Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed.

AFMBioMed is the founding name under which international conferences and summer schools are organized around the application of atomic force microscopy in life sciences and nanomedicine. From its inception at the Atomic Energy Commission in Marcoule near 2004 to its creation in 2007 and to its 10th anniversary conference in Krakow, a brief narrative history of its birth and rise will demonstrate how and what such an organization brings to laboratories and the AFM community. With the current planning of the next AFMBioMed conference in Münster in 2019, it will be 15 years of commitment to these events.

Conformational dynamics of individual antibodies using computational docking and AFM.

Molecular recognition between a receptor and a ligand requires a certain level of flexibility in macromolecules. In this study, we aimed at analyzing the conformational variability of receptors portrayed by monoclonal antibodies that have been individually imaged using atomic force microscopy (AFM). Individual antibodies were chemically coupled to activated mica surface, and they have been imaged using AFM in ambient conditions. The resulting topographical surface of antibodies was used to assemble the three subunits constituting antibodies: two antigen-binding fragments and one crystallizable fragment using a surface-constrained computational docking approach. Reconstructed structures based on 10 individual topographical surfaces of antibodies are presented for which separation and relative orientation of the subunits were measured. When compared with three X-ray structures of antibodies present in the protein data bank database, results indicate that several arrangements of the reconstructed subunits are comparable with those of known structures. Nevertheless, no reconstructed structure superimposes adequately to any particular X-ray structure consequence of the antibody flexibility. We conclude that high-resolution AFM imaging with appropriate computational reconstruction tools is adapted to study the conformational dynamics of large individual macromolecules deposited on mica.

Tobacco mosaic virus as an AFM tip calibrator.

The study of high-resolution topographic surfaces of isolated single molecules is one of the applications of atomic force microscopy (AFM). Since tip-induced distortions are significant in topographic images the exact AFM tip shape must be known in order to correct dilated AFM height images using mathematical morphology operators. In this work, we present a protocol to estimate the AFM tip apex radius using tobacco mosaic virus (TMV) particles. Among the many advantages of TMV, are its non-abrasivity, thermal stability, bio-compatibility with other isolated single molecules and stability when deposited on divalent ion pretreated mica. Compared to previous calibration systems, the advantage of using TMV resides in our detailed knowledge of the atomic structure of the entire rod-shaped particle. This property makes it possible to interpret AFM height images in term of the three-dimensional structure of TMV. Results obtained in this study show that when a low imaging force is used, the tip is sensing viral protein loops whereas at higher imaging force the tip is sensing the TMV particle core. The known size of the TMV particle allowed us to develop a tip-size estimation protocol which permits the successful erosion of tip-convoluted AFM height images. Our data shows that the TMV particle is a well-adapted calibrator for AFM tips for imaging single isolated biomolecules. The procedure developed in this study is easily applicable to any other spherical viral particles.

Single and multiple bonds in (strept)avidin-biotin interactions.

Thanks to Dynamic Force Spectroscopy (DFS) and developments of massive data analysis tools, such as YieldFinder, Atomic Force Microscopy (AFM) becomes a powerful method for analyzing long lifetime ligand-receptor interactions. We have chosen the well-known system, (strept)avidin-biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum (Walton et al., 2008). We present new measurements of force-displacement curves for the (strept)avidin-biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures ( <1 Å), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin-biotin interactions can be reconciled by taking into account multiple parallel bonds.

Deciphering the energy landscape of the interaction uranyl-DCP with antibodies using dynamic force spectroscopy.

Previous studies on molecular recognition of uranyl-DCP (dicarboxy-phenanthroline chelator) compound by two distinct monoclonal antibodies (Mabs U04S and U08S) clearly showed the presence of a biphasic shape in Bell-Evans' plots and an accentuated difference in slopes at the high loading rates. To further explore the basis in the slope difference, we have performed complementary experiments using antibody PHE03S, raised against uranyl-DCP but, presenting a strong cross-reactivity toward the DCP chelator. This work allowed us to obtain a reallocation of the respective contributions of the metal ion itself and that of the chelator. Results led us to propose a 2D schematic model representing two energy barriers observed in the systems Mabs U04S- and U08S-[UO(2)-DCP] where the outer barrier characterizes the interaction between UO(2) and Mab whereas the inner barrier characterizes the interaction between DCP and Mab. Using dynamic force spectroscopy, it is thus possible to dissect molecular interactions during the unbinding between proteins and ligands.

Surface response methodology for the study of supported membrane formation.

We report on the investigations of the formation of the tethered lipid bilayer by vesicle deposition on amine-functionalized surfaces. The tethered bilayer was created by the deposition of egg-PC vesicles containing 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly-(ethyleneglycol)-N-hydroxysuccinimide as anchoring molecules on an amine-coated surface. This approach is an easy route for the formation of a biomimetic-supported membrane. A Doelhert experimental design was applied to determine the conditions leading to the formation of a continuous and defect-free tethered bilayer on different surfaces (gold and glass). Doehlert designs allow modeling of the experimental responses by second-order polynomial equations as a function of experimental factors. Four factors expected to influence bilayer formation were studied: the lipid concentration in the vesicle suspension, the mass percentage of anchoring molecules in the vesicles, the contact time between the vesicles and the surface, and the resting time of the membrane after buffer rinse. The optimization of the membrane preparation parameters was achieved by monitoring lipid assembly formation using surface plasmon resonance spectroscopy on gold and by fluorescence recovery after photobleaching on glass. Three characteristic responses were systematically measured: the bilayer thickness, the lipid diffusion coefficient, and the lipid mobile fraction. The simultaneous inspection of the three characteristics revealed that a restricted experimental domain leads to properties that are in accordance with a bilayer presence. The factors of this domain are a lipid concentration from 0.1 to 1 mg/mL, 4-8% of anchoring molecules in the vesicles, 1-4 h of contact time between vesicles and surface, and 21-24 h of resting time after buffer rinse. Under these conditions, a membrane having a lipid mass per surface between 545 +/- 5 and 590 +/- 10 ng/cm2, a diffusion coefficient of between 2.5 +/- 0.3 x 10(-8) and 3.60 +/- 0.5 x 10(-8) cm2/s, and a mobile fraction between 94 +/- 2 and 99 +/- 1% was formed. These findings were confirmed by atomic force microscopy observations, which showed the presence of a continuous and homogeneous bilayer in the determined experimental domain. This formation procedure presents many advantages; it provides an easily obtainable biomimetic membrane model for proteins studies and offers a versatile tethered bilayer because it can be adapted easily to various types of supports.

Standardized Nanomechanical Atomic Force Microscopy Procedure (SNAP) for Measuring Soft and Biological Samples.

We present a procedure that allows a reliable determination of the elastic (Young's) modulus of soft samples, including living cells, by atomic force microscopy (AFM). The standardized nanomechanical AFM procedure (SNAP) ensures the precise adjustment of the AFM optical lever system, a prerequisite for all kinds of force spectroscopy methods, to obtain reliable values independent of the instrument, laboratory and operator. Measurements of soft hydrogel samples with a well-defined elastic modulus using different AFMs revealed that the uncertainties in the determination of the deflection sensitivity and subsequently cantilever's spring constant were the main sources of error. SNAP eliminates those errors by calculating the correct deflection sensitivity based on spring constants determined with a vibrometer. The procedure was validated within a large network of European laboratories by measuring the elastic properties of gels and living cells, showing that its application reduces the variability in elastic moduli of hydrogels down to 1%, and increased the consistency of living cells elasticity measurements by a factor of two. The high reproducibility of elasticity measurements provided by SNAP could improve significantly the applicability of cell mechanics as a quantitative marker to discriminate between cell types and conditions.

HiPIP in Rubrivivax gelatinosus is firmly associated to the membrane in a conformation efficient for electron transfer towards the photosynthetic reaction centre.

High potential iron-sulfur protein (HiPIP), a small soluble redox protein, has been shown to serve in vivo as electron donor to the photosynthetic reaction centre (RC) in Rubrivivax gelatinosus [Biochemistry 34 (1995) 11736]. The results of time-resolved optical spectroscopy on membrane-fragments from this organism indicates that the photooxidized RC is re-reduced by HiPIP even in the absence of the soluble fraction. This implies that a significant fraction of HiPIP can firmly bind to the membrane in a conformation able to interact with the RCs. Salt treatment of the membrane-fragments abolishes these re-reduction kinetics, demonstrating the presence of HiPIP on the membrane due to association with the RC rather than due to simple trapping in hypothetical chromatophores. The existence of such a functional complex in membranes is confirmed and its structure further examined by electron paramagnetic resonance (EPR) performed on membrane-fragments. Orientation-dependent EPR spectra of HiPIP were recorded on partially ordered membranes, oxidized either chemically or photochemically. Whereas hardly any preferential orientation of the HiPIP was seen in the chemically oxidised sample, a subpopulation of HiPIP showing specific orientations could be photooxidised. This fraction arises from the electron transfer complex between HiPIP and the RC.

Nanoscale structural features determined by AFM for single virus particles.

In this work, we propose "single-image analysis", as opposed to multi-image averaging, for extracting valuable information from AFM images of single bio-particles. This approach allows us to study molecular systems imaged by AFM under general circumstances without restrictions on their structural forms. As feature exhibition is a resolution correlation, we have performed AFM imaging on surfaces of tobacco mosaic virus (TMV) to demonstrate variations of structural patterns with probing resolution. Two AFM images were acquired with the same tip at different probing resolutions in terms of pixel width, i.e., 1.95 and 0.49 nm per pixel. For assessment, we have constructed an in silico topograph based on the three-dimensional crystal structure of TMV as a reference. The prominent artifacts observed in the AFM-determined shape of TMV were attributed to tip convolutions. The width of TMV rod was systematically overestimated by ~10 nm at both probing resolutions of AFM. Nevertheless, the effects of tip convolution were less severe in vertical orientation so that the estimated height of TMV by AFM imaging was in close agreement with the in silico X-ray topograph. Using dedicated image processing algorithms, we found that at low resolution (i.e., 1.95 nm per pixel), the extracted surface features of TMV can be interpreted as a partial or full helical repeat (three complete turns with ~7.0 nm in length), while individual protein subunits (~2.5 nm) were perceivable only at high resolution. The present study shows that the scales of revealed structural features in AFM images are subject to both probing resolution and processing algorithms for image analysis.

Computational reconstruction of multidomain proteins using atomic force microscopy data.

Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally based test cases: Tobacco mosaic virus, a rod-shaped virus; and Aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multicomponent structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures.

Past, present and future of atomic force microscopy in life sciences and medicine.

To introduce this special issue of the Journal of Molecular Recognition dedicated to the applications of atomic force microscopy (AFM) in life sciences, this paper presents a short summary of the history of AFM in biology. Based on contributions from the first international conference of AFM in biological sciences and medicine (AFM BioMed Barcelona, 19-21 April 2007), we present and discuss recent progress made using AFM for studying cells and cellular interactions, probing single molecules, imaging biosurfaces at high resolution and investigating model membranes and their interactions. Future prospects in these different fields are also highlighted.

On molecular recognition of an uranyl chelate by monoclonal antibodies.

The energy landscape of the uranyl (UO2) chelate dissociated from a monoclonal antibody U08S was investigated using dynamic force spectroscopy (DFS). The uranyl ion (UO2(2+)) is chelated with the ligand dicarboxy-phenanthroline (DCP). The monoclonal antibody U08S was raised against UO2-DCP and does not cross-react with DCP alone. The results of plotting the most probable force against the logarithm of the loading rate show two distinguished values of slopes of multiple fitting lines, as observed in our previous study on that system with monoclonal antibody U04S (Odorico et al., 2007a. Biophys. J. 93: 645-654.). It indicates an unbinding process undergoing at least two activation states. We have generated the histogram of unbinding events with respect to the composite stiffness of the complex between the protein and the uranyl compound. Combining the model of Bell and Evans with that of Williams, we have estimated the number of parallel bonds involved in the unbinding process and determined the value of stiffness for individual bonds. We propose that the uranyl compound binds to the two antibodies U04S and U0c at structurally equivalent locations and forms the interaction with similar coordination modes. In addition, the unbinding process goes through two steps; the first weakens the bonding of the central metal with AspL50 of the antibody and the second breaks other non-bonded interactions of the compound with the antibody.

Energy landscape of chelated uranyl: antibody interactions by dynamic force spectroscopy.

We used dynamic force spectroscopy (DFS) to explore the energy landscape of interactions between a chelated uranyl compound and a monoclonal antibody raised against the uranyl-dicarboxy-phenanthroline complex. We estimated the potential energy barrier widths and the relevant thermodynamic rate constants along the dissociation coordinate. Using atomic force microscopy, four different experimental setups with or without the uranyl ion in the chelate ligand, we have distinguished specific and nonspecific binding in the binding affinity of the uranyl compound to the antibody. The force loading rates for our system were measured from 15 to 26,400 pN/s. The results showed two regimes in the plot of the most probable unbinding force versus the logarithm of the loading rate, revealing the presence of two (at least) activation barriers. Analyses of DFS suggest parallel multivalent binding present in either regime. We have also built a molecular model for the variable fragment of the antibody and used computational graphics to dock the chelated uranyl ion into the binding pocket. The structural analysis led us to hypothesize that the two regimes originate from two interaction modes: the first one corresponds to an energy barrier with a very narrow width of 0.5 +/- 0.2 A, inferring dissociation of the uranyl ion from its first coordination shell (Asp residue); the second one with a broader energy barrier width (3.9 +/- 0.3 A) infers the entire chelate compound dissociated from the antibody. Our study highlights the sensitivity of DFS experiments to dissect protein-metal compound interactions.

Two distinct binding sites for high potential iron-sulfur protein and cytochrome c on the reaction center-bound cytochrome of Rubrivivax gelatinosus.

The photosynthetic cyclic electron transfer of the purple bacterium Rubrivivax gelatinosus, involving the cytochrome bc(1) complex and the reaction center, can be carried out via two pathways. A high potential iron-sulfur protein (HiPIP) acts as the in vivo periplasmic electron donor to the reaction center (RC)-bound cytochrome when cells are grown under anaerobic conditions in the light, while cytochrome c is the soluble electron carrier for cells grown under (8)aerobic conditions in the dark. A spontaneous reversion of R. gelatinosus C244, a defective mutant in synthesis of the RC-bound cytochrome by insertion of a Km(r) cassette leading to gene disruption with a slow growth rate, restores the normal photosynthetic growth. This revertant, designated C244-P1, lost the Km(r) cassette but synthesized a RC-bound cytochrome with an external 77-amino acid insertion derived from the cassette. We characterized the RC-bound cytochrome of this mutant by EPR, time-resolved optical spectroscopy, and structural analysis. We also investigated the in vivo electron transfer rates between the two soluble electron donors and this RC-bound cytochrome. Our results demonstrated that the C244-P1 RC-bound cytochrome is still able to receive electrons from HiPIP, but it is no longer reducible by cytochrome c(8). Combining these experimental and theoretical protein-protein docking results, we conclude that cytochrome c(8) and HiPIP bind the RC-bound cytochrome at two distinct but partially overlapping sites.