D. Alastair Smith

Pulling geometry defines the mechanical resistance of a β-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-β 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.

Hierarchical Assembly of β2-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.

Force mode atomic force microscopy as a tool for protein folding studies

Abstract The advent of a new class of force microscopes designed specifically to “pull” biomolecules has allowed non-specialists to use force microscopy as a tool to study single-molecule protein unfolding. This powerful new technique has the potential to explore regions of the protein energy landscape that are not accessible in conventional bulk studies. It has the added advantage of allowing direct comparison with single-molecule simulation experiments. However, as with any new technique, there is currently no well described consensus for carrying out these experiments. Adoption of standard schemes of data selection and analysis will facilitate comparison of data from different laboratories and on different proteins. In this review, some guidelines and principles, which have been adopted by our laboratories, are suggested. The issues associated with collecting sufficient high quality data and the analysis of those data are discussed. In single-molecule studies, there is an added complication since an element of judgement has to be applied in selecting data to analyse; we propose criteria to make this process more objective. The principal sources of error are identified and standardised methods of selecting and analysing the data are proposed. The errors associated with the kinetic parameters obtained from such experiments are evaluated. The information that can be obtained from dynamic force experiments is compared, both quantitatively and qualitatively to that derived from conventional protein folding studies.

Protein folding mechanisms: new methods and emerging ideas.

During the past year, advances in our understanding of folding mechanisms have been made through detailed experimental and theoretical studies of a number of proteins. The development of new methods has allowed the earliest events in folding to be probed and the measurement of folding at the level of individual molecules is now possible, opening the door to exciting new experiments.

A theoretical study of the structure and vibrations of 2,4,6-trinitrotolune

Abstract Theoretical calculations of the structure, internal rotations and vibrations of 2,4,6-trinitrotolune, TNT, in the gas phase were performed at the B3LYP/6-31G* and B3LYP/6-311+G** levels of theory. Two genuine energy minimum structures were found. In both structures the 4-nitro group is planar to the phenyl ring, while the 2,6-nitro groups are slightly out of plane with the phenyl ring due to steric interaction with the methyl group. The two structures are related by internal rotations of the methyl and 2, or 6-nitro group. The lowest energy route for interconversion between them is a concerted motion of the methyl group and 2 or 6 nitro group in a ‘cog wheel’ type of mechanism. The geometry of the low energy structure A is closest to that observed in the crystal structures of TNT, where all three nitro groups are out of plane with the phenyl ring. FTIR and Raman spectra of solid TNT and 13C, 15N enriched TNT are presented and assigned with the help of the B3LYP/6-311+G** calculations on A. The lower level B3LYP/6-31G* calculation fails to predict the correct vibrational coupling between the nitro and phenyl groups. The B3LYP/6-311+G** calculation gives a good prediction of the nitro vibrations and the isotopic shifts observed for TNT isotopomers.

Calibration of silicon atomic force microscope cantilevers

We present a comparison of three different methods to calibrate the spring constant of two different types of silicon beam shaped atomic force microscope (AFM) cantilevers to determine each methods accuracy, ease of use and potential destructiveness. The majority of research in calibrating AFM cantilevers has been concerned with contact mode levers. The two types of levers we have studied are used in force modulation and tapping mode in air. Not only can these types of cantilevers have spring constants an order of magnitude greater than contact mode levers, but also their geometries can be quite different from the standard V-shape contact lever. In this work we experimentally determine the correction factors for two of the calibration methods when applied to the tapping mode cantilevers and also demonstrate that the force modulation levers can be calibrated easily and accurately using these same techniques.

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)5is 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.

The atomic force microscope as a tool for studying phase separation in lipid membranes (Review)

Atomic force microscopy has developed into a powerful tool in the study of phase separation in lipid bilayers. Its ability to image a semi-fluid surface under buffer at nanometre lateral resolution and Angstrom resolution vertically allows us to distinguish phase separated lipid domains, models of the elusive rafts postulated to exist as functional platforms in the cellular membrane, which may only rise 0.3 nm above the surrounding membrane. This review charts the history of this development, and includes a description of sample preparation techniques, factors affecting image contrast mechanisms, its use in the investigation of the pre-transition ripple phase, and in the localization of cell surface proteins.

Physico-chemical properties of crystal surfaces in matrix-mineral interactions during mammalian biomineralisation

Surfaces of developing enamel crystals were shown to comprise of alternating domains of positive and less positive (perhaps even negative) charge density which directly bind a number of matrix proteins via electrostatic interactions. Studies using synthetic mineral crystals demonstrated stereo-specific docking of charged residues with crystal lattice sites. Preferential binding of matrix proteins to specific crystal faces related to interfacial hydrophobicity/hydrophobicity, was shown to control crystal habit.

Development of a scanning near-field optical probe for localised Raman spectroscopy

Abstract The scanning near-field optical probe technique has made it possible to perform conventional optical spectroscopies with unprecedented spatial resolution. We present the development of an instrument for scanning near-field Raman spectroscopy and present near-field Raman linescans and spectra of a range of materials with subwavelength resolution.