Surface Tethering Research Articles

Feedback-controlled electro-kinetic traps for single-molecule spectroscopy

A principal limitation of single-molecule spectroscopy in solution is the diffusion-limited residence time of a given molecule within the detection volume. A common solution to this problem is to immobilize molecules of interest on a passivated glass surface for extending the observation time to obtain reliable data statistics. However, surface tethering of molecules often introduces artifacts, particularly when studying the structural dynamics of biomolecules. To circumvent this limitation, we investigated alternative ways to extend single-molecule observation times in solution without surface immobilization. Among various possibilities, the so-called anti-Brownian electro-kinetic trap (or ABEL trap) seems best suited to achieve this goal. The essential part of that trap is a feedback-controlled electro-kinetic steering of a molecule’s position in reaction to its diffusive Brownian motion which is monitored by fluorescence, thus keeping the molecule within a sub-micron sized detection volume. Fluorescence trace recordings of over thousands of milliseconds duration on individual dye molecules within an ABEL trap have been reported. In this short review, we shall briefly discuss the principle and some results of ABEL trapping of individual molecules with possible extensions to future works.

Synthesis, Antimicrobial Activity, and Membrane Permeabilizing Properties of C-Terminally Modified Nisin Conjugates Accessed by CuAAC

Functionalization of the lantibiotic nisin with fluorescent reporter molecules is highly important for the understanding of its mode of action as a potent antimicrobial peptide. In addition to this, multimerization of nisin to obtain multivalent peptide constructs and conjugation of nisin to bioactive molecules or grafting it on surfaces can be attractive methods for interference with bacterial growth. Here, we report a convenient method for the synthesis of such nisin conjugates and show that these nisin derivatives retain both their antimicrobial activity and their membrane permeabilizing properties. The synthesis is based on the Cu(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) as a bioorthogonal ligation method for large and unprotected peptides in which nisin was C-terminally modified with propargylamine and subsequently efficiently conjugated to a series of functionalized azides. Two fluorescently labeled nisin conjugates together with a dimeric nisin construct were prepared while membrane insertion as well as antimicrobial activity were unaffected by these modifications. This study shows that C-terminal modification of nisin does not deteriorate biological activity in sharp contrast to N-terminal modification and therefore C-terminally modified nisin analogues are valuable tools to study the antibacterial mode of action of nisin. Furthermore, the ability to use stoichiometric amounts of the azide containing molecule opens up possibilities for surface tethering and more complex multivalent structures.

Effect of hyaluronic acid incorporation method on the stability and biological properties of polyurethane–hyaluronic acid biomaterials

The high failure rate of small diameter vascular grafts continues to drive the development of new materials and modification strategies that address this clinical problem, with biomolecule incorporation typically achieved via surface-based modification of various biomaterials. In this work, we examined whether the method of biomolecule incorporation (i.e., bulk versus surface modification) into a polyurethane (PU) polymer impacted biomaterial performance in the context of vascular applications. Specifically, hyaluronic acid (HA) was incorporated into a poly(ether urethane) via bulk copolymerization or covalent surface tethering, and the resulting PU-HA materials characterized with respect to both physical and biological properties. Modification of PU with HA by either surface or bulk methods yielded materials that, when tested under static conditions, possessed no significant differences in their ability to resist protein adsorption, platelet adhesion, and bacterial adhesion, while supporting endothelial cell culture. However, only bulk-modified PU-HA materials were able to fully retain these characteristics following material exposure to flow, demonstrating a superior ability to retain the incorporated HA and minimize enzymatic degradation, protein adsorption, platelet adhesion, and bacterial adhesion. Thus, despite bulk methods rarely being implemented in the context of biomolecule attachment, these results demonstrate improved performance of PU-HA upon bulk, rather than surface, incorporation of HA. Although explored only in the context of PU-HA, the findings revealed by these experiments have broader implications for the design and evaluation of vascular graft modification strategies.

Conserved Regions of Gonococcal TbpB Are Critical for Surface Exposure and Transferrin Iron Utilization

The transferrin-binding proteins TbpA and TbpB enable Neisseria gonorrhoeae to obtain iron from human transferrin. The lipoprotein TbpB facilitates, but is not strictly required for, TbpA-mediated iron acquisition. The goal of the current study was to determine the contribution of two conserved regions within TbpB to the function of this protein. Using site-directed mutagenesis, the first mutation we constructed replaced the lipobox (LSAC) of TbpB with a signal I peptidase cleavage site (LAAA), while the second mutation deleted a conserved stretch of glycine residues immediately downstream of the lipobox. We then evaluated the resulting mutants for effects on TbpB expression, surface exposure, and transferrin iron utilization. Western blot analysis and palmitate labeling indicated that the lipobox, but not the glycine-rich motif, is required for lipidation of TbpB and tethering to the outer membrane. TbpB was released into the supernatant by the mutant that produces TbpB LSAC. Neither mutation disrupted the transport of TbpB across the bacterial cell envelope. When these mutant TbpB proteins were produced in a strain expressing a form of TbpA that requires TbpB for iron acquisition, growth on transferrin was either abrogated or dramatically diminished. We conclude that surface tethering of TbpB is required for optimal performance of the transferrin iron acquisition system, while the presence of the polyglycine stretch near the amino terminus of TbpB contributes significantly to transferrin iron transport function. Overall, these results provide important insights into the functional roles of two conserved motifs of TbpB, enhancing our understanding of this critical iron uptake system.

Interplay between native topology and non-native interactions in the folding of tethered proteins

We assessed the interplay of native topology and non-native interactions on surface-tethered protein folding via extensive Monte Carlo simulations of a simple lattice model. In particular, we investigated the thermodynamics and kinetics of protein-like sequences enclosing different amounts of non-native interactions to protein energetics, and which were designed to fold to distinct native topologies. Our results show that the high-contact order (CO) structure renders a folding transition that is robust to (external) steric constraints and non-native interactions. On the other hand, the folding process of the simple low-CO topology can be easily hampered by the presence of a nearby chemically inert plane. In this case, if non-native interactions are highly conspicuous during folding they can actually drive chain collapse into a very native-like trapped state, which impedes the formation of the native structure. The analysis of folding kinetics reveals that the empirical correlation between folding rate and CO may not apply to surface-tethered protein folding. Indeed, results reported here show that depending on the native environment of the tethered chain terminus the folding rate of a low-CO topology can become so drastically small that the high-CO topology actually folds faster under the same conditions. We predict that complex topologies are more likely to conserve their bulk folding mechanism upon surface tethering.

How difficult is it to fold a knotted protein? In silico insights from surface-tethered folding experiments.

We explore the effect of surface tethering on the folding process of a lattice protein that contains a trefoil knot in its native structure via Monte Carlo simulations. We show that the outcome of the tethering experiment depends critically on which terminus is used to link the protein to a chemically inert plane. In particular, if surface tethering occurs at the bead that is closer to the knotted core the folding rate becomes exceedingly slow and the protein is not able to find the native structure in all the attempted folding trajectories. Such low folding efficiency is also apparent from the analysis of the probability of knot formation, pknot, as a function of nativeness. Indeed, pknot increases abruptly from ∼0 to ∼1 only when the protein has more than 80% of its native contacts formed, showing that a highly compact conformation must undergo substantial structural re-arrangement in order to get effectively knotted. When the protein is surface tethered by the bead that is placed more far away from the knotted core pknot is higher than in the other folding setups (including folding in the bulk), especially if conformations are highly native-like. These results show that the mobility of the terminus closest to the knotted core is critical for successful folding of trefoil proteins, which, in turn, highlights the importance of a knotting mechanism that is based on a threading movement of this terminus through a knotting loop. The results reported here predict that if this movement is blocked, knotting occurs via an alternative mechanism, the so-called spindle mechanism, which is prone to misfolding. Our simulations show that in the three considered folding setups the formation of the knot is typically a late event in the folding process. We discuss the implications of our findings for co-translational folding of knotted trefoils.

Functional reconstitution and osmoregulatory properties of the ProU ABC transporter fromEscherichia coli

The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., the substrate-binding protein ProX, the nucleotide-binding protein ProV and the transmembrane protein ProW, and reconstituted the full transporter complex in liposomes. We engineered a lipid anchor to ProX for surface tethering of this protein to ProVW-containing proteoliposomes. We show that glycine betaine binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner. The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system.

Vesicular Structures Self‐Assembled from Oligonucleotide‐Polymer Hybrids: Mechanical Prevention of Bacterial Colonization Upon their Surface Tethering Through Hybridization

AbstractIn order to design soft coatings, surface tethering of vesicular structures self‐assembled from oligonucleotide‐polymer hybrids is achieved through hybridization. Watson‐Crick base‐pairing occurs between the nucleotide sequences involved in the self‐assembly and their surface‐tethered complementary sequences. Combining the quartz crystal microbalance and in situ observations using confocal laser scanning microscopy, it is evidenced that the vesicles retain their morphology even under flow stress. Surprisingly, these soft surfaces prevent bacterial colonization.

Nanochannel-Based Single Molecule Recycling

We present a method for measuring the fluorescence from a single molecule hundreds of times without surface immobilization. The approach is based on the use of electroosmosis to repeatedly drive a single target molecule in a fused silica nanochannel through a stationary laser focus. Single molecule fluorescence detected during the transit time through the laser focus is used to repeatedly reverse the electrical potential controlling the flow direction. Our method does not rely on continuous observation and therefore is less susceptible to fluorescence blinking than existing fluorescence-based trapping schemes. The variation in the turnaround times can be used to measure the diffusion coefficient on a single molecule level. We demonstrate the ability to recycle both proteins and DNA in nanochannels and show that the procedure can be combined with single-pair Förster energy transfer. Nanochannel-based single molecule recycling holds promise for studying conformational dynamics on the same single molecule in solution and without surface tethering.

Enzyme Reactions in Nanoporous, Picoliter Volume Containers

Advancements in nanoscale fabrication allow creation of small-volume reaction containers that can facilitate the screening and characterization of enzymes. A porous, ∼19 pL volume vessel has been used in this work to carry out enzyme reactions under varying substrate concentrations. Assessment of small-molecule and green fluorescent protein diffusion from the vessels indicates that pore sizes on the order of 10 nm can be obtained, allowing capture of proteins and diffusive exchange of small molecules. Glucose oxidase and horseradish peroxidase can be contained in these structures and diffusively fed with a solution containing glucose and the fluorogenic substrate amplex red through the engineered nanoscale pore structure. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Kinetic characteristics of the enzyme (K(m) and V(max)) were evaluated and compared with results from conventional scale reactions. These picoliter, nanoporous containers can facilitate quick determination of enzyme kinetics in microfluidic systems without the requirement of surface tethering and can be used for applications in drug discovery, clinical diagnostics, and high-throughput screening.

Esophageal Cancer Related Gene-4 Is a Choroid Plexus-Derived Injury Response Gene: Evidence for a Biphasic Response in Early and Late Brain Injury

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24–72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6–8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation.

Surface-Grafted, Covalently Cross-Linked Hydrogel Brushes with Tunable Interfacial and Bulk Properties

Tethering macromolecules to surfaces represents a versatile approach for functionalizing, protecting, and structuring both organic and inorganic materials. In this study, thin films of poly(acrylamide) (PAAm) brushes and covalently cross-linked hydrogel brushes were grown from iniferter-functionalized silicon substrates by UVLED-initiated photopolymerization and their properties subsequently studied by means of a variety of analytical methods. The employed photografting method allowed the controlled fabrication of very thick films (up to 1 μm) in an aqueous environment, over a period of less than 1 h of polymerization and in the absence of side reactions. PAAm covalently cross-linked hydrogel brushes were prepared by feeding trace amounts of the cross-linker bis(acrylamide) (up to 1.0 wt % of monomer solution) into the reaction vessel. Both bulk and interfacial properties of these polymer films were found to be strongly influenced by lateral cross-linking of the grafted polymer chains. In agreement with theoretical expectations, the decrease of polymer-brush conformational freedom with increasing cross-link density resulted in a substantial increase of film wettability with water. The swelling ratio of the hydrogel brushes, as measured by ellipsometry and atomic force microscopy (AFM), also confirmed the formation of grafted networks and was found to be directly related to the amount of cross-linker in the monomer feed. In addition, the Young’s moduli and tribological properties of PAAm brushes and hydrogel brushes were tuned by adjusting the cross-linker concentration. Because of the additional constraint given by the surface tethering of each chain end, intermolecular cross-linking generated very high mechanical stresses within the brush structure. Covalently cross-linked hydrogel brushes thus displayed higher Young’s moduli and coefficients of friction, when compared to the grafted polymer-brush analogues. These hydrogel brushes present an opportunity for readily tailoring physical properties, especially as they allow tuning of the physical characteristics of surfaces while maintaining the interfacial chemical composition nearly constant.

Versatile Polymer Microspheres for Injection Therapy: Aspects of Fluoroscopic Traceability and Biofunctionalization

Synthesis and characterization of a series of novel microspheres featuring (i) radiopacity (i.e., clear fluoroscopic traceability) and (ii) an outer surface exposing aldehyde groups are reported. The aldehydes allowed us to tether proteins onto the particles' surface under mild conditions, under which the protein conformation and, hence, structural motifs for biorecognition are preserved. Essential monomer building blocks were (i) 4-iodobenzoyl-2-oxo-ethylmethacrylate (4-IEMA) for radiopacity and (ii) propenal for surface tethering of proteins. The particles demonstrated good X-ray visibility and cytocompatibility. Procedures to couple proteins onto the surface were optimized using fluorescent bovine serum albumin (FITC-BSA) or collagen (FITC-collagen). Furthermore, radiopaque microparticles with unlabeled bovine collagen type I were produced. The presence of immobilized collagen was verified with narrow-scan X-ray photoelectron spectroscopy. Fibroblasts readily adhere to and grow on the collagen-modified surfaces, whereas this was much less the case for the unmodified controls. The results led us to suggest that immobilized nondenatured collagen may transform filler particles from passive space-occupying objects to particles that cross-talk with surrounding tissues.

Synthesis, Characterization, and Surface Tethering of Sulfide-Functionalized Ti16-oxo-alkoxy Cages

The parent cage [Ti16O16(OEt)32] readily undergoes ligand exchange with a range of primary alcohols to yield species of the type [Ti16O16(OEt)32−x(OR′)x], with R′ = Me, n-Pr, and n-Bu with x of up to 8. Attempted ligand exchange using the thiol [HO(CH2)4SH] in an attempt to produce functionalized cages suitable for tethering to Au surfaces failed, resulting only in the polymerization of the Ti oxo-cores. However, the use of the thioether [HO(CH2)4SCH3] resulted in successful thio-functionalization and preservation of the Ti16 cage core, likely due to methyl protection on the sulfide which precludes further intermolecular reaction with other cage molecules. ESI-MS and NMR showed that the resulting substituted cage [Ti16O16(OEt)24{O(CH2)4SCH3}8] contained eight methylthio-n-butoxy ligands in two groups of four pseudoequivalent positions. High resolution XPS and STM demonstrated that this sulfide-functionalized cage underwent covalent tethering to Au surfaces involving five sulfur linkages per cage, forming a monolayer of adsorbed species in which the molecular integrity had been preserved. In contrast, the parent oxo-alkoxy cage underwent extensive decomposition, rendering it useless as a building block for specific surface architectures.

Internal Structure of Ultrathin Diblock Copolymer Brushes

The internal structure of as-deposited, high grafting density, ultrathin (thickness < 25 nm) diblock copolymer brushes (DCBs) is resolved using neutron reflectivity (NR) and grazing incidence small-angle X-ray scattering (GISAXS). DCBs of various thicknesses containing deuterated polystyrene (dPS) blocks and poly(methyl acrylate) (PMA) blocks with dPS (dPS-b-PMA) or with PMA (PMA-b-dPS) adjacent to the substrate were synthesized by atom transfer radical polymerization (ATRP). For the thinnest films, a model of two layers with a smooth interfacial gradient provides a good description of the data. For thicker dPS-b-PMA samples of sufficiently asymmetric composition, a third layer must be included. This is consistent with the presence of lateral ordering in the center of the brush, as evidenced by GISAXS data. For the thinnest DCBs, the gradient in composition perpendicular to the surface extends through nearly the entire thickness of the brush, consistent with the conjecture that the gradient is imposed by the presence of the surface field and tethering on a material that, in the absence of tethering, would be disordered. The interface widths for brushes with a PMA block tethered to the substrate are smaller than for brushes with a dPS block tethered to the substrate. In general, the region adjacent to the substrate is found to have a substantial composition of the “top” block in contrast to expectations from theory. Experimental interface width values are consistent with expectations from self-consistent field theory for brushes with a dPS bottom block. A scaling theory for the interfacial width in a DCB identifies a crossover as (d/Rg)2 > χN from the classical Helfand−Tagami regime, where w≈b/(χ1/2), to a new stretched interface regime, where w ≈ d/(χN). The scaling theory provides insight into how interface width in the DCBs should vary with grafting density, interaction parameter, and chain molecular weight and is qualitatively consistent with the experimental data and suggests directions for further work.

Effects of Water and Temperature on Conformational Order in Model Nylon Thin Films

Nylon 6,6 thin films have been examined by Raman spectroscopy to determine how chain conformation is influenced by environmental parameters such as exposure to water and temperature variations. The motivation for this work is to elucidate how interactions between water and the model polymers mediate polymer structures in applications such as the removal of salt from water in reverse osmosis membranes. Raman spectra show that model self-assembled monolayers containing Nylon 6,6 chains are semicrystalline under ambient conditions. The native chains adopt an unusual kinked and folded conformation related to that found in γ-Nylon 6,6. The regular chain deformations allow adjacent tethered chains to maximize hydrogen-bonding between neighboring amide groups under the constraints imposed by surface tethering. With increasing temperature, the films undergo a phase transition associated with the disruption of hydrogen bonds leading to structures containing more linear regions that are closer to the “all trans” case. Similar structural changes are observed on exposing the Nylon films to water. The salt content of the water does not appear to have a significant impact on the structure or phase transition in the Nylon 6,6. These results suggest that while inclusion of water has a profound effect on the polymer structure, either salt is excluded from the polymer or there is sufficient free volume within the films to accommodate hydrated ions without inducing further structural changes.

Salt Effects on Surface-Tethered Peptides in Solution

The capability to manipulate proteins/peptide fragments at liquid-solid interfaces has led to tremendous applications in detectors and biotechnology. Therefore, understanding the detailed molecular behavior of proteins and peptides tethered on a hard material surface is an interesting and important topic. The inhomogeneity presented by surfaces as well as ions in the solution plays an important role in the thermodynamics and kinetics of the tethered proteins. In this study, we perform a series of molecular dynamics simulations of a pentapeptide RHSVV, a p53 epitope, tethered on a prepared microarray surface in various salt concentrations (0, 0.14, 0.5, and 1 M NaCl), as well as free in ionic solution (0, 0.5, and 1 M). The conformational space the tethered peptide visits largely overlaps with the free peptide in solution. However, surface tethering as well as the salt concentration changes both the thermodynamics and kinetics of the peptide. Frequent conformational changes are observed during the simulations and tend to be slowed down by both increasing the salt concentration and surface tethering. The local composition of ions at different salt concentrations is also compared between the tethered and free peptide.

The effect of surface tethering on the folding of the src-SH3 protein domain

The effect of surface tethering on the folding mechanism of the src-SH3 protein domain was investigated using a coarse-grained Gō-type protein model. The protein was tethered at various locations along the protein chain and the thermodynamics and kinetics of folding were studied using replica exchange and constant temperature Langevin dynamics. Our simulations reveal that tethering in a structured part of the transition state can dramatically alter the folding mechanism, while tethering in an unstructured part leaves the folding mechanism unaltered as compared to bulk folding. Interestingly, there is only modest correlation between the tethering effect on the folding mechanism and its effect on thermodynamic stability and folding rates. We suggest locations on the protein at which tethering could be performed in single-molecule experiments so as to leave the folding mechanism unaltered from the bulk.

A microcontact printing approach to the immobilization of oligonucleotide brushes

Solution hybridized oligonucleotides were immobilized onto surfaces via micro-contact printing. Besides micro-patterning of the substrate, sequential dehybridization and rehybridization were monitored via laser scanning microscopy, which assess the surface tethering of the oligonucleotides into a brush.

Monovalent and Divalent Promoted GAAA Tetraloop-Receptor Tertiary Interactions from Freely Diffusing Single-Molecule Studies

Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg2+ and Na+ concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na+] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34±2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg2+] levels, which agrees quantitatively with the 32±1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the KD value and Hill coefficient for [Mg2+]-dependent docking decrease significantly for [Na+]=25mM vs. 125mM, indicating Mg2+ and Na+ synergy in the RNA folding process.