurs as a density-driven first-order phase transition. The minimum biotin density required for the 2D crystallization of streptavidin is found to be remarkably close to the density of the ligand-binding sites in the protein crystal. Moreover, both above and below this transition, the observed biotin-density dependence of protein adsorption is well described by the binding of biotin-bearing lipids at both of the two available sites per streptavidin molecule. These results imply that even in the low-density noncrystalline phase, the bound proteins share a common, fixed orientation relative to the surface normal, and that the 2D crystallization occurs when the lateral protein density reaches 50–70% of the 2D crystal density. This study demonstrates that in addition to a well-defined molecular orientation, high lateral packing density is essential to the 2D crystallization of proteins.

Cryo-EM Structure of Functional BK Channels in Lipid Bilayers

A natural way to preserve the native conformation of membrane proteins for structural study is to reconstitute them into liposomes (lipid vesicles). Here we applied this method to single-particle cryo-EM imaging of the hSlo large-conductance voltage- and Ca2+-activated potassium channel (BK channel). In an assay using the JC-1 voltage-sensitive dye, proteoliposomes develop a membrane potential that is equal to that produced by valinomycin, and to that of empty liposomes when the BK channels are blocked by barium (A). For cryo-EM imaging the reconstituted BK proteoliposomes were tethered to a 2D streptavidin crystal (B). From this image the periodic crystal information was removed (C) and a liposome model of electron scattering was used to subtract the lipid bilayers (D). For 3D reconstruction, single-particle images (boxes) were oriented with constraints based on the spherical vesicle geometry. A structure based on these particle images will be presented. Scale bar in the figures is 50 nm.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Modulating Colloidal Adsorption on a Two-Dimensional Protein Crystal

The geometric and physicochemical properties of the protein streptavidin make it a useful building block in the construction and manipulation of nanoscale structures and devices. However, one requirement in exploiting streptavidin for "bottom-up" assembly is the capability to modulate protein-nanoparticle interactions. This work examines the effects of pH and the biotin-streptavidin interaction on the adsorption of colloidal gold onto a two-dimensional streptavidin crystal. Particle deposition was carried out below (pH 6), at (pH 7), and above (pH 8) the protein's isoelectric point with both biotinylated and nonbiotinylated nanoparticles. Particle surface coverage depends on deposition time and pH, and increases by 1.4-10 times when biotin is incorporated onto the particle surface. This coverage is highest for both particle types at pH 6 and decreases monotonically with increasing pH. Calculations of interparticle potentials based on Derjaguin-Landau-Verwey-Overbeek (DLVO) theory demonstrate that this trend in surface coverage is most likely due to alterations in particle-surface electrostatic interactions and not a result of changes in interparticle electrostatic repulsion. Furthermore, post-adsorption alterations in pH demonstrate that electrostatically adsorbed particles can be selectively desorbed from the surface. Evaluation of the nonspecifically adsorbed fraction of biotinylated particles indicates that the receptor-ligand adsorption mechanism gives a higher rate of attachment to the substrate than nonspecific, electrostatic adsorption. This results in faster adsorption kinetics and higher coverages for biotinylated particles relative to the nonbiotinylated case.

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.

Two-Dimensional Protein Crystals on a Solid Substrate: Effect of Surface Ligand Concentration

Proteins imbedded in solid-supported lipid bilayers can serve as model systems for investigations of cellular membranes and protein behavior on surfaces. We have investigated the self-assembly of streptavidin on mica-supported bilayer membranes. Using fluorescence microscopy and atomic force microscopy, our studies reveal that the concentration of surface ligand influences the molecular packing of the resulting protein arrays, which in turn affects overall crystal morphology. Two-dimensional streptavidin crystals are obtained when the biotinylated lipid density on the substrate reaches 1.5% mole fraction, yielding high-aspect morphologies that comprise primarily of crystals with P1 symmetry. At 3% and above, crystals with C222 symmetry are formed and result in H-shaped and confluent structures. In intermediate densities between 2 and 3%, a coexistence of P1 and C222 crystal forms is observed. The relationship between macroscopic morphology and molecular configuration is similar to previously reported data obtained at the air/water interface. This suggests that, under our experimental conditions, protein interactions with the supporting substrate are less significant for defining self-assembly behavior than interactions between protein molecules. Ligand-inhibition and fluorescence recovery after photobleaching were used to elucidate the concentration-dependent mechanism for the divergent crystal forms. We have measured the diffusion coefficient of molecules in P1-forming conditions to be approximately twice that of molecules in C222-forming concentrations, which is consistent with proteins bound to the surface through one and two ligands, respectively. The differential flexibility associated with the binding state is therefore likely to alter the subtle protein interactions involved in crystallization.

Immobilization of biotinylated DNA on 2-D streptavidin crystals

The structural study of transient nucleoprotein complexes by electron microscopy is hampered by the coexistence of multiple interaction states leading to an heterogeneous image population. To tackle this problem, we have investigated the controlled immobilization of double stranded DNA molecules and of nucleoprotein complexes onto a support suitable for cryo-electron microscopy observation. The DNA was end-labeled with a biotin moiety in order to decorate, or to be incorporated into, two-dimensional streptavidin crystals formed in contact of a biotinylated lipid layer. The binding specificity and efficiency were examined by radioactively labeled oligonucleotides and by direct visualization of unstained and hydrated nucleic acid molecules in cryo-electron microscopy. By using RNA polymerase we further show that, once immobilized, femtomolar amounts of DNA template are suitable to interact with the enzyme. The image analysis of the RNA polymerase–DNA complexes showed that a three-dimensional model can be retrieved from such samples.