Examination of EmrE conformational differences in various membrane mimetic environments

2003 ◽  
Vol 81 (2) ◽  
pp. 61-70 ◽  
Author(s):  
Sandra L Federkeil ◽  
Tara L Winstone ◽  
Glen Jickling ◽  
Raymond J Turner
Keyword(s):  
Multidrug Resistance ◽  
Structural Information ◽  
Sodium Dodecyl ◽  
Tryptophan Fluorescence ◽  
Acrylamide Quenching ◽  
Small Multidrug Resistance ◽  
Membrane Mimetic ◽  
Tryptophan Residues ◽  
Helical Content ◽  
Lipid Environment

Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance in Escherichia coli to a diverse group of lipophilic cations. Research is beginning to elucidate structural information as well as substrate binding and extrusion mechanisms for this protein. However, the choice of membrane mimetic environment to perform structural studies needs to be made. In this study EmrE was solubilized in different membrane mimetic environments to investigate the influence of environment on the structure and dynamics of the protein by comparing the fluorescence properties of emission maxima, peak shifts, relative intensities, acrylamide quenching constants, and polarization. Taken together, the different fluorescence observations on EmrE in the various membrane mimetic systems tested suggest that the tryptophan residues in EmrE are present in the most flexible and exposed state when solubilized in methanol, followed by sodium dodecyl sulfate and urea. The two detergents N-dodecyl-β-D-maltoside (DM) and polyoxyethylene(8)dodecyl ether, for the most part, only display subtle differences between the spectral properties with DM best representing the lipid environment. The conformation of EmrE is clearly more open and dynamic in detergent relative to being reconstituted in small unilamellar vesicles. The fluorescence observations of EmrE solubilized in trifluoroethanol shows an environment that is similar to that of EmrE solubilized in detergents. Additionally, secondary structure was monitored by circular dichroism (CD). The CD spectra were similar among the different solubilizing conditions, suggesting little difference in α-helical content. This work establishes groundwork for the choice of solubilizing conditions for future structural, folding, and ligand binding studies.Key words: SMR, EmrE, tryptophan fluorescence, membrane proteins, detergent solubilization, membrane mimetic.

scholarly journals Crystal structures of bacterial Small Multidrug Resistance transporter EmrE in complex with structurally diverse substrates

2022 ◽  
Author(s):  
Ali A Kermani ◽  
Olive E. Burata ◽  
B Ben Koff ◽  
Akiko Koide ◽  
Shohei Koide ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Binding Site ◽  
Quaternary Ammonium ◽  
Structural Information ◽  
Structural Description ◽  
Small Multidrug Resistance ◽  
Novel Approach ◽  
Backbone Structure ◽  
Multidrug Resistance Transporter ◽  
Ammonium Compounds

Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

scholarly journals Overlapping Substrate Specificities in the Small Multidrug Resistance (SMR) Family of Transporters

2020 ◽  
Vol 118 (3) ◽  
pp. 130a
Author(s):  
Christian B. Macdonald ◽  
Ali A. Kermani ◽  
Randy B. Stockbridge
Keyword(s):  
Multidrug Resistance ◽  
Substrate Specificities ◽  
Small Multidrug Resistance

The interaction of bovine milk caseins with the detergent sodium dodecyl sulphate. II. The effect of detergent binding on spectral properties of caseins

1970 ◽  
Vol 37 (2) ◽  
pp. 259-267 ◽  
Author(s):  
G. C. Cheeseman ◽  
Dorothy J. Knight
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Difference Spectrum ◽  
Temperature Dependent ◽  
Difference Spectra ◽  
Negative Change ◽  
Tryptophan Residues ◽  
Hydrophobic Binding ◽  
The Difference

SummaryThe dissociation of casein aggregates by the detergent sodium dodecyl sulphate (SDS) gave rise to difference spectra and these spectra were characteristic for each of the different types of casein. Increase in absorption by the chromophore groups, tyrosine and tryptophan, when αs1- and β-casein aggregates were dissociated indicated binding of the detergent at regions of the molecule containing these residues. A decrease in absorption when κ-casein was dissociated indicated that the tyrosine and tryptophan residues were not in the region of the molecule to which the detergent was bound and that in the κ-casein aggregate these residues were in a more hydrophobic environment. Peaks on the difference spectra were obtained at 280 and 288 nm for αs1-casein and 284 and 291 nm for β-casein and troughs at 278 and 286 nm for κ-casein. The difference spectrum reached a maximum value when the αsl- and β-casein aggregates were dissociated and the further binding of SDS did not alter this value. The large negative change in the difference spectrum of κ-casein did not occur until after most of the aggregates were dissociated and did not reach a maximum until binding with SDS was complete. The value obtained for ΔOD was found to be temperature-dependent for β-casein-SDS interaction, but not for αs1- and κ-casein. Changes in spectra were also observed when αs1- and κ-casein interacted to form aggregates. The data obtained confirmed the importance of hydrophobic binding in casein aggregate formation and indicated the possible involvement of tyrosine and tryptophan residues in this binding.

Fluorescent Properties of Firefly Luciferases and Their Complexes with Luciferin

2000 ◽  
Vol 20 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Ekaterina I. Dementieva ◽  
Elena A. Fedorchuk ◽  
Lubov Yu. Brovko ◽  
Alexander P. Savitskii ◽  
Natalya N. Ugarova
Keyword(s):  
Energy Transfer ◽  
Tryptophan Fluorescence ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Photinus Pyralis ◽  
Fluorescent Properties ◽  
Effective Energy Transfer ◽  
Tryptophan Residues ◽  
Luciola Mingrelica

Fluorescence of luciferases from Luciola mingrelica (single tryptophanresidue, Trp-419) and Photinus pyralis (two tryptophan residues, Trp-417,Trp-426) was studied. Analysis of quenching of tryptophan fluorescenceshowed that the tryptophan residue conserved in all luciferases is notaccessible for charged quenchers, which is explained by the presence ofpositively and negatively charged amino acid residues in the close vicinityto it. An effective energy transfer from tryptophan to luciferin wasobserved during quenching of tryptophan fluorescence of both luciferaseswith luciferin. From the data on the energy transfer, the distance betweenthe luciferin molecule and Trp-417 (419) in the luciferin–luciferasecomplex was calculated: 11–15 Å for P. pyralis and 12–17Å for L. mingrelica luciferases. The role of the conserved Trp residuein the catalysis is discussed.

Structure and chemical composition of insoluble filamentous components of sperm flagellar microtubules

1982 ◽  
Vol 58 (1) ◽  
pp. 1-22
Author(s):  
R.W. Linck ◽  
G.L. Langevin
Keyword(s):  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Resistant Material ◽  
Remarkable Feature ◽  
Electrophoretic Mobilities ◽  
Helical Content ◽  
Isoelectric Points ◽  
Flagellar Proteins ◽  
Final Fraction

By progressive solvent extraction, we have obtained a series of subfragments of flagellar microtubules. Mild treatment gives rise to ribbons that contain longitudinally arranged protofilaments. Further extraction leaves a distinctive residue containing thinner ribbons, of three and eventually two protofilaments. Finally, filaments 2–3 nm in diameter and fibrous ribbons apparently containing 6 or more 2 nm subfibrils are found. This latter solvent-resistant material is consistently enriched in a characteristic set of polypeptides, which are found in flagella of several different species, including echinoderms and a mollusc. These polypeptides appear different from alpha- and beta-tubulin on the basis of their solubilities, isoelectric points and electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels; these conclusions are reinforced by peptide mapping after limited proteolytic digestion, although the latter method reveals certain similarities between these unique flagellar proteins, tubulin, chicken gizzard desmin and rabbit actin. A remarkable feature of the protein in the final fraction is the high alpha-helical content: 71% as measured by circular dichroism. We consider the possible origins of these filaments in the microtubule, in particular the possibility that microtubule protofilaments are heterogeneous in protein composition, and we discuss some of the implications of our findings.

scholarly journals Aggregation equilibria of tyrosinase of Harding-Passey mouse melanoma

1985 ◽  
Vol 228 (1) ◽  
pp. 95-101 ◽  
Author(s):  
J C Garcia-Borron ◽  
F Solano ◽  
J L Iborra ◽  
J A Lozano
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Aqueous Environment ◽  
Fluorescence Studies ◽  
Mouse Melanoma ◽  
Low Protein ◽  
Tryptophan Residues ◽  
Sample Dilution

The purification of two isoenzymes of tyrosinase has been carried out in Harding-Passey mouse melanoma. One is found in the cytosol and the other one bound to melanosomes. Both migrate as single bands on sodium dodecyl sulphate/polyacrylamide gels, having an apparent Mr of 58 000. Solubilized particulate tyrosinase showed an aggregation equilibrium involving a monomer, tetramer, octamer and a high-Mr micellar form with Brij 35, the solubilizing agent. H.p.l.c. studies indicated a interconversion between those species, the monomer contribution increasing with the sample dilution. The tetramer and the octamer probably represent the predominant forms in vivo. Soluble tyrosinase showed a simpler aggregation equilibrium, involving two forms, monomer and tetramer, with the same interconversion pattern. Fluorescence studies suggested that tryptophan residues were exposed to the aqueous environment when tyrosinase was dissociated by dilution. Tyrosinase shows a tendency to aggregate, at low protein concentration, and a resistance to dissociation by urea or SDS so remarkable that gel-permeation chromatography in 4M-urea does not affect the equilibrium, and the band obtained on SDS/polyacrylamide-gel electrophoresis is a dimer.

scholarly journals Selective Hydrolysis of Transferrin Promoted by Zr-Substituted Polyoxometalates

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3472
Author(s):  
Laura S. Van Rompuy ◽  
Nada D. Savić ◽  
Alvaro Rodriguez ◽  
Tatjana N. Parac-Vogt
Keyword(s):  
Nmr Spectroscopy ◽  
31P Nmr ◽  
Sodium Dodecyl ◽  
Tryptophan Fluorescence ◽  
31P Nmr Spectroscopy ◽  
Selective Hydrolysis ◽  
Buffer Solution ◽  
Structure Changes ◽  
Complementary Techniques ◽  
Hydrolysis Of

The hydrolysis of the iron-binding blood plasma glycoprotein transferrin (Tf) has been examined at pH = 7.4 in the presence of a series of Zr-substituted polyoxometalates (Zr-POMs) including Keggin (Et2NH2)10[Zr(PW11O39)2]∙7H2O (Zr-K 1:2), (Et2NH2)8[{α-PW11O39Zr-(μ-OH) (H2O)}2]∙7H2O (Zr-K 2:2), Wells-Dawson K15H[Zr(α2-P2W17O61)2]·25H2O (Zr-WD 1:2), Na14[Zr4(α-P2W16O59)2(μ3-O)2(μ-OH)2(H2O)4]·57H2O (Zr-WD 4:2) and Lindqvist (Me4N)2[ZrW5O18(H2O)3] (Zr-L 1:1), (nBu4N)6[(ZrW5O18(μ–OH))2]∙2H2O (Zr-L 2:2)) type POMs. Incubation of transferrin with Zr-POMs resulted in formation of 13 polypeptide fragments that were observed on sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), but the hydrolysis efficiency varied depending on the nature of Zr-POMs. Molecular interactions between Zr-POMs and transferrin were investigated by using a range of complementary techniques such as tryptophan fluorescence, circular dichroism (CD), 31P-NMR spectroscopy, in order to gain better understanding of different efficiency of investigated Zr-POMs. A tryptophan fluorescence quenching study revealed that the most reactive Zr-WD species show the strongest interaction toward transferrin. The CD results demonstrated that interaction of Zr-POMs and transferrin in buffer solution result in significant secondary structure changes. The speciation of Zr-POMs has been followed by 31P-NMR spectroscopy in the presence and absence of transferrin, providing insight into stability of the catalysts under reaction condition.

scholarly journals The conformational changes of α2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes

1987 ◽  
Vol 243 (1) ◽  
pp. 47-54 ◽  
Author(s):  
L J Larsson ◽  
P Lindahl ◽  
C Hallén-Sandgren ◽  
I Björk
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Cross Linking ◽  
Bait Region ◽  
Close Proximity ◽  
Tryptophan Residues ◽  
Thioester Bond ◽  
Bond Region

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent ‘bait’ region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

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