Philip M. Williams

Single-Molecule Studies of Protein Folding

Although protein-folding studies began several decades ago, it is only recently that the tools to analyze protein folding at the single-molecule level have been developed. Advances in single-molecule fluorescence and force spectroscopy techniques allow investigation of the folding and dynamics of single protein molecules, both at equilibrium and as they fold and unfold. The experiments are far from simple, however, both in execution and in interpretation of the results. In this review, we discuss some of the highlights of the work so far and concentrate on cases where comparisons with the classical experiments can be made. We conclude that, although there have been relatively few startling insights from single-molecule studies, the rapid progress that has been made suggests that these experiments have significant potential to advance our understanding of protein folding. In particular, new techniques offer the possibility to explore regions of the energy landscape that are inaccessible to classical ensemble measurements and, perhaps, to observe rare events undetectable by other means.

Hidden complexity in the mechanical properties of titin

Individual molecules of the giant protein titin span the A-bands and I-bands that make up striated muscle. The I-band region of titin is responsible for passive elasticity in such muscle, and contains tandem arrays of immunoglobulin domains. One such domain (I27) has been investigated extensively, using dynamic force spectroscopy and simulation. However, the relevance of these studies to the behaviour of the protein under physiological conditions was not established. Force studies reveal a lengthening of I27 without complete unfolding, forming a stable intermediate that has been suggested to be an important component of titin elasticity. To develop a more complete picture of the forced unfolding pathway, we use mutant titins—certain mutations allow the role of the partly unfolded intermediate to be investigated in more depth. Here we show that, under physiological forces, the partly unfolded intermediate does not contribute to mechanical strength. We also propose a unified forced unfolding model of all I27 analogues studied, and conclude that I27 can withstand higher forces in muscle than was predicted previously.

Spatially controlled cell engineering on biodegradable polymer surfaces

Controlling receptor‐mediated interactions between cells and template surfaces is a central principle in many tissue engineering procedures (1–3). Biomaterial surfaces engineered to present cell adhesion ligands undergo integrin‐mediated molecular interactions with cells (1, 4, 5), stimulating cell spreading, and differentiation (6–8). This provides a mechanism for mimicking natural cell‐to‐matrix interactions. Further sophistication in the control of cell interactions can be achieved by fabricating surfaces on which the spatial distribution of ligands is restricted to micron‐scale pattern features (9–14). Patterning technology promises to facilitate spatially controlled tissue engineering with applications in the regeneration of highly organized tissues. These new applications require the formation of ligand patterns on biocompatible and biodegradable templates, which control tissue regeneration processes, before removal by metabolism. We have developed a method of generating micron‐scale patterns of any biotinylated ligand on the surface of a biodegradable block copolymer, polylactide‐poly(ethylene glycol). The technique achieves control of biomolecule deposition with nanometer precision. Spatial control over cell development has been observed when using these templates to culture bovine aortic endothelial cells and PC12 nerve cells. Furthermore, neurite extension on the biodegradable polymer surface is directed by pattern features composed of peptides containing the IKVAV sequence (15, 16), suggesting that directional control over nerve regeneration on biodegradable biomaterials can be achieved.—Patel, N., Padera, R., Sanders, G. H. W., Cannizzaro, S. M., Davies, M. C., Langer, R., Roberts, C. J., Tendler, S. J. B., Williams, P. M., and Shakesheff, K. M. Spatially controlled cell engineering on biodegradable polymer surfaces. FASEB J. 12, 1447–1454 (1998)

Analytical descriptions of dynamic force spectroscopy: behaviour of multiple connections

Exploration of the stressed lifetime of a single bond can reveal details of hidden transition states along the unbonding coordinate [Faraday Discuss. 111 (1998) 1]. Such experiments with single molecules are, however, not easy. To measure the force between two molecules requires manipulation of the contact so that two and only two molecules interact. This is achieved by reducing the probability of bond formation on contact through the control of surface chemistry, molecular density, contact force and time. When the contact area and surface chemistry cannot be controlled multiple interactions may dominate. The fundamental question arises whether quantitative information pertinent to the single interaction can be extracted from measurements of multiple simultaneous detachments. Various statistical methods have been adopted in an attempt to elucidate the single-molecule event from the rupture of multiple attachments [Biophys. J. 70 (5) (1996) 2437; Biochemistry 36 (24) (1997) 7457; Langmuir 12 (5) (1996) 1291]. Here, I aim to qualify and validate, if possible, such approaches. Whilst the analysis shows that the dynamics of loading multiple attachments precludes an accurate inference of the single unbinding event, the complexity in behaviour could be exploited to construct materials with novel dynamic mechanical properties.

Interpretation of tapping mode atomic force microscopy data using amplitude-phase-distance measurements

Abstract Vibrating mode force measurements, or amplitude–phase–distance measurements, have been used to experimentally investigate contrast mechanisms in tapping mode atomic force microscopy. Gelatin adsorbed on polystyrene and mica surfaces have been taken as examples to show that the amplitude–phase–distance curves and amplitude–energy loss–distance curves enable the interpretation of artifacts in height images and contrast in phase images. The principles are applicable in general to tapping mode imaging, and are discussed in the context of previously proposed theoretical models, i.e., those based on solution of equations of motion or on energy conservation.

Characterization of Particle-Interactions by Atomic Force Microscopy: Effect of Contact Area

AbstractPurpose. The purpose of this work was to compare adhesion forces, contact area, and work of adhesion of salbutamol sulphate particles produced using micronization and a supercritical fluid technique (solution-enhanced dispersion by supercritical fluids - SEDS™) using atomic force microscopy (AFM). Methods. Adhesion forces of individual particles of micronized and SEDS™ salbutamol against a highly orientated pyrolytic graphite surface were acquired in a liquid environment consistent with that of a pressurized metered dose inhaler. The forces were then related to contact area and work of adhesion. Results. The raw adhesion force data for the micronized and SEDS™ material were 14.1 nN (SD 2.5 nN) and 4.2 nN (SD 0.8 nN), respectively. After correction for contact area, the forces per unit area were 13 mN/μm2 (SD 2.3 mN/μm2) and 3 mN/μm2 (SD 0.6 mN/μm2). The average work of adhesion was calculated using the Johnson-Kendall-Roberts theory and was found to be 19 mJm−2 (SD 3.4 mJm−2) for the micronized particle and 4 mJm−2 (SD 0.8 mJm−2) for the SEDS™ particle. Conclusions. It is possible to produce a three-dimensional representation of the contact area involved in the interaction and make quantitative comparisons between different particles. There was a lower force per unit area and work of adhesion observed for the SEDS™ material, possibly because of its lower surface free energy.

An Atomic Force Microscopy Study of the Effect of Nanoscale Contact Geometry and Surface Chemistry on the Adhesion of Pharmaceutical Particles

AbstractPurpose. To understand differences in particle adhesion observed with increasing humidity between samples of salbutamol sulfate prepared by two different methods. Methods. Atomic force microscopy (AFM) force measurements were performed as a function of humidity (<10% to 65% RH) using two systems. The first system used clean AFM tips against compressed disks of micronized and solution enhanced dispersion by supercritical fluid (SEDS) salbutamol. The second system involved particles of both salbutamol samples mounted onto the apexes of AFM cantilevers, and force measurements being performed against a highly orientated pyrolytic graphite (HOPG) substrate. Following these measurements, the contact asperities of the tips were characterized. Results. The first system showed a maximum in the observed adhesion at 22% relative humidity (RH) for the SEDS salbutamol compared to 44% RH for the micronized salbutamol. The second system showed a mix of peaks and continual increases in adhesion with humidity. The predicted Johnson-Kendall-Roberts forces were calculated and divided by the actual forces in order to produce a ratio. Conclusions. By relating the nature of the asperities to the force measurements, we propose a model in which adhesion scenarios range from single asperity nanometer-scale contact in which peaks in the adhesion were observed, to multiasperity contact where a continuous increase in adhesion was seen with humidity.

Optimizing phase imaging via dynamic force curves

Abstract Tapping mode (TM, also called intermittent contact mode) atomic force microscopy (AFM) has been routinely used in many laboratories. However, consistent or deliberate control of measuring conditions and interpretation of results are often difficult. In this article, we demonstrate how measurement parameters (drive frequency, cantilever stiffness and oscillation amplitude) affect the tapping tips state. This has been done by systematic dynamic force measurements performed on mica and polystyrene surfaces together with computer simulations. Our study shows the following results. (1) Weaker cantilevers, smaller amplitude and higher drive frequency (around the resonance) lead to an extension of the attractive region (greater phase lag) in amplitude–phase–distance curves and thus can help to achieve stable high-setpoint TM imaging with minimal tip–sample pressure. (2) Bistability of tapping tips often exists and may cause height artefacts if the setpoint falls in the bistable region. (3) Tapping tips with high vibrating energy (stiff cantilevers and large amplitude) driven at resonance are only slightly perturbed by tip–sample interactions and usually remain monostable during the sweep of the scanner position. This can help to achieve good phase contrast without significant artefacts when the setpoint falls in a continuous negative–positive phase shift transition region. (4) Low energy cantilevers (compliant cantilevers and small amplitude) usually result in large phase shift and can be used to acquire large phase contrast images. However, height artefacts will occur when the setpoint falls in the bistable region usually existing for such cantilevers. (5) Computer simulations are useful in understanding the bistability in dynamic force curves and determining either material properties or the optimal imaging parameters.

Printing patterns of biospecifically-adsorbed protein.

The advancement of elastomeric patterning techniques in recent years has significantly enhanced our ability to spatially control biomaterial surface chemistry at the micrometre level. The application of this technology to the patterning of biomolecules onto solid surfaces has created many potential applications including the development of advanced biosensors, combinatorial library screening and the formation of tissue engineering templates. In this paper, we describe the direct patterning of protein by microcontact printing. An important consideration for the fabrication of protein micropatterns intended for these applications is the nature of the protein immobilization to a substrate. To date, the patterning of proteins by direct microcontact printing (μCP) has relied on the non-covalent adsorption to a substrate. Ideally, the proteins need to be firmly anchored onto a surface without adversely effecting their activity. Here, the high affinity avidin-biotin receptor-ligand interaction has been exploited to form arrays of avidin molecules onto a polymeric substrate expressing biotin moieties. This has created a generic technique by which any biotinylated species can be subsequently immobilized into defined patterns. Utilizing atomic force microscopy (AFM), the patterned surfaces have been characterized to molecular resolution. The micropatterned sample supported cell adhesion when biotin-(G)11-GRGDS was bound to the avidin bearing arrays.

Dynamic Force Spectroscopy

Weak-noncovalent interactions govern structural cohesion and mediate most of life’s functions from the outer membrane surface to the interior nucleus of a cell. On laboratory time scales, the energy landscape of a weak bond is fully explored by Brownian-thermal excitations, and energy barriers along its dissociation pathway(s) become encoded in a rate of unbonding that can range from ∼l/μs to 1/year. When pulled apart with a ramps of force, the dissociation kinetics become transformed into a dynamic spectrum of unbonding force as a function of the steepness of the force ramps (loading rates). Expressed on a logarithmic scale in loading rate, the spectrum of breakage forces begins first with a crossover from near equilibrium to far from equilibrium unbonding and then rises through ascending regimes of strength. These regimes expose the prominent energy barriers traversed along the dissociation pathway. Labelled as dynamic force spectroscopy [7,10], this approach is being used to probe the inner world of biomolecular interactions [7, 8, 13, 14, 23, 24, 26, 30] and reveals energy barriers that are difficult or impossible to access by solution assays of near-equilibrium kinetics. These hidden barriers are crucial for specialized dynamic functions of molecules.