Ralf P. Richter

Hearing what you cannot see and visualizing what you hear: interpreting quartz crystal microbalance data from solvated interfaces.

Over the last 2 decades, the quartz crystal microbalance (QCM or QCM-D) has emerged as a versatile tool for investigating soft and solvated interfaces between solid surfaces and bulk liquids because it can provide a wealth of information about key structural and functional parameters of these interfaces. In this Feature, we offer QCM users a set of guidelines for interpretation and quantitative analysis of QCM data based on a synthesis of well-established concepts rooted in rheological research of the last century and of new results obtained in the last several years.

Analysis of CD44-hyaluronan interactions in an artificial membrane system: insights into the distinct binding properties of high and low molecular weight hyaluronan

CD44 is a major cell surface receptor for the large polydisperse glycosaminoglycan hyaluronan (HA). Binding of the long and flexible HA chains is thought to be stabilized by the multivalent nature of the sugar molecule. In addition, high and low molecular weight forms of HA provoke distinct proinflammatory and anti-inflammatory effects upon binding to CD44 and can deliver either proliferative or antiproliferative signals in appropriate cell types. Despite the importance of such interactions, however, neither the stoichiometry of multivalent HA binding at the cell surface nor the molecular basis for functional distinction between different HA size categories is understood. Here we report on the design of a supported lipid bilayer system that permits quantitative analysis of multivalent binding through presentation of CD44 in a stable, natively oriented manner and at controlled density. Using this system in combination with biophysical techniques, we show that the amount of HA binding to bilayers that are densely coated with CD44 increases as a function of HA size, with half-maximal saturation at ∼30 kDa. Moreover, reversible binding was confined to the smaller HA species (molecular weight of ≤10 kDa), whereas the interaction was essentially irreversible with larger polymers. The amount of bound HA decreased with decreasing receptor surface density, but the stability of binding was not affected. From a physico-chemical perspective, the binding properties of HA share many similarities with the typical behavior of a flexible polymer as it adsorbs onto a homogeneously attractive surface. These findings provide new insight into the multivalent nature of CD44-HA interactions and suggest a molecular basis for the distinct biological properties of different size fractions of hyaluronan.

The Inflammation-associated Protein TSG-6 Cross-links Hyaluronan via Hyaluronan-induced TSG-6 Oligomers

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).

Solvation Effects in the Quartz Crystal Microbalance with Dissipation Monitoring Response to Biomolecular Adsorption. A Phenomenological Approach

Quartz crystal microbalance with dissipation monitoring (QCM-D) has become a popular tool to investigate biomolecular adsorption phenomena at surfaces. In contrast to optical mass-sensitive techniques, which commonly detect the adsorbed nonhydrated mass, the mechanically coupled mass measured by QCM-D includes a significant amount of water. A mechanistic and quantitative picture of how the surrounding liquid couples to the deposited solutes has so far been elusive for apparently simple phenomena like the random adsorption of nanometer-sized particles on a planar surface. Using a setup that enables simultaneous measurements by reflectometry and QCM-D on the same support, we have quantified the variations in coupled water, as sensed by the QCM frequency response, as a function of coverage for the formation of monolayers of globular proteins, virus particles, and small unilamellar vesicles. We found a close-to-linear relationship between the surface coverage and the relative contribution of water to the frequency response for these adsorption scenarios. The experimental hydration curves could be reproduced quantitatively using a theoretical model that assigns a pyramid-shaped hydration coat to each adsorbed particle and that accounts for the random distribution of adsorbents on the surface. This simple model fits the experimental data well and provides insight into the parameters that affect hydration.

Model-Independent Analysis of QCM Data on Colloidal Particle Adsorption

Quartz crystal microbalance (QCM) is widely used for studying soft interfaces in liquid environment. Many of these interfaces are heterogeneous in nature, in the sense that they are composed of discrete, isolated entities adsorbed at a surface. When characterizing such interfaces, one is interested in determining parameters such as surface coverage and size of the surface-adsorbed entities. The current strategy is to obtain this information by fitting QCM data--shifts in resonance frequency, DeltaF, and bandwidth, DeltaGamma--with the model derived for smooth, homogeneous films using the film acoustic thickness and shear elastic moduli as fitting parameters. Investigating adsorption of liposomes and icosahedral virus particles on inorganic surfaces of titania and gold, we demonstrate that the predictions of this model are at variance with the experimental observations. In particular, while the model predicts that the ratio between the bandwidth and frequency shifts, DeltaGamma/DeltaF (the Df ratio), should increase with both surface coverage and particle size, we observe that this ratio increases with increasing particle size but decreases with increasing surface coverage, demonstrating that QCM response in heterogeneous films, such as those composed of adsorbed colloidal particles, does not conform with the predictions of the homogeneous film model. Employing finite element method (FEM) calculations, we show that hydrodynamic effects are the cause of this discrepancy. Finally, we find that the size of the adsorbed colloidal particles can be recovered from a model-independent analysis of the plot of the DeltaGamma/DeltaF ratio versus the frequency shift on many overtones.

Dissipation in Films of Adsorbed Nanospheres Studied by Quartz Crystal Microbalance (QCM)

The quartz crystal microbalance (QCM) has become a popular method to study the formation of surface-confined films that consist of discrete biomolecular objects--such as proteins, phospholipid vesicles, virus particles--in liquids. The quantitative interpretation of QCM data--frequency and bandwidth (or, equivalently, dissipation) shifts--obtained with such films is limited by the lack of understanding of the energy dissipation mechanisms that operate in these films as they are sheared at megahertz frequencies during the QCM experiment. Here, we investigate dissipation mechanisms in such films experimentally and by finite-element method (FEM) calculations. Experimentally, we study the adsorption of globular proteins and virus particles to surfaces with various attachment geometries: direct adsorption to the surface, attachment via multiple anchors, or attachment via a single anchor. We find that the extent of dissipation caused by the film and the evolution of dissipation as a function of surface coverage is not dependent on the internal properties of these particles but rather on the geometry of their attachment to the surface. FEM calculations reproduce the experimentally observed behavior of the dissipation. In particular, a transient maximum in dissipation that is observed experimentally is reproduced by the FEM calculations, provided that the contact zone between the sphere and the surface is narrow and sufficiently soft. Both a small-angle rotation of the sphere in the flow field of the background fluid (rocking) and a small-amplitude slippage (sliding) contribute to the dissipation. At high coverage, lateral hydrodynamic interactions between neighboring spheres counteract these modes of dissipation, which results in a maximum in dissipation at intermediate adsorption times. These results highlight that, in many scenarios of biomolecular adsorption, the dissipation is not primarily determined by the adsorbate itself, but rather by the link by which it is bound to the substrate.

QCM-D and Reflectometry Instrument: Applications to Supported Lipid Structures and Their Biomolecular Interactions

A novel setup was recently developed, combining quartz crystal microbalance with dissipation monitoring (QCM-D) and optical reflectometry for measurements on one and the same surface of, for example, biomolecular adlayers and interactions ( Rev. Sci. Instr. 2008 , 79 075107 ). This combination was chosen on the basis of prior experience of using QCM-D and optical techniques in separate instruments, which showed both the advantage of employing multiple techniques and the disadvantage of not working with the same surface and (flow) cell. The new instrument provides, for example, information about associated water and structural changes of the adlayers that would often pass unnoticed or be hard to interpret or quantify, using either technique alone. The triple response instrument (QCM-D frequency and dissipation and reflectometry) is here applied to four model systems: (A) formation of supported lipid bilayers (SLBs), (B) lipid exchange between a SLB and transiently adsorbed vesicles, (C) binding of a hydrated peptide on a functionalized SLB, and (D) streptavidin coupling to a biotinylated SLB, followed by attachment of biotinylated vesicles. The results demonstrate three major advantages of the combination instrument: (i) much faster data collection because the experiments are done on one surface for all signals, (ii) a common time axis and the same relative importance of surface kinetics and mass transport because the same liquid sample and the same transport conditions apply, and (iii) new features are discovered about the studied system that would be difficult to unravel in separate instruments.

Ultrathin nucleoporin phenylalanine-glycine repeat films and their interaction with nuclear transport receptors

Nuclear pore complexes (NPCs) are highly selective gates that mediate the exchange of all proteins and nucleic acids between the cytoplasm and the nucleus. Their selectivity relies on a supramolecular assembly of natively unfolded nucleoporin domains containing phenylalanine–glycine (FG)‐rich repeats (FG repeat domains), in a way that is at present poorly understood. We have developed ultrathin FG domain films that reproduce the mode of attachment and the density of FG repeats in NPCs, and that exhibit a thickness that corresponds to the nanoscopic dimensions of the native permeability barrier. By using a combination of biophysical characterization techniques, we quantified the binding of nuclear transport receptors (NTRs) to such FG domain films and analysed how this binding affects the swelling behaviour and mechanical properties of the films. The results extend our understanding of the interaction of FG domain assemblies with NTRs and contribute important information to refine the model of transport across the permeability barrier.

Vesicles surfing on a lipid bilayer: Self-induced haptotactic motion

Haptotaxis is a mechanism proposed at the end of the 1960s to explain cell motility. It describes cell movement induced by an adhesion gradient. In this work, we present evidence for self-induced haptotaxis using negatively charged giant vesicles interacting with positively charged supported lipid bilayers, which has not been previously described. Depending on the charge of the vesicle, we observed different behaviors. At low charge, no adhesion occurs. At high charge, the vesicle adheres but does not move. In a restricted range of intermediate charge densities, we found that the vesicle moves spontaneously with velocities of the order of a few micrometers per second over distances of >100 μm. We show that a local lipid transfer between the giant vesicle and the supported lipid bilayer takes place during the adhesion, breaking the symmetry and inducing a lateral charge gradient. This charge gradient polarizes the giant vesicle and induces its motion. To explain our observations, we propose a scaling model that relates the adhesion energy to the velocity of vesicle motion and to the characteristic lipid transfer time. Our measurements indicate that the effective adhesion energy is strongly reduced by counterions, which are dynamically trapped between the vesicle and the supported bilayer.

Binding of a model regulator of complement activation (RCA) to a biomaterial surface: Surface-bound factor H inhibits complement activation

The complement system is an important inflammatory mediator during procedures such as cardiopulmonary bypass and hemodialysis when blood is exposed to large areas of biomaterial surface. This contact between blood and the biomaterials of implants and extracorporeal circuits leads to an inflammatory response mediated by the complement system. The aim of this study was to assess the ability of a complement regulator (factor H) immobilised on a biomaterial surface to inhibit complement cascade mediated inflammatory responses. The cross-linker N-succinimidyl 3-(2-pyridyldithio) propionate was used to immobilise factor H on a model biomaterial surface without affecting the biological activity of the inhibitor. Binding of factor H was then characterised using quartz crystal microbalance-dissipation (QCM-D) and enzyme immunoassays for products of complement activation: bound C3 fragments and soluble C3a, sC5b-9, and C1s-C1INA. Immobilised factor H reduced the amount C3 fragments deposited on the biomaterial surface after incubation with serum, plasma. or whole blood. In addition, lower levels of soluble C3a and sC5b-9 were generated after incubation with whole blood. In summary, we have demonstrated that complement activation on a highly activating model surface can be inhibited by immobilised factor H and have defined prerequisites for the preparation of future biomaterial surfaces with immobilised regulators of complement activation.