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Biomedical subjects

D Alastair Smith

Publications and source records attributed to D Alastair Smith.

At least 19 recordsLinked to original sources
PubMed2006

Entropy and barrier-controlled fluctuations determine conformational viscoelasticity of single biomolecules.

Biological macromolecules have complex and nontrivial energy landscapes, endowing them with a unique conformational adaptability and diversity in function. Hence, understanding the processes of elasticity and dissipation at the nanoscale is important to molecular biology and emerging fields such as nanotechnology. Here we analyze single molecule fluctuations in an atomic force microscope, using a generic model of biopolymer viscoelasticity that includes local "internal" conformational dissipation. Comparing two biopolymers, dextran and cellulose (polysaccharides with and without local bistable transitions), demonstrates that signatures of simple conformational change are minima in both the elastic and internal friction constants around a characteristic force. A novel analysis of dynamics on a bistable energy landscape provides a simple explanation: an elasticity driven by the entropy, and friction by a barrier-controlled hopping time of populations between states, which is surprisingly distinct to the well-known relaxation time. This nonequilibrium microscopic analysis thus provides a means of quantifying new dynamical features of the energy landscape of the glucopyranose ring, revealing an unexpected underlying roughness and information on the shape of the barrier of the chair-boat transition in dextran. The results presented herein provide a basis toward probing the viscoelasticity of macromolecular conformational transitions on more complex energy landscapes, such as during protein folding.

Biopolymers↗
PubMed2006

Viscoelastic properties of single poly(ethylene glycol) molecules.

The viscoelastic properties of single poly(ethylene glycol) (PEG) molecules were measured by analysis of thermally and magnetically driven oscillations of an atomic force microscope (AFM) cantilever/molecule system. The molecular and monomer stiffness and friction of the PEG polymer were derived using a simple harmonic oscillator (SHO) model. Excellent agreement between the values of these two parameters obtained by the two approaches indicates the validity of the SHO model under the experimental regimes and the excellent reproducibility of the techniques. A sharp minimum in the monomeric friction is seen at around 180 pN applied force which we propose is due to a force induced change in the shape of the energy landscape describing the conformational transition of PEG from a helical to a planar state, which in turn affects the timescale of the transition and therefore modifies the measured internal friction. A knowledge of the viscoelastic response of PEG monomers is particularly important since PEG is widely used as a linker molecule for tethering groups of interest to the AFM tip in force spectroscopy experiments, and we show here that care must be exercised because of the force-dependent viscoelastic properties of these linkers.

Biophysics↗
PubMed2006

Urea-induced unfolding of the immunity protein Im9 monitored by spFRET.

We have studied the urea-induced unfolding of the E colicin immunity protein Im9 using diffusion single-pair fluorescence resonance energy transfer. Detailed examination of the proximity ratio of the native and denatured molecules over a wide range of urea concentrations suggests that the conformational properties of both species are denaturant-dependent. Whereas native molecules become gradually more expanded as urea concentration increases, denatured molecules show a dramatic dependence of the relationship between proximity ratio and denaturant concentration, consistent with substantial compaction of the denatured ensemble at low denaturant concentrations. Analysis of the widths of the proximity ratio distributions for each state suggests that whereas the native state ensemble is relatively narrow and homogeneous, the denatured state may possess heterogeneity in mildly denaturing conditions.

Bacterial Proteins↗
PubMed2006

Affinity of molecular interactions in the bacteriophage phi29 DNA packaging motor.

DNA packaging in the bacteriophage phi29 involves a molecular motor with protein and RNA components, including interactions between the viral connector protein and molecules of pRNA, both of which form multimeric complexes. Data are presented to demonstrate the higher order assembly of pRNA together with the affinity of pRNA:pRNA and pRNA:connector interactions, which are used to propose a model for motor function. In solution, pRNA can form dimeric and trimeric multimers in a magnesium-dependent manner, with dissociation constants for multimerization in the micromolar range. pRNA:connector binding is also facilitated by the presence of magnesium ions, with a nanomolar apparent dissociation constant for the interaction. From studies with a mutant pRNA, it appears that multimerization of pRNA is not essential for connector binding and it is likely that connector protein is involved in the stabilization of higher order RNA multimers. It is proposed that magnesium ions may promote conformational change that facilitate pRNA:connector interactions, essential for motor function.

Bacillus Phages↗
PubMed2006

Viscoelastic study of the mechanical unfolding of a protein by AFM.

We have applied a dynamic force modulation technique to the mechanical unfolding of a homopolymer of immunoglobulin (Ig) domains from titin, (C47S C63S I27)5, [(I27)5] to determine the viscoelastic response of single protein molecules as a function of extension. Both the stiffness and the friction of the homopolymer system show a sudden decrease when a protein domain unfolds. The decrease in measured friction suggests that the system is dominated by the internal friction of the (I27)5 molecule and not solvent friction. In the stiffness-extension spectrum we detected an abrupt feature before each unfolding event, the amplitude of which decreased with each consecutive unfolding event. We propose that these features are a clear indication of the formation of the known unfolding intermediate of I27, which has been observed previously in constant velocity unfolding experiments. This simple force modulation AFM technique promises to be a very useful addition to constant velocity experiments providing detailed viscoelastic characterization of single molecules under extension.

Animals↗
PubMed2006

Evidence for direct amelogenin-target cell interactions using dynamic force spectroscopy.

Increasing evidence suggests that amelogenin, long held to be a structural protein of developing enamel matrix, may also have cell signaling functions. However, a mechanism for amelogenin cell signaling has yet to be described. The aim of the present study was to use dynamic chemical force spectroscopy to measure amelogenin interactions with possible target cells. Full-length amelogenin (rM179) was covalently attached to silicon nitride AFM tips. Synthetic RGD peptides and unmodified AFM tips were used as controls. Amelogenin-RGD cell binding force measurements were carried out using human periodontal ligament fibroblasts (HPDF) from primary explants and a commercially available osteoblast-like human sarcoma cell line as the targets. Results indicated a linear logarithmic dependence between loading rate and unbinding force for amelogenin-RGD target cells across the range of loading rates used. For RGD controls, binding events measured at 5.5 nN s-1 force loading rate resulted in a mean force of 60 pN. Values for amelogenin-fibroblast and amelogenin-osteoblast-like cell unbinding forces, measured at similar loading rates, were 50 and 55 pN, respectively. These data suggest that amelogenin interacts with potential target cells with forces characteristic of specific ligand-receptor binding, suggesting a direct effect for amelogenin at target cell membranes.

Amelogenin↗
PubMed2005

Competing pathways determine fibril morphology in the self-assembly of beta2-microglobulin into amyloid.

Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of beta2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.

Amyloid↗
PubMed2005

Viscoelastic measurements of single molecules on a millisecond time scale by magnetically driven oscillation of an atomic force microscope cantilever.

The dynamical nature of biomolecular systems means that knowledge of their viscoelastic behavior is important in fully understanding function. The linear viscoelastic response can be derived from an analysis of Brownian motion. However, this is a slow measurement and technically demanding for many molecular systems of interest. To address this issue, we have developed a simple method for measuring the full linear viscoelastic response of single molecules based on magnetically driven oscillations of an atomic force microscope cantilever. The cantilever oscillation frequency is periodically swept through the system resonance in less than 200 ms allowing the power spectrum to be obtained rapidly and analyzed with a suitable model. The technique has been evaluated using dextran, a polysaccharide commonly used as a test system for single molecule mechanical manipulation experiments. The monomer stiffness and friction constants were compared with those derived from other methods. Excellent agreement is obtained indicating that the new method accurately and, most importantly, rapidly provides the viscoelastic response of a single molecule between the tip and substrate. The method will be a useful tool for studying systems that change their structure and dynamic response on a time scale of 100-200 ms, such as protein folding and unfolding under applied force.

Biophysics↗
PubMed2005

Mechanically unfolding the small, topologically simple protein L.

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.

Bacterial Proteins↗
PubMed2005

Ultraviolet resonance Raman studies reveal the environment of tryptophan and tyrosine residues in the native and partially folded states of the E colicin-binding immunity protein Im7.

Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.

Colicins↗
PubMed2004

Viscoelastic properties of single polysaccharide molecules determined by analysis of thermally driven oscillations of an atomic force microscope cantilever.

We report on single molecule measurements of the viscoelastic properties of the polysaccharide dextran using a new approach which involves acquiring the power spectral density of the thermal noise of an atomic force microscope cantilever while holding the single molecule of interest under force-clamp conditions. The attractiveness of this approach when compared with techniques which use forced oscillations under constant loading rate conditions is that it is a near-equilibrium measure of mechanical response which provides a more relevant probe of thermally driven biomolecular dynamics. Using a simple harmonic oscillator model of the cantilever-molecule system and by subtracting the response of the free cantilever taking into account hydrodynamic effects, the effective damping zetamol and elastic constant kmol of a single molecule are obtained. The molecular elasticity measured by this new technique shows a dependence on applied force that reflects the chair-boat conformational transition of the pyranose rings of the dextran molecule which is in good agreement with values obtained directly from the gradient of a conventional constant loading rate force-extension curve. The molecular damping is also seen to follow a nontrivial dependence on loading which we suggest indicates that it is internal friction and not work done on the solvent that is the dominant dissipative process.

Elasticity↗
PubMed2004

Microsecond folding dynamics of the F13W G29A mutant of the B domain of staphylococcal protein A by laser-induced temperature jump.

The small size (58 residues) and simple structure of the B domain of staphylococcal protein A (BdpA) have led to this domain being a paradigm for theoretical studies of folding. Experimental studies of the folding of BdpA have been limited by the rapidity of its folding kinetics. We report the folding kinetics of a fluorescent mutant of BdpA (G29A F13W), named F13W*, using nanosecond laser-induced temperature jump experiments. Automation of the apparatus has permitted large data sets to be acquired that provide excellent signal-to-noise ratio over a wide range of experimental conditions. By measuring the temperature and denaturant dependence of equilibrium and kinetic data for F13W*, we show that thermodynamic modeling of multidimensional equilibrium and kinetic surfaces is a robust method that allows reliable extrapolation of rate constants to regions of the folding landscape not directly accessible experimentally. The results reveal that F13W* is the fastest-folding protein of its size studied to date, with a maximum folding rate constant at 0 M guanidinium chloride and 45 degrees C of 249,000 s(-1). Assuming the single-exponential kinetics represent barrier-limited folding, these data limit the value for the preexponential factor for folding of this protein to at least approximately 2 x 10(6) s(-1).

Amino Acid Substitution↗
PubMed2003

Pulling geometry defines the mechanical resistance of a beta-sheet protein.

Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.

Anisotropy↗
PubMed2003

Hierarchical assembly of beta2-microglobulin amyloid in vitro revealed by atomic force microscopy.

The kinetics of spontaneous assembly of amyloid fibrils of wild-type beta(2)-microglobulin (beta(2)M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In parallel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and negative stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that disappear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states.

Amyloid↗
PubMed2003

Unfolding dynamics of proteins under applied force.

Understanding the mechanisms of protein folding is a major challenge that is being addressed effectively by collaboration between researchers in the physical and life sciences. Recently, it has become possible to mechanically unfold proteins by pulling on their two termini using local force probes such as the atomic force microscope. Here, we present data from experiments in which synthetic protein polymers designed to mimic naturally occurring polyproteins have been mechanically unfolded. For many years protein folding dynamics have been studied using chemical denaturation, and we therefore firstly discuss our mechanical unfolding data in the context of such experiments and show that the two unfolding mechanisms are not the same, at least for the proteins studied here. We also report unexpected observations that indicate a history effect in the observed unfolding forces of polymeric proteins and explain this in terms of the changing number of domains remaining to unfold and the increasing compliance of the lengthening unstructured polypeptide chain produced each time a domain unfolds.

Hydrogen Bonding↗
PubMed2002

The effect of core destabilization on the mechanical resistance of I27.

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.

Amino Acid Sequence↗
PubMed2002

Mechanically unfolding proteins: the effect of unfolding history and the supramolecular scaffold.

The mechanical resistance of a folded domain in a polyprotein of five mutant I27 domains (C47S, C63S I27)(5)is shown to depend on the unfolding history of the protein. This observation can be understood on the basis of competition between two effects, that of the changing number of domains attempting to unfold, and the progressive increase in the compliance of the polyprotein as domains unfold. We present Monte Carlo simulations that show the effect and experimental data that verify these observations. The results are confirmed using an analytical model based on transition state theory. The model and simulations also predict that the mechanical resistance of a domain depends on the stiffness of the surrounding scaffold that holds the domain in vivo, and on the length of the unfolded domain. Together, these additional factors that influence the mechanical resistance of proteins have important consequences for our understanding of natural proteins that have evolved to withstand force.

Computer Simulation↗