Lipid Bilayer Strengthens the Side Chain Interaction Network of a Membrane Protein

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron–electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli’s lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

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Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606776113 ◽

2016 ◽

Vol 113 (38) ◽

pp. 10559-10564 ◽

Cited By ~ 16

Author(s):

Karin Öjemalm ◽

Takashi Higuchi ◽

Patricia Lara ◽

Erik Lindahl ◽

Hiroaki Suga ◽

...

Keyword(s):

Amino Acids ◽

Membrane Protein ◽

Side Chain ◽

Transmembrane Helices ◽

Transmembrane Segments ◽

Charged Amino Acids ◽

Protein Biogenesis ◽

Quantitative Basis ◽

Apparent Free Energy ◽

Integration Efficiency

Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (ΔGapp) of “snorkeling” of charged amino acids toward the lipid–water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on ΔGapp. These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells.

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Membrane protein simulations with a united-atom lipid and all-atom protein model: lipid–protein interactions, side chain transfer free energies and model proteins

Journal of Physics Condensed Matter ◽

10.1088/0953-8984/18/28/s07 ◽

2006 ◽

Vol 18 (28) ◽

pp. S1221-S1234 ◽

Cited By ~ 105

Author(s):

D Peter Tieleman ◽

Justin L MacCallum ◽

Walter L Ash ◽

Christian Kandt ◽

Zhitao Xu ◽

...

Keyword(s):

Membrane Protein ◽

Protein Interactions ◽

Chain Transfer ◽

Side Chain ◽

Free Energies ◽

Protein Model ◽

Lipid Protein Interactions

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Pyridyl-Ala Modified Cyclic Hexapeptides: In-Vitro and In-Vivo Profiling for Oral Bioavailability

International Journal of Peptide Research and Therapeutics ◽

10.1007/s10989-019-09935-y ◽

2019 ◽

Vol 26 (3) ◽

pp. 1383-1397 ◽

Cited By ~ 1

Author(s):

Thomas Vorherr ◽

Ian Lewis ◽

Joerg Berghausen ◽

Felix Huth ◽

Michael Schaefer ◽

...

Keyword(s):

Amino Acid ◽

Oral Bioavailability ◽

Side Chain ◽

Pyridyl Nitrogen ◽

Oral Uptake ◽

Cyclic Hexapeptides ◽

Chain Interaction

Abstract We and others have been aiming at modifications to maintain or to enhance solubility while enabling permeability for cyclic hexapeptides. Especially, the 2-pyridyl-Ala modification was investigated, since in this case, the pyridyl-nitrogen is able to form an H-bond to the NH of the same residue. The hypothesis of a backbone side-chain interaction was demonstrated by NMR experiments, and further results obtained on a variety of pyridyl-Ala derivatives, studied systematically in the context of permeability, are presented in this contribution. Thus, this study sheds some more light on the pyridyl-Ala modification, which had been reported earlier. In addition to the in vitro profiling, the extent of oral bioavailability was assessed in rats. In principle, the pyridyl-Ala residue can be considered as an amino acid supporting oral uptake. Graphic Abstract

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Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder

AJP Cell Physiology ◽

10.1152/ajpcell.1991.261.1.c143 ◽

1991 ◽

Vol 261 (1) ◽

pp. C143-C153 ◽

Cited By ~ 10

Author(s):

H. W. Harris ◽

M. L. Zeidel ◽

C. Hosselet

Keyword(s):

Membrane Proteins ◽

Membrane Protein ◽

Lipid Bilayer ◽

Apical Membrane ◽

Water Channel ◽

Protein S ◽

Water Channels ◽

Toad Bladder ◽

Western Blots ◽

Vesicle Protein

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.

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Antidepressants are modifiers of lipid bilayer properties

The Journal of General Physiology ◽

10.1085/jgp.201812263 ◽

2019 ◽

Vol 151 (3) ◽

pp. 342-356 ◽

Cited By ~ 7

Author(s):

Ruchi Kapoor ◽

Thasin A. Peyear ◽

Roger E. Koeppe ◽

Olaf S. Andersen

Keyword(s):

Membrane Protein ◽

Lipid Bilayer ◽

Conformational Changes ◽

Partition Coefficients ◽

Selective Serotonin Reuptake Inhibitors ◽

Conformational Transition ◽

Hydrophobic Interactions ◽

Energetic Cost ◽

Titration Calorimetry ◽

Channel Activity

The two major classes of antidepressants, tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs), inhibit neurotransmitter reuptake at synapses. They also have off-target effects on proteins other than neurotransmitter transporters, which may contribute to both desired changes in brain function and the development of side effects. Many proteins modulated by antidepressants are bilayer spanning and coupled to the bilayer through hydrophobic interactions such that the conformational changes underlying their function will perturb the surrounding lipid bilayer, with an energetic cost (ΔGdef) that varies with changes in bilayer properties. Here, we test whether changes in ΔGdef caused by amphiphilic antidepressants partitioning into the bilayer are sufficient to alter membrane protein function. Using gramicidin A (gA) channels to probe whether TCAs and SSRIs alter the bilayer contribution to the free energy difference for the gramicidin monomer⇔dimer equilibrium (representing a well-defined conformational transition), we find that antidepressants alter gA channel activity with varying potency and no stereospecificity but with different effects on bilayer elasticity and intrinsic curvature. Measuring the antidepressant partition coefficients using isothermal titration calorimetry (ITC) or cLogP shows that the bilayer-modifying potency is predicted quite well by the ITC-determined partition coefficients, and channel activity is doubled at an antidepressant/lipid mole ratio of 0.02–0.07. These results suggest a mechanism by which antidepressants could alter the function of diverse membrane proteins by partitioning into cell membranes and thereby altering the bilayer contribution to the energetics of membrane protein conformational changes.

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