Lactose carrier protein of Escherichia coli: interaction with galactosides and protons

Jk, Wright; Riede, Isolde; Overath, Peter

doi:10.1021/bi00525a019

Cited by 74 publications

(64 citation statements)

References 44 publications

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“…We found that precipitated LPase does not show the slow MalNPyr fluctuations. (ii) Below the lipid-phase transition the transport rate is decreased drastically (7)(8)(9), although binding is unaltered in consonance with unaltered secondary structure (6). Here, we found a reduced relaxation rate of the slow MalNPyr fluctuations together with a reduced amplitude.…”

Section: Analysis and Discussionsupporting

confidence: 53%

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Internal dynamics of lactose permease.

Dornmair

Jähnig

1989

Proc. Natl. Acad. Sci. U.S.A.

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The transport protein lactose permease was reconstituted in vesicles of dimyristoylphosphatidylcholine, and the internal dynamics were studied by measuring the fluorescence anisotropy decay of the tryptophan residues and of a covalently bound pyrene label. For the tryptophans three relaxation processes and for the pyrene two relaxation processes with relaxation times in the nanosecond range were observed. What is still under debate is the relevance of such fluctuations to protein function. Are they just random unavoidable thermal motions of the protein or are they correlated in a specific way to the slow conformational changes observed in substrate binding and catalytic processes? A model has been proposed in which the fluctuations and conformational changes obey a hierarchical order (2): the fastest fluctuations give rise to slightly slower ones, which in turn give rise to still slower ones and so on until, finally, the slowest fluctuations lead to a conformational change. In an attempt to give an experimental answer to the above question we investigated the fluctuations of the transport protein lactose permease (LPase) under conditions where the protein is known to be functionally either active or inactive.LPase is a protein of the cytoplasmic membrane of Escherichia coli, which catalyzes the transport of galactosides in symport with a proton across the membrane (for reviews, see refs. 3 and 4). The protein consists of 417 amino acid residues and acts as a monomer. Based on spectroscopic data and structural predictions, a model for the folding of LPase within the membrane was proposed: ten membrane-spanning ahelices form a ring the interior ofwhich is filled with relatively hydrophilic amino acid residues suited to provide the sugarbinding site (5, 6).To investigate a putative correlation between internal fluctuations and function, a membrane protein offers two distinct advantages over soluble proteins. (i) The activity of the membrane protein can easily be switched on and off by passing through the phase transition of the surrounding lipids (7-9).(ii) On the time scale of nanoseconds relevant for internal fluctuations, a membrane protein is immobilized, whereas a soluble protein undergoes rotational diffusion that renders detection of such internal fluctuations difficult, if not impossible.The technique we used to study internal fluctuations is fluorescence anisotropy decay (FAD) (for review, see ref.10). This technique permits detection of orientational fluctuations of intrinsic or extrinsic fluorophores with characteristic times in the range of picoseconds and nanoseconds.MATERIALS AND METHODS Sample Preparation. For measurements of the tryptophan fluorescence of LPase, cytoplasmic membrane vesicles were prepared from the permease-overproducing strain E. coli T206 (11) and used directly for purification and reconstitution of LPase. Control samples were prepared identically from the permease-deficient strain E. coli T184. For measurements on pyrene-labeled permease, cytoplasmic membranes vesicles of ...

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Section: Analysis and Discussionsupporting

confidence: 53%

“…3). This condition is fulfilled in our case because the temperature dependence of the transport rate obeys an Arrhenius relation (9,28). A transport cycle involves two conformational changes, one with bound substrate and one without.…”

Section: Analysis and Discussionmentioning

confidence: 82%

Internal dynamics of lactose permease.

Dornmair

Jähnig

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…The activity of membrane proteins often exhibits a break at Tt with a low activation energy above Tt and a high activation energy below Tt (30,31). One is tempted to interprete this behavior by postulating that protein activity requires a fluid lipid environment.…”

Section: Total Free Energy and Enthalpy Changementioning

confidence: 99%

Thermodynamics and kinetics of protein incorporation into membranes.

Jähnig¹

1983

Proc. Natl. Acad. Sci. U.S.A.

116

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The free energy and enthalpy of protein incorporation into membranes are calculated with special emphasis on the hitherto neglected effects of immobilization of protein and perturbation of lipid order in the membrane. The free energy change is found to be determined by the hydrophobic effect as the driving force for incorporation and the protein immobilization effect which leads to a considerable reduction of the free energy gained from the hydrophobic effect. For incorporation of a hydrophobic, bilayer-spanning a-helix, the free energy change obtained is of the order of -15 kcal/mol (1 cal = 4.184 J) in agreement with experimental results. The lipid perturbation effect yields only a small contribution to the free energy change due to an energy/entropy compensation inherent in lipid order. This effect dominates the enthalpy change, giving rise to values on the order of 100 kcal/mol with a pronounced temperature dependence around the lipid phase transition as observed experimentally. The kinetics of protein incorporation are even more strongly affected by the lipid perturbation effect, leading to an abrupt decrease of the rate of incorporation below the lipid phase transition.It is commonly accepted that the driving force for spontaneous incorporation of proteins into membranes is the hydrophobic effect. However, a number of other effects may support or counteract the hydrophobic effect. In general, the free energy of protein incorporation includes four contributions which arise from a change of (i) water structure, (ii) protein state, (iii) lipid state, and (iv) bonds between protein and water or lipid molecules.The change of water structure represents the hydrophobic effect which originates in the reduction of the mobility of water molecules surrounding the protein. The change of the protein state comprises the external translational and rotational degrees of freedom which become immobilized upon incorporation of the protein and the internal degrees of freedom whose changes involve conformational changes and changes of internal bonds. The change of the lipid state accounts for the reduction of the mobility of the lipid molecules in a fluid membrane. This parallels the reduction of the mobility of water molecules in the hydrophobic effect and may therefore be called lipophobic effect of proteins. The change of bonds at the protein surface concerns mainly polar interactions such as hydrogen bonds and electrostatic interactions between polar amino acid residues and water molecules because the interaction energy between apolar residues and water or lipid molecules is relatively small.The gain in free energy from the hydrophobic effect has been investigated thoroughly in the past (1-4). Estimates are also available for the free energy arising from the formation or breakage of hydrogen bonds and electrostatic bonds (3, 4) as well as from the change of protein conformation (5, 6). The protein immobilization effect has not been taken into account in previous work. This effect was studied in the context of enzy...

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“…In addition, the membranes become leaky, components leak that are prerequisite for life. With quasi-crystalline membranes life is not possible [1] [2]. Cells regulate the transition temperature of their membranes with variation of components.…”

Section: Discussionmentioning

confidence: 99%

Membrane Fluidity: About the Origin of Autoimmunity

Riede¹

2014

OJI

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Abstract/HypothesisFluidity of cellular membranes is essential for life. Two possibilities are known to keep human membranes fluid: unsaturated fatty acids and cholesterol. Whereas liver cells can synthesize cholesterol, unsaturated fatty acids are essential. Life style in Western civilization leads to deprivation of essential fatty acids, to elevated serum-cholesterol-levels and to autoimmunity. Here the hypothesis is presented, and explains the relationship: deprivation of essential fatty acids lead to imminent quasi-crystallization of the membrane. Serum cholesterol-levels are elevated. Incorporation of cholesterol into membranes enhancing fluidity again, is able to repair the effect. At saturation, repair fails. Quasi-crystallization occurs. Proteins tilt into another conformation. This has not been learned during the "self" recognition process of the immune system during the embryonic phase. Immune system attacks the new conformation as "non-self", autoimmunity emerges.

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Lactose carrier protein of Escherichia coli: interaction with galactosides and protons

Cited by 74 publications

References 44 publications

Internal dynamics of lactose permease.

Internal dynamics of lactose permease.

Thermodynamics and kinetics of protein incorporation into membranes.

Membrane Fluidity: About the Origin of Autoimmunity

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