Determination of the depth of bromine atoms in bilayers formed from bromolipid probes

McIntosh, T. O.; Holloway, Peter W.

doi:10.1021/bi00380a042

Cited by 186 publications

(168 citation statements)

References 38 publications

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“…6, A and B, three planes of atoms are introduced that are perpendicular to the membrane normal and schematically identify the locations of terminal methyl carbons of the acyl chains of glycerophospholipids (membrane center), the sn-1 carbonyl oxygens, and the phosphorus atoms of lipid phosphate groups. The z coordinates of these three planes are z ϭ 0, z ϭ 14.5 Å, and z ϭ 20 Å, respectively, which is based on x-ray diffraction data on POPC bilayers (55).…”

Section: Resultsmentioning

confidence: 99%

“…The z coordinates of the imidazole nitrogens of the catalytic His 48 , which is located just slightly above the membrane surface, are 22.6 Å. Considering that the sn-2 ester groups of POPC and 1,2-dioleoyl-sn-glycero-3-phosphocholine are at ϳ15.5 Å from the membrane center (55,59), the phospholipid molecule has to travel only ϳ7 Å in order to reach the catalytic center of the enzyme, in contrast to the earlier conjectures assuming an ϳ15 Å distance between the catalytic center of membrane-bound PLA 2 and the lipid cleavage site (6 -8, 56, 58).…”

Section: Resultsmentioning

confidence: 99%

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Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

Qin

Pande

Nemec

et al. 2005

Journal of Biological Chemistry

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The phospholipase A 2 (PLA 2 ) enzymes are activated by binding to phospholipid membranes. Although the N-terminal ␣-helix of group I/II PLA 2 s plays an important role in the productive mode membrane binding of the enzymes, its role in the structural aspects of membrane-induced activation of PLA 2 s is not well understood. In order to elucidate membrane-induced conformational changes in the N-terminal helix and in the rest of the PLA 2 , we have created semisynthetic human group IB PLA 2 in which the N-terminal decapeptide is joined with the 13 C-labeled fragment, as well as a chimeric protein containing the N-terminal decapeptide from human group IIA PLA 2 joined with a 13 C-labeled fragment of group IB PLA 2 . Infrared spectral resolution of the unlabeled and 13 C-labeled segments suggests that the N-terminal helix of membranebound IB PLA 2 has a more rigid structure than the other helices. On the other hand, the overall structure of the chimeric PLA 2 is more rigid than that of the IB PLA 2 , but the N-terminal helix is more flexible. A combination of homology modeling and polarized infrared spectroscopy provides the structure of membrane-bound chimeric PLA 2 , which demonstrates remarkable similarity but also distinct differences compared with that of IB PLA 2 . Correlation is delineated between structural and membrane binding properties of PLA 2 s and their N-terminal helices. Altogether, the data provide evidence that the N-terminal helix of group I/II PLA 2 s acts as a regulatory domain that mediates interfacial activation of these enzymes.Enzymes of the phospholipase A 2 (PLA 2 ) 2 family hydrolyze the sn-2 ester bond of diacylglycerophospholipids and thus initiate the biosynthesis of lipid-derived mediators, such as eicosanoids and platelet-activating factor, which are potent mediators of inflammation, allergy, and tumorigenesis (1-3). These enzymes are divided into several groups and subgroups based on their amino acid sequences, tissue distribution, and functional features (1, 2).PLA 2 s gain their full activity only when they bind to phospholipid micelles or membranes, an effect known as interfacial activation (4 -6). The molecular mechanism of interfacial activation has been the subject of debate over several decades. Initially, similarities of crystal structures of PLA 2 s with and without bound substrate mimics argued against conformational changes in the enzymes during interfacial activation (4). Later, the interfacially activated form of group IB PLA 2 was modeled by anion-mediated dimers (7), which revealed significant conformational changes compared with "inactive" monomeric forms, mainly involving the loop that has the functionally important Tyr 69 , and the presence of an assisting water molecule (8). Several spectroscopic studies also identified conformational changes in group IB and IIA PLA 2 s upon binding to phospholipid micelles or membranes. Fluorescence studies indicated that the N-terminal helix of porcine group IB PLA 2 becomes rigid upon enzyme-substrate complex formation at the m...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

Qin

Pande

Nemec

et al. 2005

Journal of Biological Chemistry

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“…For each mutant, these time courses were monitored with five different samples, four of which contained 30 mol % of 4,5-, 6,7-, 9,10-, and 11,12-DiBrPC, respectively, in DOPC and a fifth reference sample which consisted of DOPC only. The distances of the quenching bromine atoms from the bilayer center (19) are given on the x-axis of each graph, and the reference without quencher is plotted on the far right of each 3-D graph. Similar graphs were also obtained for the Trp-102 mutant (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Outer Membrane Protein A of Escherichia coli Inserts and Folds into Lipid Bilayers by a Concerted Mechanism

et al. 1999

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Unfolded outer membrane protein A (OmpA) of Escherichia coli spontaneously inserts and refolds into lipid bilayers upon dilution of denaturing urea. In the accompanying paper, we have developed a new technique, time-resolved distance determination by fluorescence quenching (TDFQ), which is capable of monitoring the translocation across lipid bilayers of fluorescence reporter groups such as tryptophan in real time [Kleinschmidt, J. H., and Tamm, L. K. (1999) Biochemistry 38, 4996−5005]. Specifically, we have shown that wild-type OmpA, which contains five tryptophans, inserts into lipid bilayers via three structurally distinct membrane-bound folding intermediates. To take full advantage of the TDFQ technique and to further dissect the folding pathway, we have made five different mutants of OmpA, each containing a single tryptophan and four phenylalanines in the five tryptophan positions of the wild-type protein. All mutants refolded in vivo and in vitro and, as judged by SDS−PAGE, trypsin fragmentation, and Trp fluorescence, their refolded state was indistinguishable from the native state of OmpA. TDFQ analysis of the translocation across the lipid bilayer of the individual Trps of OmpA yielded the following results: Below 30 °C, all Trps started from a far distance from the bilayer center and then gradually approached a distance of approximately 10 Å from the bilayer center. In a narrow temperature range between 30 and 35 °C, Trp-15, Trp-57, Trp-102, and Trp-143 were detected very close to the center of the lipid bilayer in the first few minutes and then moved to greater distances from the center. When monitored at 40 °C, which resolved the last steps of OmpA refolding, these four tryptophans crossed the center of the bilayer and approached distances of approximately 10 Å from the center after refolding was complete. In contrast Trp-7 approached the 10 Å distance from a far distance at all temperatures and was never detected to cross the center of the lipid bilayer. The translocation rates of Trp-15, Trp-57, Trp-102, and Trp-143 which are each located in different outer loop regions of the four β-hairpins of the eight-stranded β-barrel of OmpA were very similar to one another. This result and the common distances of these Trps from the membrane center observed in the third membrane-bound folding intermediate provide strong evidence for a synchronous translocation of all four β-hairpins of OmpA across the lipid bilayer and suggest that OmpA inserts and folds into lipid bilayers by a concerted mechanism.

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“…The thickness of the hydrophobic region was approximated to be ϳ29 Å (27). For the calculation of immersion depth, the data collected for the 0.4 molar fraction quencher were used.…”

Section: Methodsmentioning

confidence: 99%

Insertion of the Membrane-proximal Region of the Neuronal SNARE Coiled Coil into the Membrane

Kweon¹,

Kim²,

Shin

2003

Journal of Biological Chemistry

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In the neuron, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins assemble into an ␣-helical coiled coil that bridges the synaptic vesicle to the plasma membrane and drives membrane fusion, a required process for neurotransmitter release at the nerve terminal. How does coiled coil formation drive membrane fusion? To investigate the structural and energetic coupling between the coiled coil and membrane, the recombinant SNARE complex in the phospholipid bilayer was studied using fluorescence quenching and site-directed spin labeling EPR. Fluorescence analysis revealed that two native Trp residues at the membrane-proximal region of the coiled coil are inserted into the membrane, tightly coupling the coiled coil to the membrane. The EPR results indicate that the coiled coil penetrates into the membrane with an oblique angle, providing a favorable geometry for the basic residues to interact with negatively charged lipids. The result supports the proposition that core complex formation directly leads to the apposition of two membranes, which could facilitate lipid mixing. Trp residues and basic residues are abundant at the membrane-proximal region of transmembrane SNARE proteins, suggesting the generality of the proposed mechanism for the SNARE complex-membrane coupling.

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Determination of the depth of bromine atoms in bilayers formed from bromolipid probes

Cited by 186 publications

References 38 publications

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

Evidence for the Regulatory Role of the N-terminal Helix of Secretory Phospholipase A2 from Studies on Native and Chimeric Proteins

Outer Membrane Protein A of Escherichia coli Inserts and Folds into Lipid Bilayers by a Concerted Mechanism

Insertion of the Membrane-proximal Region of the Neuronal SNARE Coiled Coil into the Membrane

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