Steric Trapping Research Articles

Regulating the Polymerization of DNA Structures via Allosteric Control of Monomers.

Regulation of self-assembly is crucial in constructing structural biomaterials, such as tunable DNA nanostructures. Traditional tuning of self-assembled DNA nanostructures was mainly conducted by introducing external stimuli after the assembly process. Here, we explored the allosteric assembly of DNA structures via introducing external stimuli during the assembly process to produce structurally heterogeneous polymerization products. We demonstrated that ethidium bromide (EB), a DNA intercalator, could increase the left-handed out-of-plane chirality of curved DNA structures. Then, EB and double strands were introduced as competing stimuli to transform monomers into allosteric conformations, leading to three different polymerization products. The steric trap between different polymerization products promoted the polymerized structures to keep their geometric properties, like chirality, under varying intensity of external stimuli. Our strategy harnesses allosteric effects for assembly of DNA-based materials and is expected to expand the design space for advanced control in synthetic materials.

Conformational diversity and contraction of the denatured state ensemble of a helical membrane protein

Defining the denatured state ensemble (DSE) of both soluble and membrane proteins is essential to understanding protein folding, thermodynamics, chaperone action, degradation, translocation, and cell signaling. While many studies have focused on water-soluble proteins, the DSE of membrane proteins is less well characterized. We reconstituted the DSE of a helical-bundle membrane protein GlpG in native-like E. coli lipid bilayers using our steric trapping method, which couples spontaneous denaturation of doubly biotinylated GlpG to the binding of two bulky streptavidin molecules.

Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E.coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Lipid Bilayer Strengthens the Side Chain Interaction Network of a Membrane Protein

The lipid bilayer in a cell membrane serves as a natural solvent for membrane proteins. The physical principle of how the lipid bilayer mediates the folding, stability, and cooperativity between residues to facilitate the functions of membrane proteins in cells is not well understood. Here, we employed steric trapping as a tool to study membrane protein folding in a native lipid environment. This method couples spontaneous denaturation of a doubly biotinylated protein to the competitive binding of bulky monovalent streptavidin.

The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network.

Intramembrane rhomboid proteases are of particular interest because of their function to hydrolyze a peptide bond of a substrate buried in the membrane. Crystal structures of the bacterial rhomboid protease GlpG have revealed a catalytic dyad (Ser201-His254) and oxyanion hole (His150/Asn154/the backbone amide of Ser201) surrounded by the protein matrix and contacting a narrow water channel. Although multiple crystal structures have been solved, the catalytic mechanism of GlpG is not completely understood. Because it is a serine protease, hydrogen bonding interactions between the active site residues are thought to play a critical role in the catalytic cycle. Here, we dissect the interaction energies among the active site residues His254, Ser201, and Asn154 of Escherichia coli GlpG, which form a hydrogen bonding network. We combine double mutant cycle analysis with stability measurements using steric trapping. In mild detergent, the active site residues are weakly coupled with interaction energies (ΔΔG Inter) of ‒1.4 kcal/mol between His254 and Ser201 and ‒0.2 kcal/mol between Ser201 and Asn154. Further, by analyzing the propagation of single mutations of the active site residues, we find that these residues are important not only for function but also for the folding cooperativity of GlpG. The weak interaction between Ser and His in the catalytic dyad may partly explain the unusually slow proteolysis by GlpG compared with other canonical serine proteases. Our result suggests that the weak hydrogen bonds in the active site are sufficient to carry out the proteolytic function of rhomboid proteases.

Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.

Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements in membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doubly-biotinylated protein to binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to an intramembrane protease GlpG of Escherichia coli, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.

General Steric Trapping Strategy Reveals an Intricate Cooperativity Network in the Intramembrane Protease GlpG under Native Conditions

Membrane proteins are assembled through balanced interactions among protein, lipids and water. Studying their folding while maintaining the native lipid environment has been a necessary but challenging. Here we present a set of methods for analyzing key elements in membrane protein folding, including thermodynamic stability, compactness of the unfolded state and unfolding cooperativity under native conditions. The methods are based on steric trapping which couples unfolding of a doubly-biotinylated protein to binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying those methods to an intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions for maintaining the stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.

Single-Molecule Measurement of Membrane Protein Stability

Our understanding of membrane proteins has been severely hindered by a dearth of techniques to investigate membrane protein folding and stability in lipid bilayers. Our lab has developed a technique to drive membrane protein unfolding under native conditions called the steric trap method, which uses a protein that preferentially binds the unfolded state such that binding drives unfolding. Previously, this method has been reliant on the use of a functionally assayable protein, such as the chromophore-containing protein bacteriorhodopsin or the membrane enzyme diacylglycerol kinase. We propose generalizing the steric trap method using single-molecule fluorescence technology. By moving the system to infinite dilution in which there is only a single protein per vesicle we can eliminate aggregation problems that plague membrane protein studies. The sensitivity of single-molecule detection permits us to use much less protein and lipid material, and the minimal light scattering allows us to observe proteins in vesicles instead of using solubilizing agents required for bulk spectroscopic assays. By circumventing the requirement for a protein-specific functional assay, we hope to expand the reach of membrane protein folding studies.

Design and characterization of a membrane protein unfolding platform in lipid bilayers.

Accurate measurement of membrane protein stability—and particularly how it may vary as a result of disease-phenotypic mutations—ideally requires a denaturant that can unfold a membrane-embedded structure while leaving the solubilizing environment unaffected. The steric trap method fulfills this requirement by using monovalent streptavidin (mSA) molecules to unfold membrane proteins engineered with two spatially close biotin tags. Here we adapted this method to an 87-residue helix-loop-helix (hairpin) construct derived from helices 3 and 4 in the transmembrane domain of the human cystic fibrosis transmembrane conductance regulator (CFTR), wherein helix-helix tertiary interactions are anticipated to confer a portion of construct stability. The wild type CFTR TM3/4 hairpin construct was modified with two accessible biotin tags for mSA-induced unfolding, along with two helix-terminal pyrene labels to monitor loss of inter-helical contacts by pyrene excimer fluorescence. A series of eight constructs with biotin tags at varying distances from the helix-terminal pyrene labels were expressed, purified and labeled appropriately; all constructs exhibited largely helical circular dichroism spectra. We found that addition of mSA to an optimized construct in lipid vesicles led to a complete and reversible loss in pyrene excimer fluorescence and mSA binding, and hence hairpin unfolding—results further supported by SDS-PAGE visualization of mSA bound and unbound species. While some dimeric/oligomeric populations persist that may affect quantitation of the unfolding step, our characterization of the design yields a promising prototype of a future platform for the systematic study of membrane protein folding in a lipid bilayer environment.

Developing a Universal Steric Trapping Strategy for Studying Folding and Stability of Helical Membrane Proteins

“Steric trapping” is a method that links binding of monovalent streptavidin (mSA) to unfolding of a biotinylated protein (MP). It allowed the measurements of high affinity protein–protein interactions and thermodynamic stability of polytopic helical MPs in a native environment, which had been difficult to achieve using more conventional methods. In the current steric trapping framework, a conformation-sensitive chromophore or an enzymatic activity in a target MP is required for monitoring mSA-induced unfolding. This target-specific approach is a limiting factor that hinders its application to various MP systems, i.e. MPs in a misfolded conformation, MPs with an assembly role, or MPs without convenient unfolding readout. To further advance the steric trapping strategy for more general application, we have developed novel tripartite probes possessing a thiol-reactive group, a biotin group and a fluorescent or paramagnetic group for sensitization of unfolding or binding of mSA. We applied the new strategy to investigating the stability and unfolding mechanism of an intramembrane protease GlpG. By combining FRET between fluorescently labeled GlpG and quencher-labeled mSA as a measure of mSA binding and the proteolytic activity of GlpG as a measure of unfolding, we proved the thermodynamic coupling between binding and unfolding, and determined the thermodynamic stability and unfolding rate of GlpG in a native micellar environment. While the stabilities were similar independent of the location of biotin pairs, the unfolding was 20 times faster when the biotin pair was placed near the proteolytic active site. This result suggests a subdomain-organization of the helical bundle architecture of GlpG. Steric trapping may serve as a useful tool for elucidating the local versus global flexibility of helical MPs.

Intracellular Transport by an Anchored Homogeneously Contracting F-Actin Meshwork

Actin-based contractility orchestrates changes in cell shape underlying cellular functions ranging from division to migration and wound healing. Actin also functions in intracellular transport, with the prevailing view that filamentous actin (F-actin) cables serve as tracks for motor-driven transport of cargo. We recently discovered an alternate mode of intracellular transport in starfish oocytes involving a contractile F-actin meshwork that mediates chromosome congression. The mechanisms by which this meshwork contracts and translates its contractile activity into directional transport of chromosomes remained open questions. Here, we use live-cell imaging with quantitative analysis of chromosome trajectories and meshwork velocities to show that the 3D F-actin meshwork contracts homogeneously and isotropically throughout the nuclear space. Centrifugation experiments reveal that this homogeneous contraction is translated into asymmetric, directional transport by mechanical anchoring of the meshwork to the cell cortex. Finally, by injecting inert particles of different sizes, we show that this directional transport activity is size-selective and transduced to chromosomal cargo at least in part by steric trapping or "sieving." Taken together, these results reveal mechanistic design principles of a novel and potentially versatile mode of intracellular transport based on sieving by an anchored homogeneously contracting F-actin meshwork.

Method to measure strong protein–protein interactions in lipid bilayers using a steric trap

Measuring high affinity protein-protein interactions in membranes is extremely challenging because there are limitations to how far the interacting components can be diluted in bilayers. Here we show that a steric trap can be employed for stable membrane interactions. We couple dissociation to a competitive binding event so that dissociation can be driven by increasing the affinity or concentration of the competitor. The steric trap design used here links monovalent streptavidin binding to dissociation of biotinylated partners. Application of the steric trap method to the well-characterized glycophorin A transmembrane helix (GpATM) reveals a dimer that is dramatically stabilized by 4-5 kcal/mol in palmitoyloleoylphosphatidylcholine bilayers compared to detergent. We also find larger effects of mutations at the dimer interface in bilayers compared to detergent suggesting that the dimer is more organized in a membrane environment. The high affinity we measure for GpATM in bilayers indicates that a membrane vesicle many orders of magnitude larger than a bacterial cell would be required to measure the dissociation constant using traditional dilution methods. Thus, steric trapping can open new biological systems to experimental scrutiny in natural bilayer environments.

Studying Membrane Protein Thermodynamics Using a Steric Trap

An understanding of the molecular forces that specify a protein's structure is essential for many of the central quests of structural biology. While there has been a larger effort to understand soluble protein folding, we know very little about membrane protein folding energetics. We are developing a novel method to study the forces that stabilize membrane proteins in lipid bilayers, which we termed the ‘Steric Trap method.’ The steric trap method couples protein unfolding to a measurable binding event by exploiting steric repulsion and the high affinity of the streptavidin/biotin interaction. To do this, we introduce two biotin tags on a target protein that are close in space and employ monovalent streptavidin (mSA) as our steric trap. A single mSA can bind without steric hindrance to the folded protein, but a second mSA can only bind when the protein unfolds due to steric overlap. Thus, the binding affinity of the second streptavidin provides a measure of unfolding free energy because binding is coupled to unfolding. We have developed this method on the water-soluble protein, dihydrofolate reductase (DHFR). When two biotin-labeling sites were rationally designed to be close to one another in space and near the enzyme active site of DHFR, enzymatic activity was reversibly abolished upon incubation with a molar excess of monovalent streptavidin. Incubation with a stabilizing ligand shifted streptavidin binding curves, confirming that the steric trapping can quantitatively detect changes in protein stability. Results on our application of the steric trap method to membrane proteins will be presented. The steric trap method could be a powerful tool for measuring protein association affinities, studying unfolding energetics and investigating membrane protein unfolded states in the context of membrane environments.

Measuring the Thermodynamic Stability of Strong Protein-Protein Interactions in Lipid Bilayers Using a Steric Trap

Protein-protein interactions within cell membranes play crucial roles in the assembly of membrane proteins and cell signaling. Although thermodynamic analysis of binding affinity is essential for understanding the stability, specificity, and function of membrane proteins, these measurements can be difficult to make for high affinity interactions in lipid bilayers. To address this problem, we have developed a steric trap method, which couples the dissociation of a membrane protein complex to another measurable binding event. The method postulates that a concomitant binding of two bulky monovalent streptavidins (mSA) to a doubly biotinylated protein complex occur only when the protein is dissociated due to the steric hindrance. This leads to an attenuated binding affinity of the second mSA, which is directly correlated to the stability of a target interaction. We tested the method using a glycophorinA transmembrane domain fusion to staphylococcal nuclease (SNGpA), which forms a stable dimer in various lipid environments. Equilibrium binding of mSA to the enzymatically biotinylated SNGpA exhibited two distinctive phases, which corresponds to the tight first mSA binding and the weaker second binding in decyl maltoside (DM) micelles and palmitoyloleoyl phophatidylcholine (POPC) bilayers. The stability of GpA dimer extracted from the second binding event at different micellar concentrations yielded the dissociation constants (Kd) of 10−8∼10−7 M, which agree well with the previous results. The stability of GpA dimer is enhanced in POPC bilayers by ∼4 orders of magnitude at comparable mole fractions. The difference free energies between wild-type and destabilized mutants in both systems correlate with the equilibrium sedimentation data measured in C8E5 micelles. Our results suggest that the steric trap method provides a powerful tool to study the strong protein-protein interactions in lipid bilayers.

Protein Unfolding with a Steric Trap

The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state. This has the undesirable consequence that the molecular forces responsible for configuring the polypeptide chain are also changed. It would therefore be useful to develop methods that can drive unfolding without the need for destabilizing solvent conditions. Here we introduce a new method to accomplish this goal, which we call steric trapping. In the steric trap method, the target protein is labeled with two biotin tags placed close in space so that both biotin tags can only be bound by streptavidin when the protein unfolds. Thus, binding of the second streptavidin is energetically coupled to unfolding of the target protein. Testing the method on a model protein, dihydrofolate reductase (DHFR), we find that streptavidin binding can drive unfolding and that the apparent binding affinity reports on changes in DHFR stability. Finally, by employing the slow off-rate of wild-type streptavidin, we find that DHFR can be locked in the unfolded state. The steric trap method provides a simple method for studying aspects of protein folding and stability in native solvent conditions, could be used to specifically unfold selected domains, and could be applicable to membrane proteins.

Removal of Pattern-breaking Sequences in Microtubule Binding Repeats Produces Instantaneous Tau Aggregation and Toxicity

Aggregated and highly phosphorylated tau protein is a pathological hallmark of Alzheimer's disease (AD) and other tauopathies. We identified motifs of alternating polar and apolar amino acids within the microtubule-binding repeats of tau which were interrupted by small breaking stretches. Minimal mutation of these breaking sequences yielded a unique instantly aggregating tau mutant containing longer stretches of polar/apolar amino acids without losing its microtubule-binding capacity. These modifications produced rapid aggregation and cytotoxicity with accompanying occurrence of pathologic tau phosphoepitopes (AT8, AT180, AT270, AT100, Ser(422), and PHF-1) and conformational epitopes (MC-1 and Alz50) in cells. Similar to pathological tau in the pretangle state, toxicity appeared to occur early without the requirement for extensive fibril formation. Thus, our mutant protein provides a novel platform for the investigation of the molecular mechanisms for toxicity and cellular behavior of pathologically aggregated tau proteins and the identification of its interaction partners.

Electrophoresis of Composite Molecular Objects. 2. Competition between Sieving and Frictional Effects in Polymer Solutions

We previously showed that labeling one end of single-stranded DNA molecules with a neutral label like streptavidin increases the interband separation of these hybrid DNA molecules when they are electrophoresed in denaturing cross-linked polyacrylamide gels. This separation process is called trapping electrophoresis because the dynamical properties of the analytes are strongly affected by random steric trapping in the gel. End-labeled ssDNA molecules can also be electrophoretically separated in free solution. This process is called end-labeled free solution electrophoresis, and the separation is then due to the extra hydrodynamic friction provided by the streptavidin label. In this article, we present a study of the capillary electrophoresis of DNA molecules end-labeled with streptavidin in the presence of non-cross-linked polymer solutions ranging from semidilute to ultradilute conditions. In the semidilute limit, sieving interactions dominate, and small DNA molecules move faster than larger ones. In the ultradilute limit, however, we recover free-solution-like separations where small molecules are more retarded than larger ones. We also observe a fascinating transition between these two regimes, for intermediate polymer concentrations and DNA sizes, where the competition between trapping, sieving, and frictional effects leads to nonmonotonic mobility−size relationships. Our theoretical analysis suggests a universal relationship between the mobility of labeled and unlabeled DNA molecules in all electrophoretic conditions.

Application of the concept of an electrophoretic ratchet.

Fractionation via a gel electrophoretic ratchet has previously succeeded for comparatively large (radius R > or = 95 nm) spheres (Serwer, P, Griess, G.A., Anal. Chim. Acta 1998, 372, 299-306). The electrical oscillations are the following electrical field pulses: high field --> low field --> high field, etc. The field is inverted after each pulse; the time-integral of the field can be zero. Response to the ratchet is caused by steric trapping in the high field-direction, but not in the low field-direction. Trapping and, therefore, response to the ratchet decrease as R decreases. The smaller spheres do not respond to the ratchet. In the present study, spheres with R values smaller than 95 nm are made, for the first time, to respond to a similar gel electrophoretic ratchet. To achieve this objective, the heterogeneity of pore size is increased for the gel used. The heterogeneity of pore size is increased by (i) forming the gel with degraded hydroxyethyl agarose, and (ii) gelling at comparatively high temperature. If a particle still does not respond to the ratchet (because the particle is too small), this particle has a net migration in the high field-direction, when the above-described pulsed field is biased in the high field-direction. If a particle does respond to the improved ratchet, the particle has a net migration in the low field-direction. Here, the R of ratchet-responding spheres is reduced to 30-50 nm. These ratchet-responding spheres include both intact bacteriophage particles (R = 30 nm) and latex spheres. The smaller ratchet-responding spheres have an electrophoretic mobility that decreases in magnitude as the electrical field increases in magnitude. A ratchet-based procedure is developed here to achieve continuous preparative gel electrophoresis.

Soft-Landing of Polyatomic Ions at Fluorinated Self-Assembled Monolayer Surfaces

A method of preparing modified surfaces, referred to as soft-landing, is described in which intact polyatomic ions are deposited from the gas phase into a monolayer fluorocarbon surface at room temperature. The ions are trapped in the fluorocarbon matrix for many hours. They are released, intact, upon sputtering at low or high energy or by thermal desorption, and their molecular compositions are confirmed by isotopic labeling and high-resolution mass measurements. The method is demonstrated for various silyl and pyridinium cations. Capture at the surface is favored when the ions bear bulky substituents that facilitate steric trapping in the matrix.

Trapping gel electrophoresis of end-labeled DNA: an analytical model for mobility and diffusion.

As shown by Ulanovsky, Drouin and Gilbert (Nature 1990, 343, 190-192), the gel electrophoretic migration of DNA is severely reduced by steric trapping when streptavidin is attached to one end of the polyelectrolyte. We present a model that allows us to calculate both the mobility and the diffusion coefficient, hence the resolution factor of the resulting separation. We compare our results to those of Défontaines and Viovy (Electrophoresis 1993, 14, 8-17) and we show that the averages over the molecular conformations must be done carefully. We also show that trapping increases diffusion substantially and that this makes constant-field trapping electrophoresis incapable of increasing the number of bases read per sequencing run. Finally, we conclude that severe trapping may lead to highly anomalous transport behavior where one cannot define a velocity or a diffusion constant.