Enhanced Facilitated Diffusion of Membrane-Associating Proteins under Symmetric Confinement.

The facilitated surface diffusion of transiently adsorbing molecules in a planar confined microenvironment (i.e., slit-like confinement) is highly relevant to biological phenomena, such as extracellular signaling, as well as numerous biotechnology systems. Here, we studied the surface diffusion of individual proteins confined between two symmetric lipid bilayer membranes, under a continuum of confinement heights, using single-molecule tracking and convex lens-induced confinement as well as hybrid, kinetic Monte Carlo simulations of a generalized continuous time random walk process. Surface diffusion was observed to vary non-monotonically with confinement height, exhibiting a maximum at a height of ∼750 nm, where diffusion was nearly 40% greater than that for a semi-infinite system. This demonstrated that planar confinement can, in fact, increase surface diffusion, qualitatively validating previous theoretical predictions. Simulations reproduced the experimental results and suggested that confinement enhancement of surface diffusion for symmetric systems is limited to cases where the adsorbate exhibits weak surface sticking.

Single molecule characterization of anomalous transport in a thin, anisotropic film.

The diffusion of small, charged molecules incorporated in an anisotropic polyelectrolyte multilayer (PEM) was tracked in three dimensions by combining single-molecule fluorescence localization (to characterize lateral diffusion) with Förster resonance energy transfer (FRET) between diffusing molecules and the supporting surface (to measure diffusion in the surface-normal direction). Analysis of the surface-normal diffusion required model-based statistical analysis to account for the inherently noisy FRET signal. Combining these distinct single-molecule methods, which are inherently sensitive to different length-scales, permitted simultaneous characterization of severely anisotropic diffusion, which was more than three orders of magnitude slower in the surface-normal direction. We hypothesize that the anomalously slow surface-normal diffusion was related to the periodic distribution of charge in the PEM, which created electrostatic barriers. The motion was strongly subdiffusive, with anomalous temporal scaling exponents in lateral and normal directions, suggesting a connection to the transient, random fractal conformation of polymer chains in the film's matrix.

Diffusion of Short Semiflexible DNA Polymer Chains in Strong and Moderate Confinement.

In many technological applications, DNA is confined within nanoenvironments that are smaller than the size of the unconfined polymer in solution. However, the dependence of the diffusion coefficient on molecular weight and characteristic confinement dimension remains poorly understood in this regime. Here, convex lens-induced confinement (CLiC) was leveraged to examine how the diffusion of short DNA fragments varied as a function of slit height by using single-molecule fluorescence tracking microscopy. The diffusion coefficient followed approximate power law behavior versus confinement height, with exponents of 0.27 ± 0.01, 0.32 ± 0.02, and 0.42 ± 0.06 for 692, 1343, and 2686 base pair chains, respectively. The weak dependence on slit height suggests that shorter semiflexible chains may adopt increasingly rodlike conformations and therefore experience weaker excluded-volume interactions as the confinement dimension is reduced. The diffusion coefficient versus molecular weight also exhibited apparent power law behavior, with exponents that varied slightly (from -0.89 to -0.85) with slit height, consistent with hydrodynamic interactions intermediate between Rouse and Zimm model predictions.

Single-Molecule Observations Provide Mechanistic Insights into Bimolecular Knoevenagel Amino Catalysis.

While single-molecule (SM) methods have provided new insights to various catalytic processes, bimolecular reactions have been particularly challenging to study. Here, the fluorogenic Knoevenagel condensation of an aromatic aldehyde with methyl cyanoacetate promoted by surface-immobilized piperazine is quantitatively characterized using super-resolution fluorescence imaging and stochastic analysis using hidden Markov modeling (HMM). Notably, the SM results suggest that the reaction follows the iminium intermediate pathway before the formation of a fluorescent product with intramolecular charge-transfer character. Moreover, the overall process is limited by the turnover rate of the catalyst, which is involved in multiple steps along the reaction coordinate.

Polyelectrolyte Multilayers Enhance the Dry Storage and pH Stability of Physically Entrapped Enzymes.

Polyelectrolyte multilayers (PEMs) are attractive materials for immobilizing enzymes due to their unique ionic environment, which can prevent unfolding. Here, we demonstrated that the stability to dry storage and elevated pH were significantly enhanced when negatively charged nitroreductase (NfsB) was embedded in a PEM by depositing alternating layers of the enzyme and polycation (PC) onto porous silica particles. The PC strength (i.e., pKa) and the surface charge of the film were varied to probe the effects that internal and surface chemistry had on the pH stability of the entrapped NfsB. All films showed enhanced activity retention at elevated pH (>6), and inactivation at reduced pH (<6) similar to NfsB in solution, indicating that the primary stabilizing effect of immobilization was achieved through ionic interactions between NfsB and the PC and not through changes to the surface charge of the NfsB. Additionally, films that were stored dry at 4 °C for 1 month retained full activity, while those stored at room temperature lost 30% activity. Remarkably, at 50 °C, above the NfsB melting temperature, 40% activity was retained after 1 month of dry storage. Our results suggest that internal film properties are significantly more important than surface charge, which had minor effects on activity. Specifically, immobilization with the weak PC, poly(l-lysine), increased the optimal pH and the activity of immobilized NfsB (which we attribute to greater permeability), relative to immobilization with the strong PC, poly(diallyldimethylammonium chloride). However, NfsB was leached from the PLL film to a greater extent. Overall, these observations demonstrate that internal ionic cross-linking is key to the stabilizing effects of PEMs and that the pH response can be tuned by controlling the number of cross-links (e.g., by changing the strength of the PC). However, this may be at the cost of reduced loading, illustrating the necessity of simultaneously optimizing enzyme loading, internal ionic cross-linking, and substrate transport.

Changes in microbubble dynamics upon adhesion to a solid surface.

The interaction between an acoustically driven microbubble and a surface is of interest for a variety of applications, such as ultrasound imaging and therapy. Prior investigations have mainly focused on acoustic effects of a rigid boundary, where it was generally observed that the wall increases inertia and reduces the microbubble resonance frequency. Here we investigate the response of a lipid-coated microbubble adherent to a rigid wall. Firm adhesion between the microbubble and a glass surface was achieved through either specific (biotin/avidin) or nonspecific (lipid/glass) interactions. Total internal reflection fluorescence microscopy was used to verify conditions leading to either adhesion or non-adhesion of the bubble to a glass or rigid polymer surface. Individual microbubbles were driven acoustically to sub-nanometer-scale radial oscillations using a photoacoustic technique. Remarkably, adherent microbubbles were shown to have a higher resonance frequency than non-adherent microbubbles resting against the wall. Analysis of the resonance curves indicates that adhesion stiffens the bubble by an apparent increase in the shell elasticity term and decrease in the shell viscosity. Based on these results, we conclude that surface adhesion is dominant over acoustic effects for low-amplitude microbubble oscillations.

Reduced Enzyme Dynamics upon Multipoint Covalent Immobilization Leads to Stability-Activity Trade-off.

The successful incorporation of enzymes into materials through multipoint covalent immobilization (MPCI) has served as the foundation for numerous advances in diverse fields, including biocatalysis, biosensing, and chemical weapons defense. Despite this success, a mechanistic understanding of the impact of this approach on enzyme stability has remained elusive, which is critical for realizing the full potential of MPCI. Here, we showed that the stabilization of lipase upon MPCI to polymer brush surfaces resulted from the rigidification of the enzyme with an increase in the number of enzyme-brush attachments. This was evident by a 10-fold decrease in the rates of enzyme unfolding and refolding as well as a reduction of the intrinsic fluctuations of the folded and unfolded states, which was measured by single-molecule (SM) Förster Resonance Energy Transfer imaging. Moreover, our results illuminate an important trade-off between stability and activity as a function of this decrease in structural dynamics of the immobilized lipase. Notably, as the thermal stability of lipase increased, as indicated by the temperature optimum for activity of the enzyme, the specific activity of lipase decreased. This decrease in activity was attributed to a reduction in the essential motions of the folded state that are required for catalytic turnover of substrate. These results provide direct evidence of this effect, which has long been a matter of speculation. Furthermore, our findings suggest that the retention of activity and stabilization of an enzyme may be balanced by tuning the extent of enzyme attachment.

Surface-Templated Nanobubbles Protect Proteins from Surface-Mediated Denaturation.

In this Letter, we report that surface-bound nanobubbles reduce protein denaturation on methylated glass by irreversible protein shell formation. Single-molecule total internal reflection fluorescence (SM-TIRF) microscopy was combined with intramolecular Förster resonance energy transfer (FRET) to study the conformational dynamics of nitroreductase (NfsB) on nanobubble-laden methylated glass surfaces, using reflection brightfield microscopy to register nanobubble locations with NfsB adsorption. First, NfsB adsorbed irreversibly to nanobubbles with no apparent desorption after 5 h. Moreover, virtually all (96%) of the NfsB molecules that interacted with nanobubbles remained folded, whereas less than 50% of NfsB molecules remained folded in the absence of nanobubbles on unmodified silica or methylated glass surfaces. This trend was confirmed by ensemble-average fluorometer TIRF experiments. We hypothesize that nanobubbles reduce protein damage by passivating strongly denaturing topographical surface defects. Thus, nanobubble stabilization on surfaces may have important implications for antifouling surfaces and improving therapeutic protein storage.

Standalone interferometry-based calibration of convex lens-induced confinement microscopy with nanoscale accuracy.

Strongly confined environments (confined dimensions between 1-100 nm) represent unique challenges and opportunities for understanding and manipulating molecular behavior due to the significant effects of electric double layers, high surface-area to volume ratios, and other phenomena at the nanoscale. Convex Lens-induced Confinement (CLiC) can be used to analyze the dynamics of individual molecules or particles confined in a planar slit geometry with continuously varying gap thickness. We describe an interferometry-based method for precise measurement of the slit pore geometry. Specifically, this approach permitted accurate characterization of separation distances as small as 5 nm, with 1 nm precision, without a priori knowledge or assumptions about the contact geometry, as well as a greatly simplified experimental setup that required only a lens, coverslip, and inverted microscope. The interferometry-based measurement of gap height offered a distinct advantage over conventional fluorescent dye-based methods; e.g., accurate interferometric height measurements were made at low gap heights regardless of solution conditions, while the concentration of fluorescent dye was significantly impacted by solution conditions such as ionic strength or pH. The accuracy of the interferometric measurements was demonstrated by comparing the experimentally measured concentration of a charged fluorescent dye as a function of gap thickness with dye concentration profiles calculated using Debye-Hückel theory. Accurate characterization of nanoscale gap thickness will enable researchers to study a variety of practical and biologically relevant systems within the CLiC geometry.

Complex Salt Dependence of Polymer Diffusion in Polyelectrolyte Multilayers.

Polyelectrolyte multilayers (PEMs) have significant potential in many technologies, yet the dynamics of the constituent polymer chains remains poorly understood. We used total internal reflection fluorescence microscopy to observe microscopic single-molecule transport of fluorescently labeled poly-l-lysine (PLL) diffusing within the bulk of a PEM composed of PLL and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) when exposed to NaCl solutions ranging in concentration from 0 to 2 M. Statistical analysis of PLL trajectories revealed motion that was nonergodic, subdiffusive, and temporally anticorrelated under all conditions. In contrast with previous macroscopic measurements of polymer diffusion within PEMs, the microscopic diffusion was 2-3 orders of magnitude faster and varied nonmonotonically with salt concentration in a way that was similar to trends previously associated with PEM swelling and viscoelastic properties. This trend in the anomalous diffusion was attributed to complex salt-dependent changes in the viscoelastic properties of the film that balanced intermolecular binding and molecular conformation.

Correlating Structural and Functional Heterogeneity of Immobilized Enzymes.

Many nanobiotechnology applications rely on stable and efficient integration of functional biomacromolecules with synthetic nanomaterials. Unfortunately, the reasons for the ubiquitous loss of activity of immobilized enzymes remain poorly understood due to the difficulty in distinguishing between distinct molecular-level mechanisms. Here, we employ complementary single-molecule fluorescence methods that independently measure the impact of immobilization on the structure and function ( i. e., substrate binding kinetics) of nitroreductase (NfsB). Stochastic statistical modeling methods were used to unambiguously quantify the effects of immobilized NfsB structural dynamics on function, allowing us to explicitly separate effects due to conformation and accessibility. Interestingly, we found that nonspecifically tethered NfsB exhibited enhanced stability compared to site-specifically tethered NfsB; however, the folded state of site-specifically tethered NfsB had faster substrate binding rates, suggesting improved active site accessibility. This demonstrated an unexpected intrinsic trade-off associated with competing bioconjugation methods, suggesting that it may be necessary to balance conformational stability versus active site accessibility. This nuanced view of the impact of immobilization will facilitate a rational approach to the integration of enzymes with synthetic nanomaterials.

Stabilization of Immobilized Enzymes via the Chaperone-Like Activity of Mixed Lipid Bilayers.

Biomimetic lipid bilayers represent intriguing materials for enzyme immobilization, which is critical for many biotechnological applications. Here, through the creation of mixed lipid bilayers, the retention of immobilized enzyme structures and catalytic activity are dramatically enhanced. The enhancement in the retention of enzyme structures, which correlated with an increase in enzyme activity, is observed using dynamic single-molecule (SM) fluorescence methods. The results of SM analysis specifically show that lipid bilayers composed of mixtures of 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl- sn-glycero-3-phospho-(1'- rac-glycerol) (DOPG) stabilize the folded state of nitroreductase (NfsB), increasing the rate of refolding relative to unfolding of enzyme molecules on the bilayer surface. Remarkably, for optimal compositions with 15-50% DOPG, over 95% of NfsB remains folded while the activity of the enzyme is increased as much as 2 times over that in solution. Within this range of DOPG, the strength of the interaction of folded and unfolded NfsB with the bilayer surface was also significantly altered, which was evident by the change in the diffusion of folded and unfolded NfsB in the bilayer. Ultimately, these findings provide direct evidence for the chaperone-like activity of mixed DOPG/DOPC lipid bilayers, which can be controlled by tuning the fraction of DOPG in the bilayer.

Grafting Density Impacts Local Nanoscale Hydrophobicity in Poly(ethylene glycol) Brushes.

Accumulated single-molecule observations of a fluorescent solvatochromic probe molecule were found to provide detailed local information about nanoscale hydrophobicity in polymer brushes. Using this approach, we showed that local hydrophobicity in poly(ethylene glycol) (PEG) brushes was spatially heterogeneous and increased with the surface grafting density of the polymer chains. These findings may provide an explanation for prior observations of the denaturation of surface-adsorbed proteins on PEG brushes with high grafting densities, which is believed to influence protein-mediated cell-surface interactions. Moreover, by employing the broad range of existing environmentally sensitive fluorophores, this approach may potentially be used to characterize nanoscale changes in a variety of physicochemical properties within polymeric materials.

Analyzing refractive index profiles of confined fluids by interferometry part II: Multilayer and asymmetric systems.

Methods for determining the substrate properties and the optical thickness of thin films or any variation in the refractive index of a fluid or film near a surface for unknown 5-layer symmetric and 3-layer asymmetric interferometers are presented. Both systems can be fully resolved without any known layer properties and without contact or confining the films. The method was tested using realistic simulated interferometer data, and was found to consistently yield accurate values for all desired properties. The method was experimentally validated through analysis of an asymmetric three layer interferometer system of linear polyethyleneimine (LPEI) adsorbed onto mica substrates of differing thickness and identical refractive index. The results were in excellent agreement with the dry polymer film properties measured using conventional SFA contact measurements. More complicated systems were also evaluated for feasibility, and any additional parameter specifications required for analysis were determined. The utility of this method is broad, as a single experiment in a laboratory setting can independently provide non-contact film properties and the effects of confinement on the film structure, which can be correlated to a simultaneously measured interaction force profile.

Density and Phase State of a Confined Nonpolar Fluid.

Measurements of the mean refractive index of a spherelike nonpolar fluid, octamethytetracylclosiloxane (OMCTS), confined between mica sheets, demonstrate direct and conclusive experimental evidence of the absence of a first-order liquid-to-solid phase transition in the fluid when confined, which has been suggested to occur from previous experimental and simulation results. The results also show that the density remains constant throughout confinement, and that the fluid is incompressible. This, along with the observation of very large increases (many orders of magnitude) in viscosity during confinement from the literature, demonstrate that the molecular motion is limited by the confining wall and not the molecular packing. In addition, the recently developed refractive index profile correction method, which enables the structural perturbation inherent at a solid-liquid interface and that of a liquid in confinement to be determined independently, was used to show that there was no measurable excess or depleted mass of OMCTS near the mica surface in bulk films or confined films of only two molecular layers.

Analyzing refractive index profiles of confined fluids by interferometry.

This work describes an interferometry data analysis method for determining the optical thickness of thin films or any variation in the refractive index of a fluid or film near a surface. In particular, the method described is applied to the analysis of interferometry data taken with a surface force apparatus (SFA). The technique does not require contacting or confining the fluid or film. By analyzing interferometry data taken at many intersurface separation distances out to at least 300 nm, the properties of a film can be quantitatively determined. The film can consist of material deposited on the surface, like a polymer brush, or variation in a fluid's refractive index near a surface resulting from, for example, a concentration gradient, depletion in density, or surface roughness. The method is demonstrated with aqueous polyethylenimine (PEI) adsorbed onto mica substrates, which has a large concentration and therefore refractive index gradient near the mica surface. The PEI layer thickness determined by the proposed method is consistent with the thickness measured by conventional SFA methods. Additionally, a thorough investigation of the effects of random and systematic error in SFA data analysis and modeling via simulations of interferometry is described in detail.

Thickness and refractive index of DPPC and DPPE monolayers by multiple-beam interferometry.

The thickness and refractive index of 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) monolayers Langmuir--Blodgett (LB) deposited on mica were measured in dry air and bulk water using multiple-beam interferometry (MBI). Measurements of thickness using atomic force microscopy (AFM) of identical monolayers, and X-ray reflectivity (XRR) of the monolayers on quartz were taken for comparison. The measurement of the properties of solid-supported monolayers in dry air allows lipid optical properties to be determined free from solvent effects. The thickness and refractive index measured by MBI were 25.5 ± 0.6 Å and 1.485 ± 0.007 for DPPE monolayers, and 23.9 ± 0.5 Å and 1.478 ± 0.006 for DPPC monolayers in dry air. These thicknesses are consistent with the other techniques used in this work as well as other measurements in the literature. The refractive indices of solid-supported lipid monolayers have not been previously measured. The values are higher than previous measurements on black lipid films done by reflectometry, which is attributed to increased lipid packing density and the absence of hydrocarbon solvents. Applying water to the monolayers had no measurable effect on their properties, indicating that any change in hydration was below detection.

Strain-induced optical changes in demineralized bone.

Bone "stress-whitens," becoming visibly white during mechanical loading, immediately prior to failure. Stress-whitening is known to make materials tougher by dissipating mechanical energy. A greater understanding of stress-whitening, both an optical and mechanical phenomenon, may help explain age-related increases in fracture risk that occur without changes in bone mineralization. In this work, we directly measure the optical properties of demineralized bone as a function of deformation and immersing fluid (with different hydrogen-bonding potentials, water, and ethanol). The change in refractive index of demineralized bone was linear: with deformation and not applied force. Changes in refractive index were likely due to pushing low-refractive-index fluid out of specimens and secondarily due to changes in the refractive index of the collagenous phase. Results were consistent with stress-whitening of demineralized bone previously observed. In ethanol, the refractive index values were lower and less sensitive to deformation compared with deionized water, corroborating the sensitivity to fluid hydration. Differences in refractive index were consistent with structural changes in the collagenous phase such as densification that may also occur under mechanical loading. Understanding bone quality, particularly stress-whitening investigated here, may lead to new therapeutic targets and noninvasive methods to assess bone quality.