Synthetic integrin antibodies discovered by yeast display reveal αV subunit pairing preferences with ß subunits.

Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes. Antibodies against individual RGD-binding integrins are desirable for investigating their specific functions, and were selected here from a synthetic yeast-displayed Fab library. We discovered 11 antibodies that exhibit high specificity and affinity toward their target integrins, i.e. αVß3, αVß5, αVß6, αVß8, and α5ß1. Of these, six are function-blocking antibodies and contain a ligand-mimetic R(G/L/T)D motif in their CDR3 sequences. We report antibody-binding specificity, kinetics, and binding affinity for purified integrin ectodomains, as well as intact integrins on the cell surface. We further used these antibodies to reveal binding preferences of the αV subunit for its 5 ß-subunit partners: ß6 = ß8 > ß3 > ß1 = ß5.

Synthetic integrin antibodies discovered by yeast display reveal αV subunit pairing preferences with ß subunits.

Eight of the 24 integrin heterodimers bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, and play essential roles in cell adhesion, migration, and homeostasis. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands including fibronectin, vitronectin, fibrinogen, nephronectin and the prodomain of the transforming growth factors to fulfill specific functions in cellular processes. Subtype-specific antibodies against RGD-binding integrins are desirable for investigating their specific functions. In this study, we discovered 11 antibodies that exhibit high specificity and affinity towards integrins αVß3, αVß5, αVß6, αVß8, and α5ß1 from a synthetic yeast-displayed Fab library. Of these, 6 are function-blocking antibodies containing an R(G/L/T) D motif in their CDR3 sequences. We report antibody binding specificity, kinetics, and binding affinity for purified integrin ectodomains as well as intact integrins on the cell surface. We further employed these antibodies to reveal binding preferences of the αV subunit for its 5 ß-subunit partners: ß6=ß8>ß3>ß1=ß5.

Kinetic Study on Enzymatic Hydrolysis of Cellulose in an Open, Inhibition-Free System.

Due to the complexity of cellulases and the requirement of enzyme adsorption on cellulose prior to reactions, it is difficult to evaluate their reaction with a general mechanistic scheme. Nevertheless, it is of great interest to come up with an approximate analytic description of a valid model for the purpose of developing an intuitive understanding of these complex enzyme systems. Herein, we used the surface plasmonic resonance method to monitor the action of a cellobiohydrolase by itself, as well as its mixture with a synergetic endoglucanase, on the surface of a regenerated model cellulose film, under continuous flow conditions. We found a phenomenological approach by taking advantage of the long steady state of cellulose hydrolysis in the open, inhibition-free system. This provided a direct and reliable way to analyze the adsorption and reaction processes with a minimum number of fitting parameters. We investigated a generalized Langmuir-Michaelis-Menten model to describe a full set of kinetic results across a range of enzyme concentrations, compositions, and temperatures. The overall form of the equations describing the pseudo-steady-state kinetics of the flow-system shares some interesting similarities with the Michaelis-Menten equation. The use of familiar Michaelis-Menten parameters in the analysis provides a unifying framework to study cellulase kinetics. The strategy may provide a shortcut for approaching a quantitative while intuitive understanding of enzymatic degradation of cellulose from top to bottom. The open system approach and the kinetic analysis should be applicable to a variety of cellulases and reaction systems to accelerate the progress in the field.

Evidence for S2 flexibility by direct visualization of quantum dot-labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro.

Myosins in muscle assemble into filaments by interactions between the C-terminal light meromyosin (LMM) subdomains of the coiled-coil rod domain. The two head domains are connected to LMM by the subfragment-2 (S2) subdomain of the rod. Our mixed kinetic model predicts that the flexibility and length of S2 that can be pulled away from the filament affects the maximum distance working heads can move a filament unimpeded by actin-attached heads. It also suggests that it should be possible to observe a head remain stationary relative to the filament backbone while bound to actin (dwell), followed immediately by a measurable jump upon detachment to regain the backbone trajectory. We tested these predictions by observing filaments moving along actin at varying ATP using TIRF microscopy. We simultaneously tracked two different color quantum dots (QDs), one attached to a regulatory light chain on the lever arm and the other attached to an LMM in the filament backbone. We identified events (dwells followed by jumps) by comparing the trajectories of the QDs. The average dwell times were consistent with known kinetics of the actomyosin system, and the distribution of the waiting time between observed events was consistent with a Poisson process and the expected ATPase rate. Geometric constraints suggest a maximum of ∼26 nm of S2 can be unzipped from the filament, presumably involving disruption in the coiled-coil S2, a result consistent with observations by others of S2 protruding from the filament in muscle. We propose that sufficient force is available from the working heads in the filament to overcome the stiffness imposed by filament-S2 interactions.

Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain.

The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys-108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin-activated steady-state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C-terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC-Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.

A Simple Flow Reactor for Continuous Synthesis of Biographene for Enzymology Studies.

The unique properties of graphene make it an intriguing platform for the attachment and enhancement of biological molecules, but it has yet to achieve its full potential in terms of biological applications. Single-layer graphene is expensive, making alternatives to this material highly desired for applications that require high-quality graphene in large quantities. In this context, we report a simple, environmentally friendly, nonlabor-intensive method for the synthesis of colloidal graphene suspensions of 3-5 layers, stabilized by bovine serum albumin, in water. The method involves a flow reactor designed to continually yield high-quality graphene colloids, synthesized, purified, and optimized all in one setup. The flow reactor is able to produce colloidal graphene sheets on a multigram scale, and these colloids were characterized by Raman spectroscopy, electron microscopy, and zeta potential studies. The average size of the sheets is 0.16µm2, each consisting of 3-5 layers of graphene with little or no sp3 defects. These graphene colloids stabilized by the protein were successfully used in protein kinetic studies as well as in surface plasmon resonance protein binding studies. The ease of synthesis of these high-quality graphene colloidal suspensions in water provides an exciting opportunity for biographene to be used on an industrial scale for electronic, thermal, and enzymology applications.

Tuning Enzyme/α-Zr(IV) Phosphate Nanoplate Interactions via Chemical Modification of Glucose Oxidase.

Using glucose oxidase (GOx) and α-Zr(IV) phosphate nanoplates (α-ZrP) as a model system, a generally applicable approach to control enzyme-solid interactions via chemical modification of amino acid side chains of the enzyme is demonstrated. Net charge on GOx was systematically tuned by appending different amounts of polyamine to the protein surface to produce chemically modified GOx(n), where n is the net charge on the enzyme after the modification and ranged from -62 to +95 electrostatic units in the system. The binding of GOx(n) with α-ZrP nanosheets was studied by isothermal titration calorimetry (ITC) as well as by surface plasmon resonance (SPR) spectroscopy. Pristine GOx showed no affinity for the α-ZrP nanosheets, but GOx(n) where n ≥ -20 showed binding affinities exceeding (2.1 ± 0.6) × 106 M-1, resulting from the charge modification of the enzyme. A plot of GOx(n) charge vs Gibbs free energy of binding (ΔG) for n = +20 to n = +65 indicated an overall increase in favorable interaction between GOx(n) and α-ZrP nanosheets. However, ΔG is less dependent on the net charge for n > +45, as evidenced by the decrease in the slope as charge increased further. All modified enzyme samples and enzyme/α-ZrP complexes retained a significant amount of folding structure (examined by circular dichroism) as well as enzymatic activities. Thus, strong control over enzyme-nanosheet interactions via modulating the net charge of enzymes may find potential applications in biosensing and biocatalysis.

Supramolecular Assembly of Comb-like Macromolecules Induced by Chemical Reactions that Modulate the Macromolecular Interactions In Situ.

Supramolecular polymerization or assembly of proteins or large macromolecular units by a homogeneous nucleation mechanism can be quite slow and require specific solution conditions. In nature, protein assembly is often regulated by molecules that modulate the electrostatic interactions of the protein subunits for various association strengths. The key to this regulation is the coupling of the assembly process with a reversible or irreversible chemical reaction that occurs within the constituent subunits. However, realizing this complex process by the rational design of synthetic molecules or macromolecules remains a challenge. Herein, we use a synthetic polypeptide-grafted comb macromolecule to demonstrate how the in situ modulation of interactions between the charged macromolecules affects their resulting supramolecular structures. The kinetics of structural formation was studied and can be described by a generalized model of nucleated polymerization containing secondary pathways. Basic thermodynamic analysis indicated the delicate role of the electrostatic interactions between the charged subunits in the reaction-induced assembly process. This approach may be applicable for assembling a variety of ionic soft matters that are amenable to chemical reactions in situ.

Enzymatic Activities of Polycatalytic Complexes with Nonprocessive Cellulases Immobilized on the Surface of Magnetic Nanoparticles.

Polycatalytic enzyme complexes made by immobilization of industrial enzymes on polymer- or nanoparticle-based scaffolds are technologically attractive due to their recyclability and their improved substrate binding and catalytic activities. Herein, we report the synthesis of polycatalytic complexes by the immobilization of nonprocessive cellulases on the surface of colloidal polymers with a magnetic nanoparticle core and the study of their binding and catalytic activities. These polycatalytic cellulase complexes have increased binding affinity for the substrate. But due to their larger size, these complexes were unable to access to the internal surfaces of cellulose and have significantly lower binding capacity when compared to those of the corresponding free enzymes. Analysis of released soluble sugars indicated that the formation of complexes may promote the prospect of having consistent, multiple attacks on cellulose substrate. Once bound to the substrate, polycatalytic complexes tend to remain on the surface with very limited mobility due to their strong, multivalent binding to cellulose. Hence, the overall performance of polycatalytic complexes is limited by its substrate accessibility as well as mobility on the substrate surface.