Pierre Parot

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.

Charge recombination at low temperature in photosynthetic bacteria reaction centers: Evidence for two conformation states

Abstract The kinetics of the decay of state P + Q − A at low temperature for bacterial reaction centers present a complex wavelength dependence around the crossing points of the light-induced difference spectrum (approx. 801 and approx. 757 nm). This phenomenon is observed for chromatophores of Rhodospirillum rubrum G9 and Rhodobacter sphaeroides R26 or 241 as well as for isolated reaction centers of the later species. For untreated reaction centers or chromatophores, the kinetics of the absorbance changes measured at wavelengths slightly above 801 nm are about three times faster than the ones measured below 801 nm. These results are taken as evidence of two conformational states for the reaction centers. Each of these two states has a characteristic decay rate, 11.5 and 36.5 ms respectively, in the case of R26 reaction centers, and a slightly different light-induced spectrum. These two states are in the ratio 60/40, the more abundant being the faster one. They are not influenced by the pH, the presence of the secondary acceptor or the removal of the accessory bacteriochlorophyll molecule of the non photoactive branch. These two conformational states are still present in LM particles, although the kinetics of their charge recombination process are slowed down by a factor of 2.5. Preillumination of reaction centers at room temperature, which lengthens the charges recombination process at low temperature (Kleinfeld, D., Okamura, M.Y. and Feher, G. (1984) Biochemistry 23, 5780–5786) affects only the kinetics of the slow decaying state. This light pretreatment leads also to a ratio of 0.5 between the fast and the slow decaying states, compared to 1.5 for dark-adapted reaction centers. The non-exponential decay of the P + Q − A state measured in the long-wavelength band at low temperature is explained by the superposition of the decays of these two conformational states of the reaction center.

The light-harvesting complexes of a thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum

Abstract Several biophysical properties (absorption, fluorescence, linear dichroism) are reported for the chromatophore membranes of the thermophilic purple sulfur bacterium, Chromatium tepidum. Like the mesophilic strain Chromatium vinosum, two types of light-harvesting complex are present: one absorbs at 800 nm and 855 nm; the second complex is equivalent to the B890 complex of C. vinosum, but absorbs at 918 nm, i.e., 30 nm higher. This is the highest absorption band observed so far for a light-harvesting complex containing bacteriochlorophyll a. In spite of the small overlap between the fluorescence and absorption bands of the two light-harvesting complexes, specially at low temperature, an efficient energy transfer occurs from the high-energy (B800–855) to the low-energy (B920) complexes. The B800–855 complexes have been isolated from the whole membrane by lauryldimethylamine N-oxide treatment, whereas only a partial purification was achieved for the B920 complexes.

Structure of Chloroflexus aurantiacus reaction center: Photoselection at low temperature

Abstract Photoselection experiments have been performed on isolated Chloroflexus aurantiacus reaction centers at 20 K. Our data show that the average angle between the ground state BPh Qy transitions and the 890 nm transition is approx. 50°. Only two BPh Qy transitions are affected by the charge separation. These two transitions are perpendicular to the long-wavelength band of the primary donor. The ground state of the 813 nm transition makes an angle of 35° with the dimer absorption band. The polarization ratio of the light-induced absorption decrease at 815 nm is not consistent with that decrease being due solely to an electrochromic bandshift of the 813 nm transition.

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.

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. Copyright

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.

Effects of low temperature and lipid rigidity on the charge recombination process in Rps. viridis and Rb. sphaeroides reaction centers

The pH dependence of the decay rate of the P + Q − A state (P is the primary electron donor, and Q A , the first stable electron acceptor) was studied at cryogenic temperatures by the absorbance change technique. This study was done in aqueous solvent, in native reaction centers from Rhodopseudomonas viridis and in reaction centers from Rhodobacter sphaeroides in which the native Q A was replaced by the 1-amino-5-chloroanthraquinone. The previously reported biphasicity of the P + Q − A decays in both types of reaction centers (Sebban, P. and Wraight, C.A. (1989) Biochim. Biophys. Acta 974, 54–65; Sebban, P. (1988) Biochim. Biophys. Acta 936, 124–132) is also observed in aqueous buffer at low temperature. At variance to room temperature, where a marked pH dependence was previously observed, the relative distributions of the two components of the decay ( A fast and A slow ) remain constant at 90 K, in the pH range 5.5–11. A fast / A slow is equal to 20:80 and 40:60, in Rhodopseudomonas viridis and in modified Rhodobacter sphaeroides reaction centers, respectively. To further study the possible influence of rigidity of the protein environment on the above parameters, we have reconstituted the reaction centers from Rps. viridis in dimyristoylphosphatidylcholine and dielaidoylphosphatidylcholine liposomes. Below the phase transition temperatures of those lipids ( T c =23°C and 9.5°C, respectively), A slow dominates similarly to what occurs at low temperature. However, as the temperature is increased above T c , i.e. in the fluid phase of the lipid, A fast becomes greater than 50%. The same viscosity effect was observed in glycerol, where the A fast / A slow ratio drops from 1.22 at 35°C to 0.43 at −10°C, decreases slightly to about 0.25 at −30°C, and stays constant until 80K. Our data support the idea of two well-defined states of the reaction centers whose relative distribution may vary depending on the physical conditions, such as low temperature or viscosity, imposed by the medium. At 90 K, the rate constants of P + Q − A charge recombination in Rps. viridis reaction centers observed in aqueous buffer vary in the pH range 5.5–11.5 in a way that is reminiscent of what has previously been observed at room temperature, but with much lower amplitude of the variations. It is suggested that proton distribution present in the dark before cooling or/and proton motion and uptake by the protein at low temperatures modulate the free energy level of the P + Q − A state.

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. Copyright

Purification and characterization of the photochemical reaction center of the thermophilic purple sulfur bacterium Chromatium tepidum

Abstract Pure reaction center preparations from the thermophilic species Chromatium tepidum have been obtained by lauryldimethylamine N-oxide treatment of chromatophores. The light-induced difference spectrum in presence of 10 mM sodium ascorbate revealed the presence of two high-potential cytochrome c hemes (α-band, 555 nm; γ-band, 422 nm). The dithionite-minus-oxidized difference spectrum in the α-band suggests the presence of additional hemes of low potential. These hemes are associated with a single polypeptide (Mr = 36 000). The reaction center pigments, probably four bacteriochorophyll a and two bacteriopheophytin a molecules, are associated with three polypeptides of apparent molecular weights equal to 33 000, 30 000 and 22 000. A carotenoid molecule is also bound to the reaction center. The three main absorption bands of this molecule are located at 480, 510 and 530 nm at liquid helium temperature. Photochemical activity is found to be stable, even after heating for 10 min at temperatures higher than 60 °C in intact chromatophore membranes. On the other hand, isolated reaction centers or chromatophores treated with 1% lauryldimethylamine N-oxide are fully inactivated after heating at temperatures higher than 50 °C. From these results, we propose that lipid-protein interactions are of prime importance in the thermal stabilization of Chromatium tepidum reaction centers.