共查询到20条相似文献,搜索用时 15 毫秒
1.
H Suga K Shirabe T Yamamoto M Tasumi M Umeda C Nishimura A Nakazawa M Nakanishi Y Arata 《The Journal of biological chemistry》1991,266(21):13537-13543
Structural changes upon binding to the membrane of a COOH-terminal channel-forming thermolytic fragment of colicin E1 have been studied by means of a variety of spectroscopic techniques. Circular dichroism measurements show that the thermolytic fragment predominantly takes a helical structure in aqueous and detergent solutions. Fourier transform infrared spectroscopic measurements indicate that the content of the beta-structure is significantly increased when the thermolytic fragment is bound to vesicles. On the basis of the result of tryptophan fluorescence measurements, we have concluded that each of the three tryptophan residues of the thermolytic fragment exists in different environments, i.e. one is buried in the lipid bilayer, one exists on the cis side of the vesicles, and one exists near the surface of the lipid bilayer. The Fourier transform infrared and fluorescence data have been used along with the crystal structure of colicin A, which is highly homologous to colicin E1 in structure and function, to propose a model of the thermolytic fragment bound to the lipid vesicles. 相似文献
2.
Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes 总被引:2,自引:0,他引:2
J A Killian R C Keller M Struyvé A I de Kroon J Tommassen B de Kruijff 《Biochemistry》1990,29(35):8131-8137
The interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with different phospholipid vesicles was investigated by fluorescence techniques, using a synthetic mutant signal peptide in which valine at position -8 in the hydrophobic sequence was replaced by tryptophan. First it was established that this mutation in the signal sequence of prePhoE does not affect in vivo and in vitro translocation efficiency and that the biophysical properties of the synthetic mutant signal peptide are similar to those of the wild-type signal peptide. Next, fluorescence experiments were performed which showed an increase in quantum yield and a blue shift of the emission wavelength maximum upon interaction of the signal peptide with lipid vesicles, indicating that the tryptophan moiety enters a more hydrophobic environment. These changes in intrinsic fluorescence were found to be more pronounced in the presence of phosphatidylglycerol (PG) or cardiolipin (CL) than with phosphatidylcholine (PC). In addition, quenching experiments demonstrated a shielding of the tryptophan fluorescence from quenching by the aqueous quenchers iodide and acrylamide upon interaction of the signal peptide with lipid vesicles, a shielding in the case of acrylamide that was more pronounced in the presence of negatively charged lipids. Finally it was found that acyl chain brominated lipids incorporated into phospholipid bilayers were able to quench the tryptophan fluorescence of the signal peptide, with the quenching efficiency in CL vesicles being much higher than in PC vesicles. The results clearly demonstrate that the PhoE signal peptide interacts strongly with different lipid vesicles.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
Fluorescence energy transfer distance measurements using site-directed single cysteine mutants. The membrane insertion of colicin A 总被引:6,自引:0,他引:6
The ion-channel-forming C-terminal fragment of colicin A binds to negatively charged lipid vesicles and provides an example of insertion of a soluble protein into a lipid bilayer. The soluble structure is known from X-ray crystallography and consists of a ten-helix bundle containing a hydrophobic helical hairpin. In this work fluorescence spectroscopy was used to study the membrane-bound structure. An extrinsic probe, N'-(iodoacetyl)-N'-(5-sulfol-naphthyl)ethylenediamine (IAEDANS) was attached to mutant proteins each of which bears a unique cysteine residue. Three mutants K39C (helix 2), T127C (between helices 6 and 7) and S16Crpt (helix 1, which bears a decapeptide repeat before the mutation) gave useful derivatives. In the soluble protein they showed emission wavelengths decreasing in the order K39C greater than T127C greater than S16Crpt and although all showed blue shifts on addition of dimyristoylphosphatidylglycerol (DMPG) this order was maintained in the membrane-bound state. These shifts were not indicative of deep membrane insertion. Polarization of IAEDANS revealed differences in mobility between mutants. The three tryptophan residues were used as a compound donor to IAEDANS in resonance energy transfer distance determinations. The values obtained for the soluble form were 1.2 A to 3.2 A longer than in the crystal structure. On addition of lipids the indicated distances increased: S16Crpt-I(AEDANS) 6.45 A (22%), K39C-I 5.45 A (18%) and T127C-I 2.4 A (14%). N-bromosuccinimide (NBS) completely abolishes the tryptophan emission from the thermolytic fragment. When lipids were added to a mixture containing ten NBS-treated channel-forming fragments to one IAEDANS labelled fragment the indicated distances increased rather more: S16Crpt-I 9.7 A (38%), K39C-I 8.1 A (36%) and T127C-I 2.5 A (16%). This showed that intermolecular transfer reduces the distance estimated in samples containing only labelled protein. The ensemble of results shows that the amphipathic helices of the C-terminal fragment open out on the surface of the lipid bilayer during the initial phase of membrane insertion. 相似文献
4.
The membrane insertion of urea-denatured colicin E1 was studied by using fluorescence spectroscopy, circular dichroism and monolayer techniques. The results showed that the denatured colicin E1 taking mainly the 'random coil' conformation may recover its orderliness to a certain extent under the induction of the phospholipid membrane and insert spontaneously into phospholipid membrane, indicating that unfolding of colicin E1 does not inhibit its membrane insertion. Among the four tryptophan residues of the membrane-bound colicin E1 molecules, at least two were accessible by the quenchers, i.e. not inserted into the membranes. Although urea-denatured colicin E1 interacted preferentially with negatively charged phospholipids, it seems less dependent on the negatively charged lipid than colicin A. The addition of urea increased the speed of the adsorption of colicin E1 to the membrane, but did not affect obviously its membrane insertion ability. 相似文献
5.
Site-site interaction in the phospholipid activation of D-beta-hydroxybutyrate dehydrogenase 总被引:1,自引:0,他引:1
D-beta-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with absolute specificity for phosphatidylcholine (PC). The enzyme devoid of lipid, the apodehydrogenase, inserts spontaneously into phospholipid vesicles where it exists as a tetramer. We now find the lipid activation to be limited by the mole fraction of PC in the total phospholipid. These studies suggest that the concentration of the enzyme-PC complex, which is essential for enzymic activity, becomes diffusion limited at lower PC concentration. The lipid activation and the tryptophan fluorescence of purified D-beta-hydroxybutyrate dehydrogenase were studied in the presence of a constant "bilayer background" of approximately 100 nonactivating phospholipid molecules/enzyme monomer. Activation by PC was half-maximal at 20 PC molecules/enzyme monomer. This value was doubled when the amount of "background" phospholipid was doubled. Activation proceeded with positive cooperativity having a Hill coefficient of approximately 2.4. These data indicate interactions between at least three PC-binding sites. The quenching of tryptophan fluorescence by the phospholipid activator, 1-palmitoyl-2-(1-pyrenyl)-decanoyl-PC (2-pyrenyl-PC), gives a saturation curve with half-maximal quenching of 6 quencher molecules/enzyme monomer. This value is equivalent to an apparent phospholipid-protein dissociation constant in the two-dimensional membrane and corresponds to approximately 6 mol % of total phospholipid. In distinct contrast to the phospholipid activation curve, the fluorescence quenching saturation curve was hyperbolic and there was no specificity for PC. The fluorescence quenching by 2-pyrenyl-PC could be diminished by using a several-fold excess of PC or other phospholipids so as to reduce the mole fraction of quencher in the bilayer. It would appear that formation of enzyme-PC complex is a dynamic process consisting of at least two discernible steps: 1) a primary interaction, as measured by tryptophan quenching, which is hyperbolic and not specific for lecithin. This interaction is independent from and precedes 2) phospholipid activation of D-beta-hydroxybutyrate dehydrogenase, which is cooperative in nature and specific for lecithin. 相似文献
6.
多肽及蛋白质的插膜机制是目前分子生物学、细胞生物学研究中十分活跃的领域之一。本文通过荧光、圆二色等波谱学技术,深入地探讨了处于不同构象状态的毒素蛋白分子与磷脂膜作用后的构象变化。结果表明:带负电荷的磷脂膜对处于不同构象状态的ColicinE1分子的二级结构有较强的诱导作用;这种作用是电荷依赖性的。处于不同构象状态的毒素蛋白分子在磷脂膜的诱导下均可不同程度恢复其天然状态下插膜时的构象。不同磷脂对ColicinE1分子诱导的强弱依次为DMPG>DMPE>DMPC。ColicinE1分子与磷脂膜的结合是紧密的,结合后的蛋白质有较强的抗变性能力。 相似文献
7.
2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
The changes in intrinsic spectral properties of protein kinase C were monitored upon association with its divalent cation and lipid activators in a model membrane system. The enzyme demonstrated changes in both its intrinsic fluorescence and far ultraviolet circular dichroism spectra upon association with lipid vesicles in the absence of calcium. The acidic phospholipid, phosphatidylserine, significantly quenched the intrinsic tryptophan fluorescence and was also the most potent lipid support for the phosphorylating activity of the enzyme. The enzyme was fully activated by a number of Ca2(+)-lipid combinations which correlated with maximal fluorescence quenching (40-50%) of available tryptophan residues in hydrophobic domains. The circular dichroism structure of the associated active-protein Ca2(+)-lipid complexes suggested different active enzyme secondary structures. However, the Ca2(+)-dependent changes in fluorescence and circular dichroism spectra were observed only after the enzyme associated with the lipid vesicles. These data suggest that protein kinase C has the properties of a complex multidomain protein and provides an additional perspective into the mechanism of protein kinase C activation. 相似文献
9.
Interaction of the precursor to mitochondrial aspartate aminotransferase and its presequence peptide with model membranes 总被引:1,自引:0,他引:1
Donate F Yañez AJ Iriarte A Martinez-Carrion M 《The Journal of biological chemistry》2000,275(44):34147-34156
The possible contribution of the mature portion of a mitochondrial precursor protein to its interaction with membrane lipids is unclear. To address this issue, we examined the interaction of the precursor to mitochondrial aspartate aminotransferase (pmAAT) and of a synthetic peptide corresponding to the 29-residue presequence peptide (mAAT-pp) with anionic phospholipid vesicles. The affinity of mAAT-pp and pmAAT for anionic vesicles is nearly identical. Results obtained by analyzing the effect of mAAT-pp or full-length pmAAT on either the permeability or microviscosity of the phospholipid vesicles are consistent with only a shallow insertion of the presequence peptide in the bilayer. Analysis of the quenching of Trp-17 fluorescence by brominated phospholipids reveals that this presequence residue inserts to a depth of approximately 9 A from the center of the bilayer. Furthermore, in membrane-bound pmAAT or mAAT-pp, both Arg-8 and Arg-28 are accessible to the solvent. These results suggest that the presequence segment lies close to the surface of the membrane and that the mature portion of the precursor protein has little effect on the affinity or mode of binding of the presequence to model membranes. In the presence of vesicles, mAAT-pp adopts considerable alpha-helical structure. Hydrolysis by trypsin after Arg-8 results in the dissociation of the remaining 21-residue C-terminal peptide fragment from the membrane bilayer, suggesting that the N-terminal portion of the presequence is essential for membrane binding. Based on these results, we propose that the presequence peptide may contain dual recognition elements for both the lipid and import receptor components of the mitochondrial membrane. 相似文献
10.
Le Rumeur E Fichou Y Pottier S Gaboriau F Rondeau-Mouro C Vincent M Gallay J Bondon A 《The Journal of biological chemistry》2003,278(8):5993-6001
Dystrophin is assumed to act via the central rod domain as a flexible linker between the amino-terminal actin binding domain and carboxyl-terminal proteins associated with the membrane. The rod domain is made up of 24 spectrin-like repeats and has been shown to modify the physical properties of lipid membranes. The nature of this association still remains unclear. Tryptophan residues tend to cluster at or near to the water-lipid interface of the membrane. To assess dystrophin rod domain-membrane interactions, tryptophan residues properties of two recombinant proteins of the rod domain were examined by (1)H NMR and fluorescence techniques in the presence of membrane lipids. F114 (residues 439-553) is a partly folded protein as inferred from (1)H NMR, tryptophan fluorescence emission intensity, and the excited state lifetime. By contrast, F125 (residues 439-564) is a folded compact protein. Tryptophan fluorescence quenching shows that both proteins are characterized by structural fluctuations with their tryptophan residues only slightly buried from the surface. In the presence of negatively charged small vesicles, the fluorescence characteristics of F125 change dramatically, indicating that tryptophan residues are in a more hydrophobic environment. Interestingly, these modifications are not observed with F114. Fluorescence quenching experiments confirm that tryptophan residues are shielded from the solvent in the complex F125 lipids by a close contact with lipids. The use of membrane-bound quenchers allowed us to conclude that dystrophin rod domain lies along the membrane surface and may be involved in a structural array comprising membrane and cytoskeletal proteins as well as membrane lipids. 相似文献
11.
HIV-1 gp41N端融合肽与脂膜作用的荧光及单分子层膜研究 总被引:1,自引:1,他引:0
我们以前的实验证明HIV-1gp41N端23肽即融合肽HIVwt能促融合,但它的突变体HIVGlu(2位V→E)却不能诱发融合。为了进一步研究多肽结构与功能的关系,我们用荧光及单分子层膜技术研究HIVwt及HIVGlu与脂的相互作用。荧光淬灭实验表明HIVwt插入带负电磷脂并插入较深;而HIVGlu虽然能与带负电脂结合但并不插膜。单层膜实验进一步证实了上述结果:HIVwt对带负电磷脂POPG的临界插膜压达到43mN/m,而HIVGlu的临界插膜压只有31mN/m。这提示HIVwt与单层膜不仅存在静电作用还有较强的疏水作用。结合上述实验结果推测HIVwt能插入脂膜的酰基链因而容易促发融合;而HIVGlu插膜很浅或根本不能入膜从而不能促融合 相似文献
12.
In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Intrinsic fluorescence of a hydrophobic myelin protein and some complexes with phospholipids 总被引:4,自引:0,他引:4
The fluorescence characteristics of lipophilin, a proteolipid apoprotein from human myelin, were determined in aqueous and lipid environments. In all cases the tryptophan residues were located in buried hydrophobic sites of uniform, but limited, accessibility to the permeant quenching agent acrylamide; only in the helicogenic solvent 2-chloroethanol were the protein fluorophores exposed to the medium. Quantum yields were dependent on the state of aggregation of the protein in aqueous solution and increased considerably on treatment with lysolecithin micelles, or when the protein was combined with phosphatidylcholine by codialysis from 2-chloroethanol into water. Fluorescence titrations indicated that lipophilin bound to lysolecithin with an association constant greater than 10(6) L/mol. Radiationless singlet excitation energy transfer from tyrosine to tryptophan residues was found to decrease markedly when the protein was combined with lipids. When the protein was introduced into dimyristoylphosphatidylcholine vesicles, the tryptophan fluorescence did not detect any solid-liquid phase change. These results were consistent with strong hydrophobic interactions between lipophilin and phospholipids, which lead to conformational adjustments in the protein, and to establishment of an immobilized layer of boundary lipid in bilayer systems. 相似文献
14.
Hsp22/HspB8 is a member of the small heat-shock protein family, whose function is not yet completely understood. Our immunolocalization studies in a human neuroblastoma cell line, SK-N-SH, using confocal microscopy show that a significant fraction of Hsp22 is localized to the plasma membrane. We therefore investigated its interactions with lipid vesicles in vitro. Intrinsic tryptophan fluorescence is quenched in the presence of lipid vesicles derived from either bovine brain lipid extract or purified lipids. Time-resolved fluorescence studies show a decrease in the lifetimes of the tryptophan residues. Both of these results indicate burial of some tryptophan residues of Hsp22 upon interaction with lipid vesicles. Membrane interactions also lead to increase in fluorescence polarization of Hsp22. Gel-filtration chromatography shows that Hsp22 binds stably with lipid vesicles; the extent of binding depends on the nature of the lipid. Hsp22 binds more strongly to vesicles made of lipids containing a phosphatidic acid, phosphatidylinositol or phosphatidylserine headgroup (known to be present in the inner leaflet of plasma membrane) compared with lipid vesicles made of a phosphatidylcholine head-group alone. Far-UV CD spectra reveal conformational changes upon binding to the lipid vesicles or in membrane-mimetic solvent, trifluoroethanol. Thus our fluorescence, CD and gel-filtration studies show that Hsp22 interacts with membrane and this interaction leads to stable binding and conformational changes. The present study therefore clearly demonstrates that Hsp22 exhibits potential membrane interaction that may play an important role in its cellular functions. 相似文献
15.
J H Lakey D Massotte F Heitz J L Dasseux J F Faucon M W Parker F Pattus 《European journal of biochemistry》1991,196(3):599-607
In order to gain some insight into the mechanism of insertion into membranes of the pore-forming domain of colicin A and the structure of its membrane-bound form, circular dichroism (in the near and far ultraviolet), fluorescence and ultraviolet spectroscopy experiments were carried out. Because the structure of the water-soluble form of this fragment has been determined by X-ray crystallography, these spectroscopic methods provided valuable information on the secondary structure and the environment of aromatic residues within the two forms of the peptide. These results strongly suggest that the pore-forming domain of colicin A does not undergo drastic unfolding upon insertion into membrane. The conformational change associated with this process is triggered by the negatively charged lipids and probably consists of a reorientation of helix pairs with respect to each other. Exposure of the aromatic residues to the aqueous phase decreases on binding to lipids whilst the exposure of the tryptophans to the membrane phase increases. This cannot occur without a reorientation of helices 3-10. All data from this study support the model presented previously in which the known crystal structure opens like an 'umbrella' inserting the hydrophobic hairpin (helix 8-9) perpendicular to the membrane plane and the helical pair 1-2 and the domain containing the three tryptophans (helices 3-7) lying more or less parallel to the membrane plane. Lipids are bound more tightly to the protein at acidic pH than at neutral pH although a similar lipid protein complex is formed with 1,2-dimyristoyl-sn-glycero(3)-phospho(1)- -sn-glycerol at both pH values. 相似文献
16.
Liver FABP (fatty-acid-binding protein) binds a variety of non-polar anionic ligands including fatty acids, fatty acyl CoAs, lysophospholipids and bile acids. Liver FABP is also able to bind to anionic phospholipid vesicles under conditions of low ionic strength, and membrane binding results in the release of bound ligand. However, the molecular interactions involved in binding to the phospholipid interface and the mechanism of ligand release are not known. Ligand release could be due to a significant conformational change in the protein at the interface or interaction of a phospholipid molecule with the ligand-binding cavity of the protein resulting in ligand displacement. Two portal mutant proteins of liver FABP, L28W and M74W, have now been used to investigate the binding of liver FABP to anionic phospholipid vesicles, monitoring changes in fluorescence and also fluorescence quenching in the presence of brominated lipids. There is a large increase in fluorescence intensity when the L28W mutant protein binds to vesicles prepared from DOPG (dioleoyl-sn-phosphatidylglycerol), but a large decrease in fluorescence intensity when the M74W mutant binds to these vesicles. The Br(4)-phospholipid prepared by bromination of DOPG dramatically quenches both L28W and M74W, consistent with the close proximity of a fatty acyl chain to the tryptophan residues. The binding of liver FABP to DOPG vesicles is accompanied by only a minimal change in the CD spectrum. Overall, the results are consistent with a molecule of anionic phospholipid interacting with the central cavity of the liver FABP, possibly involving the phospholipid molecule in an extended conformation. 相似文献
17.
18.
Background
Protein transport across cellular membranes is an important aspect of toxin biology. Escherichia coli cell killing by nuclease colicins occurs through DNA (DNases) or RNA (RNases) hydrolysis and to this end their cytotoxic domains require transportation across two sets of membranes. In order to begin to unravel the molecular mechanisms underlying the membrane translocation of colicin nuclease domains, we have analysed the membrane association of four DNase domains (E9, a charge reduction E9 mutant, E8, and E7) and one ribosomal RNase domain (E3) using a biomembrane model system.Principal Results
We demonstrate, through the use of large unilamellar vesicles composed of synthetic and E. coli lipids and a membrane surface potential sensor, that the colicin nuclease domains bind anionic membranes only, with micromolar affinity and via a cooperative binding mechanism. The evaluation of the nuclease bilayer insertion depth, through a fluorescence quenching analysis using brominated lipids, indicates that the nucleases locate to differential regions in the bilayer. Colicin DNases target the interfacial region of the lipid bilayer, with the DNase E7 showing the deepest insertion, whereas the ribosomal RNase E3 penetrates into the hydrophobic core region of the bilayer. Furthermore, the membrane association of the DNase E7 and the ribosomal RNase E3 induces vesicle aggregation, lipid mixing and content leakage to a much larger extent than that of the other DNases analysed.Conclusions/Significance
Our results show, for the first time, that after the initial electrostatically driven membrane association, the pleiotropic membrane effects induced by colicin nuclease domains relate to their bilayer insertion depth and may be linked to their in vivo membrane translocation. 相似文献19.
Harrington A Darboe N Kenjale R Picking WL Middaugh CR Birket S Picking WD 《Biochemistry》2006,45(2):626-636
Shigella flexneri causes dysentery after invading the epithelial cells of the human colon. Enterocyte invasion is induced by the bacterial effector IpaC (invasion plasmid antigen C), which triggers Shigella entry into epithelial cells by a rather poorly understood mechanism. IpaC is also involved in pathogen escape into the host cell cytoplasm following uptake, and this property may be reflected in its ability to disrupt phospholipid vesicles in vitro. Purified recombinant IpaC interacts with liposome vesicles to cause the release of small molecules trapped inside. This interaction requires that the liposomes possess an acidic phospholipid component. To better understand the events involved in the disruption of liposomes by IpaC, single tryptophan mutants were generated to permit the use of intrinsic fluorescence, circular dichroism, and ultraviolet absorption spectroscopies to examine the effect that phospholipid membrane association has on IpaC structure and stability. These mutants were also used to determine how amino acid substitutions within specific regions of IpaC influence its activity in vivo. The outcomes of this study include findings that cholesterol greatly impacts IpaC association with phospholipid membranes, tryptophan incorporation into specific regions of IpaC (especially near the C-terminus) can greatly impact its in vivo activity, and interaction with phospholipid membranes causes differing degrees of change in the fluorescence of tryptophan residues introduced at specific sites within IpaC. These data, together with fluorescence quenching analyses, provide new functional and structural information concerning IpaC and its insertion into phospholipid membranes. 相似文献
20.
The molecular aggregate size of the closed state of the colicin E1 channel was determined by fluorescence resonance energy transfer experiments involving a fluorescence donor (three tryptophans, wild-type protein) and a fluorescence acceptor (5-(((acetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (AEDANS), Trp-deficient protein). There was no evidence of energy transfer between the donor and acceptor species when bound to membrane large unilamellar vesicles. These experiments led to the conclusion that the colicin E1 channel is monomeric in the membrane-bound closed channel state. Experiments were also conducted to study the membrane topology of the closed colicin channel in membrane large unilamellar vesicles using acrylamide as the membrane-impermeant, nonionic quencher of tryptophan fluorescence in a battery of single tryptophan mutant proteins. Furthermore, additional fluorescence parameters, including fluorescence emission maximum, fluorescence quantum yield, and fluorescence decay times, were used to assist in mapping the topology of the closed channel. Results suggest that the closed channel comprises most of the polypeptide of the channel domain and that the hydrophobic anchor domain does not transverse the membrane bilayer but nonetheless is deeply embedded within the hydrocarbon core of the membrane. Finally, a model is proposed which features at least two states that are in rapid equilibrium with each other and in which one state is more heavily populated than the other. 相似文献