Pierre Panine

Dimension, Shape, and Conformational Flexibility of a Two Domain Fungal Cellulase in Solution Probed by Small Angle X-ray Scattering

Cellulase Cel45 from Humicola insolens has a modular structure with a catalytic module and a cellulose-binding module (CBM) separated by a 36 amino acid, glycosylated, linker peptide. The solution conformation of the entire two domain Cel45 protein as well as the effect of the length and flexibility of the linker on the spatial arrangement of the constitutive modules were studied by small angle x-ray scattering combined with the known three-dimensional structure of the individual modules. The measured dimensions of the enzyme show that the linker exhibits an extended conformation leading to a maximum extension between the two centers of mass of each module corresponding to about four cellobiose units on a cellulose chain. The glycosylation of the linker is the key factor defining its extended conformation, and a five proline stretch mutation on the linker was found to confer a higher rigidity to the enzyme. Our study shows that the respective positioning of the catalytic module and the CBM onto the insoluble substrate is most likely influenced by the linker structure and flexibility. Our results are consistent with a model where cellulases can move on the surface of cellulose with a caterpillar-like displacement with free energy restrictions.

Fibrillogenesis in Dense Collagen Solutions: A Physicochemical Study

Fibrillogenesis, the formation of collagen fibrils, is a key factor in connective tissue morphogenesis. To understand to what extent cells influence this process, we systematically studied the physicochemistry of the self-assembly of type I collagen molecules into fibrils in vitro. We report that fibrillogenesis in solutions of type I collagen, in a high concentration range close to that of living tissues (40-300 mg/ml), yields strong gels over wide pH and ionic strength ranges. Structures of gels were described by combining microscopic observations (transmission electron microscopy) with small- and wide-angle X-ray scattering analysis, and the influence of concentration, pH, and ionic strength on the fibril size and organization was evaluated. The typical cross-striated pattern and the corresponding small-angle X-ray scattering 67-nm diffraction peaks were visible in all conditions in the pH 6 to pH 12 range. In reference conditions (pH 7.4, ionic strength=150 mM, 20 degrees C), collagen concentration greatly influences the overall macroscopic structure of the resultant fibrillar gels, as well as the morphology and structure of the fibrils themselves. At a given collagen concentration, increasing the ionic strength from 24 to 261 mM produces larger fibrils until the system becomes biphasic. We also show that fibrils can form in acidic medium (pH approximately 2.5) at very high collagen concentrations, beyond 150 mg/ml, which suggests a possible cholesteric-to-smectic phase transition. This set of data demonstrates how simple physicochemical parameters determine the molecular organization of collagen. Such an in vitro model allows us to study the intricate process of fibrillogenesis in conditions of molecular packing close to that which occurs in biological tissue morphogenesis.

Skeletal muscle resists stretch by rapid binding of the second motor domain of myosin to actin

A shortening muscle is a machine that converts metabolic energy into mechanical work, but, when a muscle is stretched, it acts as a brake, generating a high resistive force at low metabolic cost. The braking action of muscle can be activated with remarkable speed, as when the leg extensor muscles rapidly decelerate the body at the end of a jump. Here we used time-resolved x-ray and mechanical measurements on isolated muscle cells to elucidate the molecular basis of muscle braking and its rapid control. We show that a stretch of only 5 nm between each overlapping set of myosin and actin filaments in a muscle sarcomere is sufficient to double the number of myosin motors attached to actin within a few milliseconds. Each myosin molecule has two motor domains, only one of which is attached to actin during shortening or activation at constant length. A stretch strains the attached motor domain, and we propose that combined steric and mechanical coupling between the two domains promotes attachment of the second motor domain. This mechanism allows skeletal muscle to resist external stretch without increasing the force per motor and provides an answer to the longstanding question of the functional role of the dimeric structure of muscle myosin.

Structural changes in the myosin filament and cross‐bridges during active force development in single intact frog muscle fibres: stiffness and X‐ray diffraction measurements

Structural and mechanical changes occurring in the myosin filament and myosin head domains during the development of the isometric tetanus have been investigated in intact frog muscle fibres at 4°C and 2.15 μm sarcomere length, using sarcomere level mechanics and X‐ray diffraction at beamline ID2 of the European Synchrotron Radiation Facility (Grenoble, France). The time courses of changes in both the M3 and M6 myosin‐based reflections were recorded with 5 ms frames using the gas‐filled RAPID detector (MicroGap Technology). Following the end of the latent period (11 ms after the start of stimulation), force increases to the tetanus plateau value (T0) with a half‐time of 40 ms, and the spacings of the M3 and M6 reflections (SM3 and SM6) increase by 1.5% from their resting values, with time courses that lead that of force by ∼10 and ∼20 ms, respectively. These temporal relations are maintained when the increase of force is delayed by ∼10 ms by imposing, from 5 ms after the first stimulus, 50 nm (half‐sarcomere)−1 shortening at the velocity (V0) that maintains zero force. Shortening at V0 transiently reduces SM3 following the latent period and delays the subsequent increase in SM3, but only delays the SM6 increase without a transient decrease. Shortening at V0 imposed at the tetanus plateau causes an abrupt reduction of the intensity of the M3 reflection (IM3), whereas the intensity of the M6 reflection (IM6) is only slightly reduced. The changes in half‐sarcomere stiffness indicate that the isometric force at each time point is proportional to the number of myosin heads bound to actin. The different sensitivities of the intensity and spacing of the M3 and M6 reflections to the mechanical responses support the view that the M3 reflection in active muscle originates mainly from the myosin heads attached to the actin filament and the M6 reflection originates mainly from a fixed structure in the myosin filament signalling myosin filament length changes during the tetanus rise.

Photochromic hybrid organic-inorganic liquid- crystalline materials built from nonionic surfactants and polyoxometalates: elaboration and structural study

This work reports the elaboration and structural study of new hybrid organic-inorganic materials constructed via the coupling of liquid-crystalline nonionic surfactants and polyoxometalates (POMs). X-ray scattering and polarized light microscopy demonstrate that these hybrid materials, highly loaded with POMs (up to 18 wt %), are nanocomposites of liquid-crystalline lamellar structure (Lalpha), with viscoelastic properties close to those of gels. The interpretation of X-ray scattering data strongly suggests that the POMs are located close to the terminal -OH groups of the nonionic surfactants, within the aqueous sublayers. Moreover, these materials exhibit a reversible photochromism associated to the photoreduction of the polyanion. The photoinduced mixed-valence behavior has been characterized through ESR and UV-visible-near-IR spectroscopies that demonstrate the presence of W(V) metal cations and of the characteristic intervalence charge transfer band in the near-IR region, respectively. These hybrid nanocomposites exhibit optical properties that may be useful for applications involving UV-light-sensitive coatings or liquid-crystal-based photochromic switches. From a more fundamental point of view, these hybrid materials should be very helpful models for the study of both the static and dynamic properties of nano-objects confined within soft lamellar structures.

Motion of myosin head domains during activation and force development in skeletal muscle

Muscle contraction is driven by a change in the structure of the head domain of myosin, the “working stroke” that pulls the actin filaments toward the midpoint of the myosin filaments. This movement of the myosin heads can be measured very precisely in intact muscle cells by X-ray interference, but until now this technique has not been applied to physiological activation and force generation following electrical stimulation of muscle cells. By using this approach, we show that the long axes of the myosin head domains are roughly parallel to the filaments in resting muscle, with their center of mass offset by approximately 7 nm from the C terminus of the head domain. The observed mass distribution matches that seen in electron micrographs of isolated myosin filaments in which the heads are folded back toward the filament midpoint. Following electrical stimulation, the heads move by approximately 10 nm away from the filament midpoint, in the opposite direction to the working stroke. The time course of this motion matches that of force generation, but is slower than the other structural changes in the myosin filaments on activation, including the loss of helical and axial order of the myosin heads and the change in periodicity of the filament backbone. The rate of force development is limited by that of attachment of myosin heads to actin in a conformation that is the same as that during steady-state isometric contraction; force generation in the actin-attached head is fast compared with the attachment step.

The structural basis of the increase in isometric force production with temperature in frog skeletal muscle

X‐ray diffraction patterns were recorded from isolated single fibres of frog skeletal muscle during isometric contraction at temperatures between 0 and 17°C. Isometric force was 43 ± 2% (mean ±s.e.m., n= 10) higher at 17°C than 0°C. The intensity of the first actin layer line increased by 57 ± 18% (n= 5), and the ratio of the intensities of the equatorial 1,1 and 1,0 reflections by 20 ± 7% (n= 10), signalling radial or azimuthal motions of the myosin head domains. The M3 X‐ray reflection from the axial repeat of the heads along the filaments was 27 ± 4% more intense at 17°C, suggesting that the heads became more perpendicular to the filaments. The ratio of the intensities of the higher and lower angle peaks of the M3 reflection (RM3) was 0.93 ± 0.02 (n= 5) at 0°C and 0.77 ± 0.02 at 17°C. These peaks are due to interference between the two halves of each myosin filament, and the RM3 decrease shows that heads move towards the midpoint of the myosin filament at the higher temperature. Calculations based on a crystallographic model of the heads indicated that the observed RM3 change corresponds to tilting of their light‐chain domains by 9 deg, producing an axial displacement of 1.4 nm, which is equal to that required to strain the actin and myosin filaments under the increased force. We conclude that the higher force generated by skeletal muscle at higher temperature can be accounted for by axial tilting of the myosin heads.

The complex phase behaviour of suspensions of goethite (α-FeOOH) nanorods in a magnetic field

In 1902, Majorana reported the magneto-optical properties of aqueous colloidal suspensions of mixed iron oxides. Oddly enough, the magnetic-field induced birefringence displayed a non-monotonic dependence upon field intensity. This behaviour was later interpreted as due to the existence in these sols of at least two different chemical species. During the course of our studies of mineral liquid crystals, we have revisited this problem by examining aqueous suspensions of pure goethite (α-FeOOH) nanorods. Although they are comprised of a single chemical species, these suspensions show the same odd behaviour reported by Majorana. Moreover, we show that, as the volume fraction increases, the suspensions have an isotropic liquid/nematic/rectangular columnar phase sequence, with first-order transitions between these phases. The non-monotonic dependence of the field-induced birefringence can be explained by the existence of a remanent magnetic moment of the nanorods and the negative anisotropy of their magnetic susceptibility. Therefore, the nanorods align parallel to a weak field but realign perpendicular to the field beyond Bc ≈ 375 mT. In addition, other interesting phenomena appear upon application of a magnetic field: the disordered (i.e. isotropic in zero-field) phase becomes highly anisotropic and difficult to distinguish from the nematic phase. Both phases then acquire not only quadrupolar order but also dipolar order. The rectangular columnar phase is strongly stabilised versus the nematic one. Our experimental observations raise new theoretical questions about the phase diagram of these suspensions with respect to volume fraction and magnetic field intensity.

A microfluidic cell for studying the formation of regenerated silk by synchrotron radiation small- and wide-angle X-ray scattering

A tube-in-square-pipe microfluidic glass cell has been developed for studying the aggregation and fiber formation from regenerated silk solution by in-situ small-angle X-ray scattering using synchrotron radiation. Acidification-induced aggregation has been observed close to the mixing point of the fibroin and buffer solution. The fibrous, amorphous material is collected in a water bath. Micro-wide-angle X-ray scattering of the dried material confirms its beta-sheet nature.

Two-dimensional camera for millisecond range time-resolved small-and wide-angle X-ray scattering

A combined small-angle and wide-angle X-ray scattering (SAXS/WAXS) camera has been implemented using area detectors suitable for real time experiments down to millisecond time range. The design is based on the existing high brilliance SAXS camera with a sample-to-detector distance variable from 1 m to 10 m and to which a two-dimensional WAXS detector is coupled. Two independent image intensified CCD detectors allow simultaneous real-time SAXS/WAXS experiments on oriented samples down to the millisecond time range. A wide scattering vector range spanning from 0.001 A-1 to 6 A-1 is covered by this combined setup. Both detectors have single photon sensitivity and the spatial resolution is about 200 m. The WAXS detector is mounted at an angle inclined to the primary beam and the process of image transformation into non-arbitrary scattering vector coordinates is demonstrated for a standard p-bromobenzoic acid sample.