ABSTRACT
Penicillin binding proteins (PBPs) catalyze essential steps in the biosynthesis of peptidoglycan, the main component of the bacterial cell wall. PBPs can harbor two catalytic domains, namely the glycosyltransferase (GT) and transpeptidase (TP) activities, the latter being the target for ß-lactam antibiotics. Despite the availability of structural information regarding bi-functional PBPs, little is known regarding the interaction and flexibility between the TP and GT domains. Here, we describe the structural characterization in solution by small angle X-ray scattering (SAXS) of PBP1b, a bi-functional PBP from Streptococcus pneumoniae. The molecule is present in solution as an elongated monomer. Refinement of internal coordinates starting from a homology model yields models in which the two domains are in an extended conformation without any mutual contact compatible with the existence of restricted mobility.
Subject(s)
Penicillin-Binding Proteins/chemistry , Streptococcus pneumoniae/metabolism , Models, Chemical , Protein Structure, Tertiary , Scattering, Small Angle , X-RaysABSTRACT
We report on small-angle x-ray scattering measurements on liquid water aimed at characterizing the pressure evolution of its large-scale structure. Diffraction profiles have been fitted assuming a Lorentzian dependence on the exchanged momentum. As a result, we observe an anomalous behavior of the diffracted intensity that tends to disappear, increasing either the pressure or the temperature. This effect is discussed in detail and imputed to the ability of hydrostatic pressure to weaken hydrogen bonds.
ABSTRACT
Myelin is a multi-lamellar membrane surrounding neuronal axons and increasing their conduction velocity. When investigated by small-angle x-ray scattering (SAXS), the lamellar quasi-periodical arrangement of the myelin sheath gives rise to distinct peaks, which allow the determination of its molecular organization and the dimensions of its substructures. In this study we report on the myelin sheath structural determination carried out on a set of human brain tissue samples coming from surgical biopsies of two patients: a man around 60 and a woman nearly 90 years old. The samples were extracted either from white or grey cerebral matter and did not undergo any manipulation or chemical-physical treatment, which could possibly have altered their structure, except dipping them into a formalin solution for their conservation. Analysis of the scattered intensity from white matter of intact human cerebral tissue allowed the evaluation not only of the myelin sheath periodicity but also of its electronic charge density profile. In particular, the thicknesses of the cytoplasm and extracellular regions were established, as well as those of the hydrophilic polar heads and hydrophobic tails of the lipid bilayer. SAXS patterns were measured at several locations on each sample in order to establish the statistical variations of the structural parameters within a single sample and among different samples. This work demonstrates that a detailed structural analysis of the myelin sheath can also be carried out in randomly oriented samples of intact human white matter, which is of importance for studying the aetiology and evolution of the central nervous system pathologies inducing myelin degeneration.
Subject(s)
Brain/diagnostic imaging , Brain/ultrastructure , Myelin Sheath/diagnostic imaging , Myelin Sheath/ultrastructure , Scattering, Small Angle , X-Ray Diffraction/methods , Humans , Molecular Conformation , RadiographyABSTRACT
Stopped-flow mixing coupled to small-angle X-ray scattering (SAXS) is an established technique for investigating structural kinetics in solution down to the millisecond range. More recently, the emphasis has shifted to the sub-millisecond range using continuous flow microfluidic mixing devices. The aim of this article is to review the present status and limitations when applying mixing techniques to a wide range of soft matter and biological systems. In the case of SAXS, special consideration of the mixing quality is necessary for a quantitative description of the scattered intensity. This is demonstrated through two representative examples involving protein refolding and micellar self-assembly.
ABSTRACT
This paper describes time-resolved x-ray diffraction data monitoring the transformation of one inverse bicontinuous cubic mesophase into another, in a hydrated lipid system. The first section of the paper describes a mechanism for the transformation that conserves the topology of the bilayer, based on the work of Charvolin and Sadoc, Fogden and Hyde, and Benedicto and O'Brien in this area. We show a pictorial representation of this mechanism, in terms of both the water channels and the lipid bilayer. The second section describes the experimental results obtained. The system under investigation was 2:1 lauric acid: dilauroylphosphatidylcholine at a hydration of 50% water by weight. A pressure-jump was used to induce a phase transition from the gyroid (Q(G)(II)) to the diamond (Q(D)(II)) bicontinuous cubic mesophase, which was monitored by time-resolved x-ray diffraction. The lattice parameter of both mesophases was found to decrease slightly throughout the transformation, but at the stage where the Q (D)(II) phase first appeared, the ratio of lattice parameters of the two phases was found to be approximately constant for all pressure-jump experiments. The value is consistent with a topology-preserving mechanism. However, the polydomain nature of our sample prevents us from confirming that the specific pathway is that described in the first section of the paper. Our data also reveal signals from two different intermediate structures, one of which we have identified as the inverse hexagonal (H(II)) mesophase. We suggest that it plays a role in the transfer of water during the transformation. The rate of the phase transition was found to increase with both temperature and pressure-jump amplitude, and its time scale varied from the order of seconds to minutes, depending on the conditions employed.
ABSTRACT
The relative effectiveness of different anions in crystallizing proteins follows a reversed Hofmeister sequence for pHSubject(s)
Proteins/chemistry
, Biomechanical Phenomena
, Hydrogen-Ion Concentration
, Ions/chemistry
, Models, Biological
, Solutions
ABSTRACT
We have studied the transient stages in the formation of unilamellar vesicles with millisecond time resolution. The self-assembly was initiated by rapid mixing of equimolar amounts of anionic and zwitterionic micelles and the transient micellar entities were probed by time-resolved small-angle x-ray scattering. Within the mixing time, original micelles transformed to disklike micelles which evolved further to a critical size and then closed to form monodisperse unilamellar vesicles within a second. Subsequent growth led to an unexpected broadening of the vesicle size distribution.
Subject(s)
Crystallization/methods , Lipid Bilayers/chemistry , Liposomes/chemistry , Membrane Fluidity , Models, Chemical , Models, Molecular , Kinetics , Macromolecular Substances/chemistry , Molecular Conformation , Particle Size , SolutionsABSTRACT
The behavior of brome mosaic virus in solution as a function of physico-chemical conditions has already been characterized by Small Angle X-ray Scattering studies. The most striking result was that the precipitates induced by the addition of polyethylene glycol were in fact made of microcrystals. This result was reinvestigated on the ID2 beamline at ESRF (Grenoble, France) to determine whether there was an amorphous state before the organized one, and measure the necessary period of latency for the microcrystals to form. The stopped-flow device associated to the high brilliance of the beamline enabled us to characterize the growth of the diffraction peaks as a function of time.
Subject(s)
Bromovirus/chemistry , Chemical Precipitation , Crystallization , Crystallography, X-Ray , Polyethylene Glycols , Scattering, Radiation , X-RaysABSTRACT
The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl(-) ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle X-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.
Subject(s)
Cytochrome c Group/chemistry , Scattering, Radiation , Animals , Circular Dichroism , Dimerization , Horses , Hydrogen-Ion Concentration , Models, Molecular , Monte Carlo Method , Myocardium/metabolism , Protein Conformation , Protein Denaturation , Protein Folding , Protein Structure, Secondary , Spectrophotometry , X-RaysABSTRACT
Procollagen C-propeptide domains direct chain association during intracellular assembly of procollagen molecules. In addition, they control collagen solubility during extracellular proteolytic processing and fibril formation and interact with cell surface receptors and extracellular matrix components involved in feedback inhibition, mineralization, cell growth arrest, and chemotaxis. At present, three-dimensional structural information for the C-propeptides, which would help to understand the underlying molecular mechanisms, is lacking. Here we have carried out a biophysical study of the recombinant C-propeptide trimer from human procollagen III using laser light scattering, analytical ultracentrifugation, and small angle x-ray scattering. The results show that the trimer is an elongated molecule, which by modeling of the x-ray scattering data appears to be cruciform in shape with three large lobes and one minor lobe. We speculate that each of the major lobes corresponds to one of the three component polypeptide chains, which come together in a junction region to connect to the rest of the procollagen molecule.
Subject(s)
Collagen Type III/chemistry , Procollagen/chemistry , Procollagen/isolation & purification , Protein Structure, Quaternary , Cell Line , Collagen Type III/metabolism , Culture Media, Serum-Free , Humans , Models, Molecular , Procollagen/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Scattering, Radiation , Solutions , UltracentrifugationABSTRACT
Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI(2)dIII(1) heterotrimer in solution, but we could find no evidence for the formation of a dI(2)dIII(2) tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotide-binding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI(2)dIII(1) heterotrimer were investigated. (a) The rate of release of NADP(+) from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI(2)dIII(1) complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.
Subject(s)
NADP Transhydrogenases/chemistry , Binding Sites , Enzyme Stability , NAD/metabolism , Protons , SolutionsABSTRACT
A combination of small angle X-ray scattering and gel techniques was used to follow the kinetics of protein crystal growth as a function of time. Hen egg white lysozyme, at different protein concentrations, was used as a model system. A new sample holder was designed, in which supersaturation is induced in the presence of salt by decreasing the temperature. It had been shown previously that a decrease in temperature and/or an increase in crystallizing agent induces an increase in the attractive interactions present in the lysozyme solutions, the lysozyme remaining monomeric. In the present paper we show that similar behaviour is observed in NaCl when agarose gels are used. During crystal growth, special attention was paid to determine whether oligomers were formed as the protein in solution was incorporated in the newly formed crystals. From these first series of experiments, we did not find any indication of oligomer formation between monomer in solution and crystal. The results obtained are in agreement with the hypothesis that lysozyme crystals in NaCl grow by addition of monomeric particles.
Subject(s)
Muramidase/chemistry , Crystallization , Egg Proteins/chemistry , Gels , Kinetics , Scattering, Radiation , Solutions , Temperature , X-RaysABSTRACT
Osmotic pressure, small-angle X-ray scattering and quasi-elastic light scattering were used to study the medium-range interaction potentials between macromolecules in solution. These potentials determine macromolecular crystallization. Calf eye lens gamma-crystallins were used as a model system with the charge, and therefore the interactions, varied with pH. The second virial coefficient was determined under the same conditions with each of the three techniques. Osmotic pressure and quasi-elastic light scattering can be used conveniently in the laboratory to rapidly test the type of interactions (either attractive or repulsive) present in the solution. The measurement is direct with osmotic pressure, whereas with quasi-elastic light scattering, the directly measured coefficient is a combination of thermodynamic and hydrodynamic terms. X-rays, which require more sophisticated equipment such as synchrotron radiation facilities, can provide more detailed information on the interparticle potentials when models are used. At low ionic strength, two potentials were found necessary to account for the temperature and pH phase diagram as a function of protein concentration. The first potential is the van der Waals attractive potential that was previously shown to account for the fluid-fluid phase separation at low temperature. The second potential is an electrostatic coulombic repulsive potential which is a function of the protein charge and thus of the pH. The interaction trail could be followed at protein concentrations as low as 10 mg ml(-1). The results as a whole are expected to be valid for all compact low molecular weight proteins at low ionic strength.