Investigating protein structure and dynamics through wide-angle X-ray solution scattering

Wide-angle X-ray scattering (WAXS) is a powerful tool that can be used to gain information on the structure and dynamics of proteins and other biomolecules in solution. Improved methods for the calculation of WAXS patterns from available or putative protein models allow to better exploit the structural information contained in the experimental data. These methods, together with recent applications of static and time-resolved WAXS, are briefly reviewed

Demonstration of a picosecond Bragg switch for hard x-rays in a synchrotron-based pump-probe experiment

We report a benchmark experiment that demonstrates shortening of hard x-ray pulses in a synchrotron-based optical pump - x-ray probe experiment. The pulse shortening device, a picosecond Bragg switch, reduces the temporal resolution of an incident x-ray pulse to 7.5 ps. We employ the Bragg switch to monitor propagating sound waves in nanometer-thin epitaxial films. With the experimental data we infer pulse duration, diffraction efficiency and switching contrast of the device. A detailed efficiency analysis shows, that the switch can deliver up to 1010 photons/sec in high-repetition rate synchrotron experiments

On the molecular structure of human neuroserpin polymers

The polymerization of serpins is at the root of a large class of diseases; the molecular structure of serpin polymers has been recently debated. Here, we study the polymerization kinetics of human neuroserpin by Fourier Transform Infra Red spectroscopy and by time-lapse Size Exclusion Chromatography. Firstly we show that two distinct neuroserpin polymers, formed at 45 and 85 °C, display the same isosbestic points in the Amide I′ band, and therefore share common secondary structure features. We also find a concentration independent polymerization rate at 45 °C, suggesting that the polymerization rate-limiting step is the formation of an activated monomeric species. The polymer structures are consistent with a model that predicts the bare insertion of portions of the reactive center loop into the A β-sheet of neighboring serpin molecule, although with different extents at 45 and 85 °C

Functional and dysfunctional conformers of human neuroserpin Characterized by optical spectroscopies and molecular dynamics

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latent serpins, we propose a structural model for latent NS, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatial distribution of intramolecular salt-bridges and hydrogen bonds

Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment

Atomistic characterization of the active-site solvation dynamics of a model photocatalyst

The interactions between the reactive excited state of molecular photocatalysts and surrounding solvent dictate reaction mechanisms and pathways, but are not readily accessible to conventional optical spectroscopic techniques. Here we report an investigation of the structural and solvation dynamics following excitation of a model photocatalytic molecular system [Ir-2(dimen)(4)](2+), where dimen is para-diisocyanomenthane. The time-dependent structural changes in this model photocatalyst, as well as the changes in the solvation shell structure, have been measured with ultrafast diffuse X-ray scattering and simulated with Born-Oppenheimer Molecular Dynamics. Both methods provide direct access to the solute-solvent pair distribution function, enabling the solvation dynamics around the catalytically active iridium sites to be robustly characterized. Our results provide evidence for the coordination of the iridium atoms by the acetonitrile solvent and demonstrate the viability of using diffuse X-ray scattering at free-electron laser sources for studying the dynamics of photocatalysis.1

The Tempered Polymerization of Human Neuroserpin

Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimer's disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate monomeric conformer, which then associates with a native monomer to yield a dimeric species. After the formation of small polymers, the aggregation proceeds via monomer addition as well as polymer-polymer association. No further secondary mechanism takes place up to very high temperatures, thus resulting in the formation of neuroserpin linear polymeric chains. Most interesting, the overall aggregation is tuned by the co-occurrence of monomer inactivation (i.e. the formation of latent neuroserpin) and by a mechanism of fragmentation. The polymerization kinetics exhibit a unique modulation of the average mass and size of polymers, which might suggest synchronization among the different processes involved. Thus, fragmentation would control and temper the aggregation process, instead of enhancing it, as typically observed (e.g.) for amyloid fibrillation