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Physical and chemical attributes of cod roeKatsiadaki, Ioanna January 1997 (has links)
No description available.
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Studies of specific molecular interactions within and between membrane bilayersSizer, P. J. H. January 1986 (has links)
No description available.
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Biophysical studies on the human erythrocyte anion transporter, band 3Taylor, Andrew Mark January 1997 (has links)
No description available.
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Modular Switches in Protein Function: A Spectroscopic ApproachMadathil, Sineej 05 January 2010 (has links) (PDF)
Understanding the molecular basis of protein function is a challenging task that
lays the foundation for the pharmacological intervention in many diseases originating
in altered structural states of the involved proteins. Dissecting a complex functional
machinery into modules is a promising approach to protein function. The motivation
for this work was to identify minimal requirements for “local” switching processes in
the function of multidomain proteins that can adopt a variety of structural substates
of different biological activity or representing intermediates of a complex reaction
path. For example, modular switches are involved in signal transduction, where
receptors respond to ligand-activation by specific conformational changes that are
allosterically transmitted to “effector recognition sites” distant from the actual
ligand-binding site. Heptahelical receptors have attracted particular attention due to
their ubiquitous role in a large variety of pharmacologically relevant processes.
Although constituting switches in their own right, it has become clear through
mutagenesis and functional studies that receptors exhibit substates of partial
active/inactive structure that can explain biological phenotypes of different levels of
activity. Here, the notion that microdomains undergo individual switching processes
that are integrated in the overall response of structurally regulated proteins is
addressed by studies on the molecular basis of proton-dependent (chemical) and
force-dependent (mechanical) conformational transitions.
A combination of peptide synthesis, biochemical analysis, and secondary
structure sensitive spectroscopy (Infrared, Circular dichroism, Fluorescence) was
used to prove the switching capability of putative functional modules derived from
three selected proteins, in which conformational transitions determine their function
in transmembrane signaling (rhodopsin), transmembrane transport
(bacteriorhodopsin) and chemical force generation (kinesin-1). The data are then
related to the phenotypes of the corresponding full length-systems. In the first two
systems the chemical potential of protons is crucial in linking proton exchange
reactions to transmembrane protein conformation. This work addresses the
hypothesized involvement of lipid protein interactions in this linkage (1). It is shown
here that the lipidic phase is a key player in coupling proton uptake at a highly
conserved carboxylic acid (DRY motif located at the C-terminus of helix 3) to conformation during activation of class-1 G protein coupled receptors (GPCRs)
independently from ligand protein interactions and interhelical contacts. The data
rationalize how evolutionary diversity underlying ligand-specifity can be reconciled
with the conservation of a cytosolic ‘proton switch’, that is adapted to the general
physical constraints of a lipidic bilayer described here for the prototypical class-1
GPCR rhodopsin (2).
Whereas the exact sequence of modular switching events is of minor
importance for rhodopsin as long as the final overall active conformation is reached,
the related heptahelical light-transducing proton pump bacteriorhodopsin (bR),
requires the precise relative timing in coupling protonation events to
conformationtional switching at the cytosolic, transmembrane, and extracellular
domains to guarantee vectorial proton transport. This study has focused on the
cytosolic proton uptake site of this retinal protein whose proton exchange reactions at
the cytosolic halfchannel resemble that of rhodopsin. It was a prime task in this work
to monitor in real time the allosteric coupling between different protein regions. A
novel powerful method based on the correlation of simultaneously recorded infrared
absorption and fluorescence emission changes during bR function was established
here (3), to study the switching kinetics in the cytosolic proton uptake domain
relative to internal proton transfer reactions at the retinal and its counter ion. Using
an uptake-impaired bR mutant the data proves the modular nature of domain
couplings and shows that the energy barrier of the conformational transition in the
cytosolic half but not its detailed structure is under the control of proton transfer
reactions at the retinal Schiff base and its counter ion Asp85 (4).
Despite the different functions of the two studied retinal proteins, the
protonation is coupled to local switching mechanisms studied here at two levels of
complexity, [a] a single carboxylic acid side chain acting as a lipid-dependent proton
switch [b] a full-length system, where concerted modular regions orchestrate the
functional coupling of proton translocation reactions. Switching on the level of an
individual amino acid is shown to rely on localizable chemical properties (charge
state, hydrophobicity, rotamer state). In contrast, switching processes involving
longer stretches of amino acids are less understood, less generalizable, and can
constitute switches of mechanical, rather than chemical nature. This applies
particularly to molecular motors, where local structural switching processes are directly involved in force generation. A controversy exists with respect to the
structural requirements for the cooperation of many molecular motors attached to a
single cargo. The mechanical properties of the Hinge 1 domain of kinesin-1 linking
the “neck” and motor domain to the “tail” were addressed here to complement single
molecule data on torsional flexibility with secondary structure analysis and thermal
stability of peptides derived from Hinge 1 (5). It is shown that the Hinge 1 exhibits
an unexpected helix-forming propensity that resists thermal forces but unfolds under
load. The data resolve the paradox that the hinge is required for motor cooperation,
whereas it is dispensable for single motor processivity, clearly emphasizing the
modular function of the holoprotein. However, the secondary-structural data reveal
the functional importance of providing high compliance by force-dependent
unfolding, i.e. in a fundamentally different way than disordered domains that are
flexible but yet do not support cooperativity.
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Modular Switches in Protein Function: A Spectroscopic ApproachMadathil, Sineej 08 December 2009 (has links)
Understanding the molecular basis of protein function is a challenging task that
lays the foundation for the pharmacological intervention in many diseases originating
in altered structural states of the involved proteins. Dissecting a complex functional
machinery into modules is a promising approach to protein function. The motivation
for this work was to identify minimal requirements for “local” switching processes in
the function of multidomain proteins that can adopt a variety of structural substates
of different biological activity or representing intermediates of a complex reaction
path. For example, modular switches are involved in signal transduction, where
receptors respond to ligand-activation by specific conformational changes that are
allosterically transmitted to “effector recognition sites” distant from the actual
ligand-binding site. Heptahelical receptors have attracted particular attention due to
their ubiquitous role in a large variety of pharmacologically relevant processes.
Although constituting switches in their own right, it has become clear through
mutagenesis and functional studies that receptors exhibit substates of partial
active/inactive structure that can explain biological phenotypes of different levels of
activity. Here, the notion that microdomains undergo individual switching processes
that are integrated in the overall response of structurally regulated proteins is
addressed by studies on the molecular basis of proton-dependent (chemical) and
force-dependent (mechanical) conformational transitions.
A combination of peptide synthesis, biochemical analysis, and secondary
structure sensitive spectroscopy (Infrared, Circular dichroism, Fluorescence) was
used to prove the switching capability of putative functional modules derived from
three selected proteins, in which conformational transitions determine their function
in transmembrane signaling (rhodopsin), transmembrane transport
(bacteriorhodopsin) and chemical force generation (kinesin-1). The data are then
related to the phenotypes of the corresponding full length-systems. In the first two
systems the chemical potential of protons is crucial in linking proton exchange
reactions to transmembrane protein conformation. This work addresses the
hypothesized involvement of lipid protein interactions in this linkage (1). It is shown
here that the lipidic phase is a key player in coupling proton uptake at a highly
conserved carboxylic acid (DRY motif located at the C-terminus of helix 3) to conformation during activation of class-1 G protein coupled receptors (GPCRs)
independently from ligand protein interactions and interhelical contacts. The data
rationalize how evolutionary diversity underlying ligand-specifity can be reconciled
with the conservation of a cytosolic ‘proton switch’, that is adapted to the general
physical constraints of a lipidic bilayer described here for the prototypical class-1
GPCR rhodopsin (2).
Whereas the exact sequence of modular switching events is of minor
importance for rhodopsin as long as the final overall active conformation is reached,
the related heptahelical light-transducing proton pump bacteriorhodopsin (bR),
requires the precise relative timing in coupling protonation events to
conformationtional switching at the cytosolic, transmembrane, and extracellular
domains to guarantee vectorial proton transport. This study has focused on the
cytosolic proton uptake site of this retinal protein whose proton exchange reactions at
the cytosolic halfchannel resemble that of rhodopsin. It was a prime task in this work
to monitor in real time the allosteric coupling between different protein regions. A
novel powerful method based on the correlation of simultaneously recorded infrared
absorption and fluorescence emission changes during bR function was established
here (3), to study the switching kinetics in the cytosolic proton uptake domain
relative to internal proton transfer reactions at the retinal and its counter ion. Using
an uptake-impaired bR mutant the data proves the modular nature of domain
couplings and shows that the energy barrier of the conformational transition in the
cytosolic half but not its detailed structure is under the control of proton transfer
reactions at the retinal Schiff base and its counter ion Asp85 (4).
Despite the different functions of the two studied retinal proteins, the
protonation is coupled to local switching mechanisms studied here at two levels of
complexity, [a] a single carboxylic acid side chain acting as a lipid-dependent proton
switch [b] a full-length system, where concerted modular regions orchestrate the
functional coupling of proton translocation reactions. Switching on the level of an
individual amino acid is shown to rely on localizable chemical properties (charge
state, hydrophobicity, rotamer state). In contrast, switching processes involving
longer stretches of amino acids are less understood, less generalizable, and can
constitute switches of mechanical, rather than chemical nature. This applies
particularly to molecular motors, where local structural switching processes are directly involved in force generation. A controversy exists with respect to the
structural requirements for the cooperation of many molecular motors attached to a
single cargo. The mechanical properties of the Hinge 1 domain of kinesin-1 linking
the “neck” and motor domain to the “tail” were addressed here to complement single
molecule data on torsional flexibility with secondary structure analysis and thermal
stability of peptides derived from Hinge 1 (5). It is shown that the Hinge 1 exhibits
an unexpected helix-forming propensity that resists thermal forces but unfolds under
load. The data resolve the paradox that the hinge is required for motor cooperation,
whereas it is dispensable for single motor processivity, clearly emphasizing the
modular function of the holoprotein. However, the secondary-structural data reveal
the functional importance of providing high compliance by force-dependent
unfolding, i.e. in a fundamentally different way than disordered domains that are
flexible but yet do not support cooperativity.
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Computational studies of talin-mediated integrin activationKalli, Antreas C. January 2013 (has links)
Integrins are large heterodimeric (αβ) cell surface receptors that play a key role in the formation of focal adhesion complexes and are involved in various signal transduction pathways. They are ‘activated’ to a high affinity state by the formation of an intracellular complex between the membrane, the integrin β-subunit tail and talin, a process known as ‘inside-out activation’. The head domain of talin, a FERM domain homologue, plays a vital role in the formation of this complex. Recent studies also suggest that kindlins act in synergy with talin to induce integrin activation. Despite much available structural and functional data, details of how talin activates integrins remain elusive. In this thesis a multiscale simulation approach (using a combination of coarse-grained and atomistic molecular dynamics simulations) together with NMR experiments were employed to study talin-mediated integrin inside-out activation. A number of novel insights emerged from these studies including: (i) the crucial role of negatively charged lipids in talin/membrane association; (ii) a novel V-shape conformation of the talin head domain which optimizes its interactions with negatively charged lipids; (iii) that interactions of talin with negatively charged moieties in the membrane orient talin to optimize interactions with the β cytoplasmic tail; (iv) that binding of talin to the β cytoplasmic tail promotes rearrangement of the integrin TM helices and weakens the integrin α/β association; and (v) that an increase in the tilt angle of the β integrin TM helix initiates a scissoring movement of the two integrin TM helices. These results, combined with experimental data, provide new insights into the mechanism of integrin inside-out activation. The same multiscale approach was used to demonstrate the crucial role of the Gx3G motif in the packing of the integrin transmembrane helices. Using recent structural data the integrin/talin complex was modelled and inserted in bilayers which resemble the biological plasma membrane. The results demonstrate the dynamic nature of the integrin receptor and suggest that the integrin/talin complex alters the lipid organization and motion in the outer and inner bilayer leaflets in an asymmetric way and that diffusion of lipids in the vicinity of the protein is slowed down. The work in this thesis demonstrates that multiscale simulations have considerable strengths when applied to investigations of structure/function relationships in membrane proteins.
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Membrane-mimetic systems : Novel methods and results from studies of respiratory enzymesNordlund, Gustav January 2013 (has links)
The processes localized to biological membranes are of great interest, both from a scientific and pharmaceutical point of view. Understanding aspects such as the detailed mechanism and regulation of these processes requires investigation of the structure and function of the membrane-bound proteins in which they take place. The study of these processes is often complicated by the need to create in vitro systems that mimic the environment in which these proteins are normally found in vivo. This thesis describes some of the methods available for membrane-protein studies in membrane-mimetic systems, as well as our work aimed at developing such systems. Furthermore, results from studies using these systems are described. In the first two studies, described in Papers I & II, we investigated the use of silica particle-supported lipid bilayers, both for membrane-protein studies and as possible drug-delivery vehicles. Successful reconstitution of a multisubunit proton-pump, cytochrome c oxidase is described and characterized. Initial attempts to develop drug-delivery systems with two different targeting peptides are also described in the thesis. The second part of this thesis revolves around our work with membraneprotein dependent pathways. Results from studies of systems where the proton- pump bo3 oxidase and ATP synthase work in concert are described. The results show a surprising lipid-composition dependence for the coupled bo3- ATP-synthase activity (Paper III). Finally, a new system utilizing synaptic vesicle-fusion proteins for coreconstitution of membrane proteins is described, showing successful coreconstitution of a small respiratory chain, delivery of soluble proteins to preformed liposomes and reconstitution of ATP synthase in native membranes (Paper IV). / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>
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Property-controlling Enzymes at the Membrane InterfaceGe, Changrong January 2011 (has links)
Monotopic proteins represent a specialized group of membrane proteins in that they are engaged in biochemical events taking place at the membrane interface. In particular, the monotopic lipid-synthesizing enzymes are able to synthesize amphiphilic lipid products by catalyzing two biochemically distinct molecules (substrates) at the membrane interface. Thus, from an evolutionary point of view, anchoring into the membrane interface enables monotopic enzymes to confer sensitivity to a changing environment by regulating their activities in the lipid biosynthetic pathways in order to maintain a certain membrane homeostasis. We are focused on a plant lipid-synthesizing enzyme DGD2 involved in phosphate shortage stress, and analyzed the potentially important lipid anchoring segments of it, by a set of biochemical and biophysical approaches. A mechanism was proposed to explain how DGD2 adjusts its activity to maintain a proper membrane. In addition, a multivariate-based bioinformatics approach was used to predict the lipid-binding segments for GT-B fold monotopic enzymes. In contrast, a soluble protein Myr1 from yeast, implicated in vesicular traffic, was also proposed to be a membrane stress sensor as it is able to exert different binding properties to stressed membranes, which is probably due to the presence of strongly plus-charged clusters in the protein. Moreover, a bacterial monotopic enzyme MGS was found to be able to induce massive amounts of intracellular vesicles in Escherichia coli cells. The mechanisms involve several steps: binding, bilayer lateral expansion, stimulation of lipid synthesis, and membrane bending. Proteolytic and mutant studies indicate that plus-charged residues and the scaffold-like structure of MGS are crucial for the vesiculation process. Hence, a number of features are involved governing the behaviour of monotopic membrane proteins at the lipid bilayer interface. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.
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The Structural Basis for Lipid-Dependent Uncoupling of the Nicotinic Acetylcholine ReceptorSun, Jiayin January 2017 (has links)
In lipid membranes lacking activating lipids, the nicotinic acetylcholine receptor adopts an uncoupled conformation that binds ligand, but does not transition into an open conformation. Understanding the mechanisms of lipid-dependent uncoupling is essential to understanding lipid-nAChR interactions, which may be implicated in pathological conditions such as nicotine addition. Here, I tested two structural features of a proposed uncoupling method to elucidate the mechanism of lipid-dependent uncoupling. First, infrared measurements and electrophysiological characterization performed in prokaryotic homologues indicate that lipid sensitivity is largely controlled by the most peripheral α-helix in the transmembrane domain, M4. My data show that tighter association of M4 with the adjacent M1 and M3 transmembrane α-helices decreases a receptor’s propensity to adopt a lipid-dependent uncoupled conformation. Second, I indirectly tested the hypothesis that uncoupling results from a conformational change at the extracellular/transmembrane domain interface that leads to an increased separation between the two domains and ultimately to a constriction of the channel pore. Finally, biophysical studies presented in this dissertation shed light on the complex binding of a number of non-competitive channel blockers to the nicotinic acetylcholine receptor channel pore in both the resting and desensitized states. The data provide further insight into the structural rearrangements that occur upon uncoupling of ligand binding and gating in the nicotinic acetylcholine receptor.
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Etude des étapes de structuration du fromage fondu : impact formulation et procédé / Processed cheese structuration : impact of formulation and processRullière-Puech, Célie 07 November 2012 (has links)
Le fromage fondu est un produit alimentaire de seconde transformation obtenu après mélange et cuisson de fromages, additionnés éventuellement d'autres ingrédients laitiers. Ce produit, dont la consommation croit dans de nombreuses régions du monde, présente une grande variété d'applications et peut être conservé plusieurs mois à température ambiante. Cependant, les mécanismes moléculaires sous-jacents à sa structuration biochimique demeurent mal connus et sa fabrication industrielle reste souvent empirique.L'objectif général de ce travail est d'améliorer la compréhension des étapes de structuration biochimique du fromage fondu de type « portion triangulaire tartinable », au cours des étapes de sa fabrication. Dans un premier temps, les propriétés physico-chimiques des sels de fonte, additifs ajoutés au fromage fondu, ont été étudiées dans des milieux modèles de complexités croissantes, de la solution aqueuse jusqu'au lait écrémé. La composition de ces sels de fonte, leur hydrolyse après traitement thermique ainsi que leur interaction avec certains constituants laitiers ont été évaluées par deux méthodes complémentaires : la chromatographie ionique et la RMN du phosphore. Par ailleurs, il a été montré que ces sels, via la chélation du calcium, induisaient la dissociation des caséines, modifiant les propriétés d'hydratation et d'émulsification de ces dernières.Dans un deuxième temps, le rôle des sels de fonte quant à la structuration des protéines, de l'eau, des minéraux et de la matière grasse a été analysé dans des matrices plus complexes : les portions triangulaires tartinables, aux étapes clés de leur fabrication. Un mécanisme d'interaction des constituants biochimiques majoritaires a été proposé, prenant en compte l'évolution des molécules sous l'effet des contraintes physiques et chimiques appliquées au cours du procédé. / Processed cheese is manufactured by the secondary processing food industry, by mixing and heating cheese, along with other dairy ingredients. This product, whose consumption grows in many parts of the world, has a wide variety of applications and can be stored for several months at room temperature. However, the molecular mechanisms underlying its structure remain poorly understood and industrial production is often empirical. The general objective of this work is to improve the understanding of biochemical steps structuring spreadable processed cheese during its manufacture. At first, the physico-chemical properties of additives used in processed cheese, i.e. emulsifying salts, have been studied in simplified environments of increasing complexity, from aqueous solutions to skimmed milk. The composition of theses salts, their hydrolysis after heat treatment and their interaction with some dairy constituents were assessed by two complementary methods: ion chromatography and phosphorus NMR. Moreover, it has been shown that these salts, through the calcium chelation, induced casein dissociation and modified their hydration and emulsifying properties. Then, the role of these salts on the structure and interactions between proteins, water, minerals and fat were analyzed in spreadable processed cheese, at different steps of its manufacture. A mechanism of interaction between the major biochemical constituents has been proposed, taking into account the evolution of molecules under the influence of physical and chemical constraints applied during the process.
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