Giovanni Dietler

Force-distance curves by atomic force microscopy

Abstract Atomic force microscopy (AFM) force-distance curves have become a fundamental tool in several fields of research, such as surface science, materials engineering, biochemistry and biology. Furthermore, they have great importance for the study of surface interactions from a theoretical point of view. Force-distance curves have been employed for the study of numerous materials properties and for the characterization of all the known kinds of surface forces. Since 1989, several techniques of acquisition and analysis have arisen. An increasing number of systems, presenting new kinds of forces, have been analyzed. AFM force-distance curves are routinely used in several kinds of measurement, for the determination of elasticity. Hamaker constants, surface charge densities, and degrees of hydrophobicity. The present review is designed to indicate the theoretical background of AFM force-distance curves as well as to present the great variety of measurements that can be performed with this tool. Section 1 is a general introduction to AFM force-distance curves. In Sections 2–4 the fundamentals of the theories concerning the three regions of force-distance curves are summarized. In particular, Section 2 contains a review of the techniques employed for the characterization of the elastic properties of materials. After an overview of calibration problems (Section 5), the different forces that can be measured with AFM force-distance curves are discussed. Capillary, Coulomb, Van der Waals, double-layer, solvation, hydration, hydrophobic, specific and steric forces are considered. For each force the available theoretical aspects necessary for the comprehension of the experiments are provided. The main experiments concerning the measurements of such forces are listed, pointing out the experimental problems, the artifacts that are likely to affect the measurement, and the main established results. Experiments up to June 1998 are reviewed. Finally, in Section 7, techniques to acquire force-distance curves sequentially and to draw bidimensional maps of different parameters are listed.

Understanding amyloid aggregation by statistical analysis of atomic force microscopy images

The aggregation of proteins is central to many aspects of daily life, including food processing, blood coagulation, eye cataract formation disease and prion-related neurodegenerative infections. However, the physical mechanisms responsible for amyloidosis-the irreversible fibril formation of various proteins that is linked to disorders such as Alzheimers, Creutzfeldt-Jakob and Huntingtons diseases-have not yet been fully elucidated. Here, we show that different stages of amyloid aggregation can be examined by performing a statistical polymer physics analysis of single-molecule atomic force microscopy images of heat-denatured beta-lactoglobulin fibrils. The atomic force microscopy analysis, supported by theoretical arguments, reveals that the fibrils have a multistranded helical shape with twisted ribbon-like structures. Our results also indicate a possible general model for amyloid fibril assembly and illustrate the potential of this approach for investigating fibrillar systems.

Rapid detection of bacterial resistance to antibiotics using AFM cantilevers as nanomechanical sensors

The widespread misuse of drugs has increased the number of multiresistant bacteria, and this means that tools that can rapidly detect and characterize bacterial response to antibiotics are much needed in the management of infections. Various techniques, such as the resazurin-reduction assays, the mycobacterial growth indicator tube or polymerase chain reaction-based methods, have been used to investigate bacterial metabolism and its response to drugs. However, many are relatively expensive or unable to distinguish between living and dead bacteria. Here we show that the fluctuations of highly sensitive atomic force microscope cantilevers can be used to detect low concentrations of bacteria, characterize their metabolism and quantitatively screen (within minutes) their response to antibiotics. We applied this methodology to Escherichia coli and Staphylococcus aureus, showing that live bacteria produced larger cantilever fluctuations than bacteria exposed to antibiotics. Our preliminary experiments suggest that the fluctuation is associated with bacterial metabolism.

Stiffness Tomography by Atomic Force Microscopy

The atomic force microscope is a convenient tool to probe living samples at the nanometric scale. Among its numerous capabilities, the instrument can be operated as a nano-indenter to gather information about the mechanical properties of the sample. In this operating mode, the deformation of the cantilever is displayed as a function of the indentation depth of the tip into the sample. Fitting this curve with different theoretical models permits us to estimate the Youngs modulus of the sample at the indentation spot. We describe what to our knowledge is a new technique to process these curves to distinguish structures of different stiffness buried into the bulk of the sample. The working principle of this new imaging technique has been verified by finite element models and successfully applied to living cells.

Interactions between NEEP21, GRIP1 and GluR2 regulate sorting and recycling of the glutamate receptor subunit GluR2.

Trafficking of AMPA‐type glutamate receptors (AMPAR) between endosomes and the postsynaptic plasma membrane of neurons plays a central role in the control of synaptic strength associated with learning and memory. The molecular mechanisms of its regulation remain poorly understood, however. Here we show by biochemical and atomic force microscopy analyses that NEEP21, a neuronal endosomal protein necessary for receptor recycling including AMPAR, is associated with the scaffolding protein GRIP1 and the AMPAR subunit GluR2. Moreover, the interaction between NEEP21 and GRIP1 is regulated by neuronal activity. Expression of a NEEP21 fragment containing the GRIP1‐binding site decreases surface GluR2 levels and delays recycling of internalized GluR2, which accumulates in early endosomes and lysosomes. Infusion of this fragment into pyramidal neurons of hippocampal slices induces inward rectification of AMPAR‐mediated synaptic responses, suggesting decreased GluR2 expression at synapses. These results indicate that NEEP21–GRIP1 binding is crucial for GluR2‐AMPAR sorting through endosomes and their recruitment to the plasma membrane, providing a first molecular mechanism to differentially regulate AMPAR subunit cycling in internal compartments.

Measurement of intrinsic properties of amyloid fibrils by the peak force QNM method.

We report the investigation of the mechanical properties of different types of amyloid fibrils by the peak force quantitative nanomechanical (PF-QNM) technique. We demonstrate that this technique correctly measures the Youngs modulus independent of the polymorphic state and the cross-sectional structural details of the fibrils, and we show that values for amyloid fibrils assembled from heptapeptides, α-synuclein, Aβ(1-42), insulin, β-lactoglobulin, lysozyme, ovalbumin, Tau protein and bovine serum albumin all fall in the range of 2-4 GPa.

Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy

Measuring the biophysical properties of macromolecular complexes at work is a major challenge of modern biology. The protein complex composed of vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa, and syntaxin 1 [soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex] is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. To better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces and dissociation kinetics of the SNAREs and led us to propose a sequence of interactions. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein neuronal Sec1 injected into the atomic force microscope chamber prevented the complex formation. Finally, we measured the effect of tetanus toxin protease on the SNARE complex and its activity by on-line registration during tetanus toxin injection. These experiments provide a basis for the functional study of protein microdomains and also suggest opportunities for sensitive screening of drugs that can modulate protein–protein interactions.

Scaling exponents and probability distributions of DNA end-to-end distance

The scaling of the average gyration radius of polymers as a function of their length can be experimentally determined from ensemble measurements, such as light scattering, and agrees with analytical estimates. Ensemble techniques, yet, do not give access to the full probability distributions. Single molecule techniques, instead, can deliver information on both average quantities and distribution functions. Here we exploit the high resolution of atomic force microscopy over long DNA molecules adsorbed on a surface to measure the average end-to-end distance as a function of the DNA length, and its full distribution function. We find that all the scaling exponents are close to the predicted 3D values (upsilon=0.589+/-0.006 and delta=2.58+/-0.77). These results suggest that the adsorption process is akin to a geometric projection from 3D to 2D, known to preserve the scaling properties of fractal objects of dimension df<2.

Active Solubilization and Refolding of Stable Protein Aggregates By Cooperative Unfolding Action of Individual Hsp70 Chaperones

Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.

Novel Mechanistic Insight into the Molecular Basis of Amyloid Polymorphism and Secondary Nucleation during Amyloid Formation

The formation of amyloid β (Aβ) fibrils is crucial in initiating the cascade of pathological events that culminates in Alzheimers disease. In this study, we investigated the mechanism of Aβ fibril formation from hydrodynamically well defined species under controlled aggregation conditions. We present a detailed mechanistic model that furnishes a novel insight into the process of Aβ42 fibril formation and the molecular basis for the different structural transitions in the amyloid pathway. Our data reveal the structure and polymorphism of Aβ fibrils to be critically influenced by the oligomeric state of the starting materials, the ratio of monomeric-to-aggregated forms of Aβ42 (oligomers and protofibrils), and the occurrence of secondary nucleation. We demonstrate that monomeric Aβ42 plays an important role in mediating structural transitions in the amyloid pathway, and for the first time, we provide evidences that Aβ42 fibrillization occurs via a combined mechanism of nucleated polymerization and secondary nucleation. These findings will have significant implications to our understanding of the molecular basis of amyloid formation in vivo, of the heterogeneity of Aβ pathology (e.g., diffuse versus amyloid plaques), and of the structural basis of Aβ toxicity.