Functional consequences of the oligomeric assembly of proteorhodopsin.

The plasma membrane is the crucial interface between the cell and its exterior, packed with embedded proteins experiencing simultaneous protein-protein and protein-membrane interactions. A prominent example of cell membrane complexity is the assembly of transmembrane proteins into oligomeric structures, with potential functional consequences that are not well understood. From the study of proteorhodopsin (PR), a prototypical seven-transmembrane light-driven bacterial proton pump, we find evidence that the inter-protein interaction modulated by self-association yields functional changes observable from the protein interior. We also demonstrate that the oligomer is likely a physiologically relevant form of PR, as crosslinking of recombinantly expressed PR reveals an oligomeric population within the Escherichia coli membrane (putatively hexameric). Upon chromatographic isolation of oligomeric and monomeric PR in surfactant micelles, the oligomer exhibits distinctly different optical absorption properties from monomeric PR, as reflected in a prominent decrease in the pKa of the primary proton acceptor residue (D97) and slowing of the light-driven conformational change. These functional effects are predominantly determined by specific PR-PR contacts over nonspecific surfactant interactions. Interestingly, varying the surfactant type alters the population of oligomeric states and the proximity of proteins within an oligomer, as determined by sparse electron paramagnetic resonance distance measurements. Nevertheless, the dynamic surfactant environment retains the key function-tuning property exerted by oligomeric contacts. A potentially general design principle for transmembrane protein function emerges from this work, one that hinges on specific oligomeric contacts that can be modulated by protein expression or membrane composition

ECUT (Energy Conversion and Utilization Technologies) program: Biocatalysis Project

Fiscal year 1987 research activities and accomplishments for the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Division are presented. The project's technical activities were organized into three work elements. The Molecular Modeling and Applied Genetics work element includes modeling and simulation studies to verify a dynamic model of the enzyme carboxypeptidase; plasmid stabilization by chromosomal integration; growth and stability characteristics of plasmid-containing cells; and determination of optional production parameters for hyper-production of polyphenol oxidase. The Bioprocess Engineering work element supports efforts in novel bioreactor concepts that are likely to lead to substantially higher levels of reactor productivity, product yields, and lower separation energetics. The Bioprocess Design and Assessment work element attempts to develop procedures (via user-friendly computer software) for assessing the economics and energetics of a given biocatalyst process

Aerospace Medicine and Biology: A continuing bibliography with indexes, supplement 199

This bibliography lists 82 reports, articles, and other documents introduced into the NASA scientific and technical information system in October 1979

Cell-free protein synthesis of membrane (1,3)-beta-D-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.Agalya Periasamy, Nadim Shadiac, Amritha Amalraj, Soňa Garajová, Yagnesh Nagarajan, Shane Waters, Haydyn D.T. Mertens, Maria Hrmov

Recent advances in smart biotechnology: Hydrogels and nanocarriers for tailored bioactive molecules depot

Over the past ten years, the global biopharmaceutical market has remarkably grown, with ten over the top twenty worldwide high performance medical treatment sales being biologics. Thus, biotech R&D (research and development) sector is becoming a key leading branch, with expanding revenues. Biotechnology offers considerable advantages compared to traditional therapeutic approaches, such as reducing side effects, specific treatments, higher patient compliance and therefore more effective treatments leading to lower healthcare costs. Within this sector, smart nanotechnology and colloidal self-assembling systems represent pivotal tools able to modulate the delivery of therapeutics. A comprehensive understanding of the processes involved in the self assembly of the colloidal structures discussed therein is essential for the development of relevant biomedical applications. In this review we report the most promising and best performing platforms for specific classes of bioactive molecules and related target, spanning from siRNAs, gene/plasmids, proteins/growth factors, small synthetic therapeutics and bioimaging probes.Istituto Italiano di Tecnologia (IIT)COST Action [CA 15107]People Program (Marie Curie Actions) of the European Union's Seventh Framework Program under REA [606713 BIBAFOODS]Portuguese Foundation for Science and Technology (FCT) [PTDC/AGR-TEC/4814/2014, IF/01005/2014]Fundacao para a Ciencia e Tecnologia [SFRH/BPD/99982/2014]Danish National Research Foundation [DNRF 122]Villum Foundation [9301]Italian Ministry of Instruction, University and Research (MIUR), PRIN [20109PLMH2]"Fondazione Beneficentia Stiftung" VaduzFondo di Ateneo FRAFRAinfo:eu-repo/semantics/publishedVersio

The Evolution of Cholesterol-Rich Membrane in Oxygen Adaption: The Respiratory System as a Model.

The increase in atmospheric oxygen levels imposed significant environmental pressure on primitive organisms concerning intracellular oxygen concentration management. Evidence suggests the rise of cholesterol, a key molecule for cellular membrane organization, as a cellular strategy to restrain free oxygen diffusion under the new environmental conditions. During evolution and the increase in organismal complexity, cholesterol played a pivotal role in the establishment of novel and more complex functions associated with lipid membranes. Of these, caveolae, cholesterol-rich membrane domains, are signaling hubs that regulate important in situ functions. Evolution resulted in complex respiratory systems and molecular response mechanisms that ensure responses to critical events such as hypoxia facilitated oxygen diffusion and transport in complex organisms. Caveolae have been structurally and functionally associated with respiratory systems and oxygen diffusion control through their relationship with molecular response systems like hypoxia-inducible factors (HIF), and particularly as a membrane-localized oxygen sensor, controlling oxygen diffusion balanced with cellular physiological requirements. This review will focus on membrane adaptations that contribute to regulating oxygen in living systems

Xylem surfactants introduce a new element to the cohesion-tension theory

Vascular plants transport water under negative pressure without constantly creating gas bubbles that would disable their hydraulic systems. Attempts to replicate this feat in artificial systems almost invariably result in bubble formation, except under highly controlled conditions with pure water and only hydrophilic surfaces present. In theory, conditions in the xylem should favor bubble nucleation even more: there are millions of conduits with at least some hydrophobic surfaces, and xylem sap is saturated or sometimes supersaturated with atmospheric gas and may contain surface-active molecules that can lower surface tension. So how do plants transport water under negative pressure? Here, we show that angiosperm xylem contains abundant hydrophobic surfaces as well as insoluble lipid surfactants, including phospholipids, and proteins, a composition similar to pulmonary surfactants. Lipid surfactants were found in xylem sap and as nanoparticles under transmission electron microscopy in pores of intervessel pit membranes and deposited on vessel wall surfaces. Nanoparticles observed in xylem sap via nanoparticle-tracking analysis included surfactant-coated nanobubbles when examined by freeze-fracture electron microscopy. Based on their fracture behavior, this technique is able to distinguish between dense-core particles, liquid-filled, bilayer-coated vesicles/liposomes, and gas-filled bubbles. Xylem surfactants showed strong surface activity that reduces surface tension to low values when concentrated as they are in pit membrane pores. We hypothesize that xylem surfactants support water transport under negative pressure as explained by the cohesion-tension theory by coating hydrophobic surfaces and nanobubbles, thereby keeping the latter below the critical size at which bubbles would expand to form embolisms

The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.

GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia

Critical Structural and Functional Roles for the N-Terminal Insertion Sequence in Surfactant Protein B Analogs

Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., approximately residues 8-25 and 63-78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1-7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary alpha-helix and secondary beta-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a "saposin-like" fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B