Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Interfacial Reactions of Ozone with Surfactant Protein B in a Model Lung Surfactant System

Oxidative stresses from irritants such as hydrogen peroxide and ozone (O_3) can cause dysfunction of the pulmonary surfactant (PS) layer in the human lung, resulting in chronic diseases of the respiratory tract. For identification of structural changes of pulmonary surfactant protein B (SP-B) due to the heterogeneous reaction with O_3, field-induced droplet ionization (FIDI) mass spectrometry has been utilized. FIDI is a soft ionization method in which ions are extracted from the surface of microliter-volume droplets. We report structurally specific oxidative changes of SP-B_(1−25) (a shortened version of human SP-B) at the air−liquid interface. We also present studies of the interfacial oxidation of SP-B_(1−25) in a nonionizable 1-palmitoyl-2-oleoyl-sn-glycerol (POG) surfactant layer as a model PS system, where competitive oxidation of the two components is observed. Our results indicate that the heterogeneous reaction of SP-B_(1−25) at the interface is quite different from that in the solution phase. In comparison with the nearly complete homogeneous oxidation of SP-B_(1−25), only a subset of the amino acids known to react with ozone are oxidized by direct ozonolysis in the hydrophobic interfacial environment, both with and without the lipid surfactant layer. Combining these experimental observations with the results of molecular dynamics simulations provides an improved understanding of the interfacial structure and chemistry of a model lung surfactant system subjected to oxidative stress

Study of Iron Ion Transit through Three-Fold Channel of Ferritin Cage

Ferritin is an iron-storage globular protein with an ability to uptake, mineralize and release iron ions in a controllable manner. The globular hollow shell allows storage of mineralized iron, with several channels responsible for the transit of ions into the shell and out of it. Understanding of the detailed molecular functioning of ferritin is required for rational design of biomimetic conjugate nano-biosystems containing ferritin-like constituents. In this work, ferritin was investigated both numerically by all-atom molecular dynamics (MD) simulations, and experimentally by Raman spectroscopy. Molecular dynamic simulations of a model system comprising iron ions (Fe2+) and a ferritin trimer expressing a three-fold channel responsible for the ion transport, have revealed a quick entering of ions in the channel. The transit of iron ions through the channel was thoroughly investigated. The transit was found to be driven by both electrostatic charge of ferritin, and interaction between the ions. Exit (expulsion) of an iron ion from the channel was observed at a condition that at least one more ion is present in the channel. Raman characterization of an iron-loaded ferritin solution revealed pronounced bands attributable to iron, as expected. However, Raman spectra of apo-ferritin, which does not contain an iron mineral, also exhibited similar bands. Based on the results of MD simulations, it was hypothesized that apo-ferritin retains iron ions in its three-fold channels, and these ions may produce the observed Raman bands. The study of molecular mechanisms involved in the iron ion transit elucidates the pathways of iron uptake and release in ferritin

Planar Refrains

My practice explores phenomenal poetic truths that exist in fissures between the sensual and physical qualities of material constructs. Magnifying this confounding interspace, my work activates specific instruments within mutable, relational systems of installation, movement, and documentation. The tools I fabricate function within variable orientations and are implemented as both physical barriers and thresholds into alternate, virtual domains. Intersecting fragments of sound and moving image build a nexus of superimposed spatialities, while material constructions are enveloped in ephemeral intensities. Within this compounded environment, both mind and body are charged as active sites through which durational, contemplative experiences can pass. Reverberation, the ghostly refrain of a sound calling back to our ears from a distant plane, can intensify our emotional experience of place. My project Planar Refrains utilizes four electro-mechanical reverb plates, analog audio filters designed to simulate expansive acoustic arenas. Historically these devices have provided emotive voicings to popular studio recordings, dislocating the performer from the commercial studio and into a simulated reverberant territory of mythic proportions. The material resonance of steel is used to filter a recorded signal, shaping the sound of a human performance into something more transformative, a sound embodying otherworldly dynamics. In subverting the designed utility of reverb plates, I am exploring their value as active surfaces extending across different spatial realities. The background of ephemeral sonic residue is collapsed into the foreground, a filter becomes sculpture, and this sculpture becomes an instrument in an evolving soundscape

Technique for normalization of cross-linked peptide ion intensity to elucidate enzymatic conformational changes

Cross-linking coupled to Liquid Chromatography/Mass Spectrometry (CXMS) has become an invaluable technique for examining protein-protein interactions and monitoring enzymatic conformational changes. Thus far, comparative quantitation of cross-linked peptides measured by their mass spectrometry (MS) ion intensity is employed to deduce residue cross-linking propensity and spatial proximity. Using two distinct conformations of a structurally well-characterized model protein, we examined the correlation of cross-linked peptide MS signals and distances, but were not able to observe any obvious correlation. Conceivably, several physiochemical factors can affect residue reactivities thus MS signal intensity of the corresponding cross-linked peptides. For NHS ester chemical cross-linkers, the NHS ester functionality often undergo hydrolysis simultaneously to produce dead-end cross-links. Importantly, these dead-end cross-links have proved to be a reliable measurement of the cross-linked lysine residues. Therefore, we propose a novel analytical method by which these dead-end cross-links provide important contextual information about the amino acid side chains involved in cross-linking reactions. Normalization of cross-linked peptide ion intensity against the cognate dead-end cross-links yields a value (proportional cross-link intensity) which can provide a basis for comparison across different biochemical conditions or enzyme interaction states. In this work, we use this method in conjunction with isotopically labeled cross-linking reagents to examine the conformational changes on the part of HtpG (the E. coli homolog of Hsp90) and a truncation mutant of one of its client proteins, Staphylococcal Nuclease. Dramatic conformational changes are observed in the client protein as HtpG transitions through its ATPase cycle. Next, we apply label-free quantitation and the proportional cross-link intensity model to the vertebrate retina enzyme Phosphodiesterase 6 (PDE6). Through pair-wise combinations of the catalytic subunits, cyclic GMP, and the inhibitory subunits, we observe conformational changes in response to allosteric binding of regulatory subunits and molecules

Deriving amino acid contact potentials from their frequencies of occurence in proteins: a lattice model study

The possibility of deriving the contact potentials between amino acids from their frequencies of occurence in proteins is discussed in evolutionary terms. This approach allows the use of traditional thermodynamics to describe such frequencies and, consequently, to develop a strategy to include in the calculations correlations due to the spatial proximity of the amino acids and to their overall tendency of being conserved in proteins. Making use of a lattice model to describe protein chains and defining a "true" potential, we test these strategies by selecting a database of folding model sequences, deriving the contact potentials from such sequences and comparing them with the "true" potential. Taking into account correlations allows for a markedly better prediction of the interaction potentials

Insights into the interaction dynamics between volatile anesthetics and tubulin through computational molecular modelling

General anesthetics, able to reversibly suppress all conscious brain activity, have baffled medical science for decades, and little is known about their exact molecular mechanism of action. Given the recent scientific interest in the exploration of microtubules as putative functional targets of anesthetics, and the involvement thereof in neurodegenerative disorders, the present work focuses on the investigation of the interaction between human tubulin and four volatile anesthetics: ethylene, desflurane, halothane and methoxyflurane. Interaction sites on different tubulin isotypes are predicted through docking, along with an estimate of the binding affinity ranking. The analysis is expanded by Molecular Dynamics simulations, where the dimers are allowed to freely interact with anesthetics in the surrounding medium. This allowed for the determination of interaction hotspots on tubulin dimers, which could be linked to different functional consequences on the microtubule architecture, and confirmed the weak, Van der Waals-type interaction, occurring within hydrophobic pockets on the dimer. Both docking and MD simulations highlighted significantly weaker interactions of ethylene, consistent with its far lower potency as a general anesthetic. Overall, simulations suggest a transient interaction between anesthetics and microtubules in general anesthesia, and contact probability analysis shows interaction strengths consistent with the potencies of the four compounds