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E London

Publications and source records attributed to E London.

At least 37 records · Page 2Linked to original sources
PubMed1998

The location of fluorescence probes with charged groups in model membranes.

The location of commonly used charged fluorescent membrane probes in membranes was determined in order to: (1) investigate the relationship between the structure of hydrophobic molecules and their depth within membranes; and (2) aid interpretation of experiments in which these fluorescent probes are used to examine membrane structure. Membrane depth was calculated using parallax analysis, a method in which the quenching induced by lipids carrying a nitroxide group at different locations in the membrane is compared. Shallow locations were found for xanthene dyes (fluorescein, eosin, Texas Red and rhodamine) both in free form and when attached either to the headgroup of phospholipids or long hydrocarbon chains. The exact structure of the xanthene and the nature of its linkage to lipid had only a modest effect on membrane location, which ranged between 19 and 24 A from the center of the bilayer in a charged state. Thus, the location of these fluorophores largely reflects their intrinsic properties rather than the nature of the groups to which they are attached. Furthermore, cationic and anionic xanthene derivatives had similar depths, indicating the type of charge does not have a large effect on depth. Consistent with this conclusion, shallow locations were also found for other hydrocarbon chain-linked cationic (acridine orange and styrylpyridinium) and anionic (coumarin, anilinonaphthalenesulfonic acid (ANS), and toluidinylnaphthalenesulfonic acid (TNS)) charged probes. These all located at 16-18 A from the bilayer center. We conclude that both anionic and cationic molecules that are otherwise hydrophobic predominantly occupy shallow locations within the polar headgroup region of the bilayer no matter how hydrophobic the molecule to which they are linked. This depth is significantly shallower than that occupied by most previously studied uncharged polar molecules that locate near the membrane surface. Consistent with this conclusion, a 2-4 A deeper location was found for xanthene probes with no net charge. In other experiments, methods to avoid chemical reactions that can distort the measurement of depth by fluorescence quenching were developed.

Binding Sites↗
PubMed1998

Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain.

The membrane topography of proteins that convert between soluble and membrane-inserted states has proven a challenging problem. In particular, it has been difficult to define both whether a transmembrane orientation is achieved and what are the boundaries of membrane-inserted segments. In this report the fluorescence of bimane-labeled Cys residues and the binding of anti-BODIPY antibodies to BODIPY-labeled Cys residues are combined to define these features for helices TH8 and TH9 of the T domain of diphtheria toxin. Using a series of labeled residues the topography of these helices was examined in both conformations of membrane-inserted T domain identified previously (Wang, Y., Malenbaum, S. E., Kachel, K., Zhan, H., Collier, R. J., and London, E. (1997) J. Biol. Chem. 272, 25091-25098). In the shallowly inserted conformation these helices are found to be aligned close to the cis surface of the bilayer all along their sequences. In contrast, in the more deeply inserted conformation most TH8 and TH9 residues examined located in a non-polar environment, with the boundaries of the membrane-inserted sequences close to residues 324 and 372-374 on the cis (insertion) side of the bilayer. It was also found that residues 348 and 349, which are in the loop connecting TH8 and TH9, reached the opposite trans side of the bilayer, but did not protrude fully into the aqueous environment. These boundaries suggest the membrane-inserted segments of TH8 and TH9 form transmembrane helices about 25 residues in length, and suggest that they are connected by a tight turn. It is concluded that this combination of fluorescent techniques can be combined to obtain transmembrane helix topography.

Boron Compounds↗
PubMed1998

Location of diphenylhexatriene (DPH) and its derivatives within membranes: comparison of different fluorescence quenching analyses of membrane depth.

The average membrane location of a series of diphenylhexatriene (DPH)-derived membrane probes was analyzed by measuring the quenching of DPH fluorescence with a series of nitroxide-labeled lipids in which the depth of the nitroxide group is varied. All DPH derivatives were located deeply within the bilayer. Some derivatives were anchored at a shallower depth than free DPH by attachment to cationic or anionic groups. However, the absolute change in DPH depth upon attachment to such groups was relatively modest (<4 A). In fact, protonated DPH fatty acid and a DPH fatty acyl group attached to a phosphatidylcholine were found to locate slightly more deeply than free DPH. The location of DPH derivatives can be explained by the length of the DPH group and its tendency to orient predominantly parallel to the fatty acyl chains of the bilayer. These factors allow a charged group attached to one end of a DPH molecule to be accommodated at the polar surface while maintaining a deep DPH location. Basically, it appears that most DPH derivatives probe the same region in the bilayer. We conclude previously reported differences in fluorescence polarization of free and anchored forms of DPH may reflect a direct effect of anchoring on motion rather than an effect on average DPH location. Other experiments showed the localization of DPH probes was found to be similar in the presence and absence of cholesterol. This implies that previously observed cholesterol-induced effects on DPH fluorescence polarization also largely reflect differences in DPH motion, not DPH location. From the quenching results it was also possible to define rules governing the location of a variety of chemical groups in membranes by comparison of the results obtained with DPH derivatives to those of similar derivatives of other fluorescent groups. Finally, an important goal of this study was to compare different methods of analysis of quenching data: parallax analysis, distribution (Gaussian) analysis (using a single Gaussian), and a second-order polynomial analysis. To evaluate the accuracy of these methods, the apparent depths of a series of fluorescence probes previously analyzed by parallax analysis was reanalyzed with all three methods. There was good agreement unless the fluorescent molecule was very shallow or very deep. In such cases, only parallax analysis gave physically reasonable results. This is likely to be due to the lack of a sufficient number of quenchers spanning a wide enough range for other analyses to compensate for deviations from ideal curves. Parallax analysis was also compared to distribution (Gaussian) analysis using a double Gaussian fit to account for quenching from the trans leaflet (Ladokhin, A. (1997) Methods Enzymol. 278, 462-473). Again more physically reasonable results were obtained from parallax analysis, likely due to non-Gaussian behavior of the depth dependence of quenching. Notwithstanding these observations, the significant number of cases where Gaussian curve fitting methods for quenching analysis are most powerful are discussed.

Cations↗
PubMed1998

Adrenal neuropeptide Y mRNA but not preproenkephalin mRNA induction by stress is impaired by aging in Fischer 344 rats.

Relatively few molecular markers of stress have been studied in aged individuals. Interactions of age and stress on adrenal neuropeptide Y (NPY) and preproenkephalin (ppENK) expression have not been reported. The purpose of these studies was to characterize the adrenal NPY and ppENK responses to stress using a common stressor, physical restraint for 2 h, in Fischer 344 rats at 7, 16 and 23 months of age. Northern blot techniques were used to evaluate induction by stress of adrenal NPY mRNA and adrenal ppENK mRNA. Two humoral responses to stress, serum glucose and corticosterone, were measured to corroborate that a stress response occurred. We observed that the induction by stress of adrenal NPY mRNA is impaired with age but the stress-induced elevation of adrenal ppENK mRNA, blood glucose, and corticosterone show no evidence of age-related impairments.

Adrenal Glands↗
PubMed1998

Groups with polar characteristics can locate at both shallow and deep locations in membranes: the behavior of dansyl and related probes.

To understand the relationship between the chemical structure of polar molecules and their membrane location, the behavior of dansyl (dimethylaminonaphthalenesulfonyl) and related polar fluorescent probes was examined. The depth of these probes in lipid bilayers was determined by parallax analysis of fluorescence quenching [Chattopadhyay and London (1987) Biochemistry 26, 39-45; Abrams & London, Biochemistry (1993) 32, 10826-10831]. Quenching was measured for dansyl groups: (1) attached to the polar headgroup of PE, (2) linked to an alkyl chain, (3) attached to the end of a fatty acyl chain, and (4) attached to the polar headgroup of PE via a spacer group. In all cases, the dansyl probes located in the polar headgroup region, 19-21 A from the bilayer center. This shows the dansyl group has a strong tendency to seek a shallow location in the polar headgroup region. The only exception to this pattern was in the case of a dialkylated dansyl, for which two populations were observed. One population was at the polar headgroup level, but the second was deeply buried in the acyl chain region. To see if the polar sulfonamide group of dansyl influences depth, a structurally related probe substituting a thiocarbamoyl linkage, dimethylaminonaphthalenethiocarbamoyl (dantyl)-labeled PE, was synthesized. Dantyl groups were located deeper than dansyl groups, 13-16 A from the bilayer center. There was an even more dramatic difference in depth between dansyl and mansyl (methylanilinonaphthalenesulfonyl) derivatives. Mansyl probes, which have an extra phenyl group relative to dansyl, were found to locate deeply within the acyl chain region of the bilayer (6-7 A from the bilayer center) when attached to the polar headgroup of PE. Thus, the membrane location of polar groups depends strongly on the details of their chemical structure, and it is possible for a polar group to locate both at shallow and deep locations. These results suggest the energy to bury a polar moiety in the hydrophobic part of the bilayer is not prohibitively high. This contrasts to the behavior of charged groups, which appear to be restricted to shallow locations in membranes. In this report, the effect of populations at two different depths on the parallax analysis is also considered.

Dansyl Compounds↗
PubMed1998

Cholesterol and sphingolipid enhance the Triton X-100 insolubility of glycosylphosphatidylinositol-anchored proteins by promoting the formation of detergent-insoluble ordered membrane domains.

Glycosylphosphatidylinositol (GPI)-anchored proteins can be isolated from both cells and sphingolipid and cholesterol-rich liposomes (SCRLs) in association with detergent-insoluble membranes. We found previously that detergent insolubility of lipids was characteristic of phases in which lipid acyl chains are ordered. We presented evidence that GPI-anchored proteins are insoluble because they associate with cholesterol and sphingolipid-rich lipid domains with properties similar to the liquid-ordered phase. Here, this model was tested by a variety of approaches. First, we demonstrated that saponin, which removes cholesterol from cell membranes and allows solubilization of GPI-anchored proteins by Triton X-100, had the same effect on the GPI-anchored protein alkaline phosphatase (PLAP) in SCRLs of appropriate lipid composition. The similarity of saponin action in cells and simple liposomes suggests that the compound disrupts protein-lipid interactions. However, direct interactions between PLAP and cholesterol were not needed for insolubility, because the protein was also insoluble in cholesterol-free liposomes containing lipid in an ordered phase. Instead, cholesterol acted by greatly enhancing the formation of a detergent-insoluble phase by sphingolipids, which have a tendency to form ordered phases. We propose that saponin solubilizes GPI-anchored proteins because the lipid composition of cell membranes (and the SCRLs used above) supports ordered phase formation in the presence but not the absence of cholesterol. Supporting this model, saponin did not promote Triton X-100 solubilization of PLAP in SCRLs with sphingolipid levels high enough to allow ordered phase formation in the absence of cholesterol. We also showed that two additional GPI-anchored proteins are detergent-insoluble in SCRLs and that detergent does not artifactually create ordered domains or cause components of solubilized membranes to associate with detergent-resistant membranes present in separate bilayers in the same lysate. We conclude that the ordered domain model explains the behavior of detergent-resistant membranes in liposomes and cells.

Animals↗
PubMed1998

Functions of lipid rafts in biological membranes.

Recent studies showing that detergent-resistant membrane fragments can be isolated from cells suggest that biological membranes are not always in a liquid-crystalline phase. Instead, sphingolipid and cholesterol-rich membranes such as plasma membranes appear to exist, at least partially, in the liquid-ordered phase or a phase with similar properties. Sphingolipid and cholesterol-rich domains may exist as phase-separated "rafts" in the membrane. We discuss the relationship between detergent-resistant membranes, rafts, caveolae, and low-density plasma membrane fragments. We also discuss possible functions of lipid rafts in membranes. Signal transduction through the high-affinity receptor for IgE on basophils, and possibly through related receptors on other hematopoietic cells, appears to be enhanced by association with rafts. Raft association may also aid in signaling through proteins anchored by glycosylphosphatidylinositol, particularly in hematopoietic cells and neurons. Rafts may also function in sorting and trafficking through the secretory and endocytic pathways.

Animals↗
PubMed1998

Membrane-inserted colicin E1 channel domain: a topological survey by fluorescence quenching suggests that model membrane thickness affects membrane penetration.

The topography of the closed-state membrane-associated, colicin E1 channel domain was examined using depth-dependent fluorescence quenching to determine the membrane location of various single Trp residues introduced into the sequence by site-directed mutagenesis. We have extended previous studies (Palmer, L. R., and Merrill, A. R. (1994) J. Biol. Chem. 269, 4187-4193) with additional single Trp residues in the helix 8/9 region, and with an additional quencher located in the polar region of the membrane to detect shallowly located Trp residues. Quenching data for seven single Trp mutants examined in the previous study, but without the shallow quencher, confirmed the previously reported depths. Mutants containing single Trp at residues 355, 460, or 507 were found to be more shallowly located than those at 404, 443, 484, or 495. In addition, analysis of fluorescence in the presence of the shallow quencher eliminated the possibility that there is a predominant population of these residues residing near the membrane-aqueous interface. The fluorescence quenching of three new single Trp at residues 478, 492, or 499 introduced into the channel domain was also evaluated. These residues were found at either medium or deep locations in the bilayer. Of special interest was the position of the Trp at residue 492 (W492), which is within the loop region connecting hydrophobic helices 8 and 9. If helices 8 and 9 were fully transmembraneous, then the predicted W492 location would have been shallow. Instead the quenching pattern demonstrated W492 to be deeply embedded in the lipid bilayer. We also studied the effect of altering bilayer width on protein conformation. Membrane width had little effect on most residues, but Trp at residues 478 and 507 were located more shallowly in thin bilayers. We also examined the effect of bilayer width on the position of Cys 505 labeled with bimane, an environmentally sensitive fluorophore. As the membrane width was decreased, C505-bimane shifted into a more nonpolar environment, as judged by fluorescence emission lambda max and quenching. Models for the conformation of helices 8/9 and the effect of membrane width on these helices are considered. We conclude that helices 8 and 9 probably do not adopt a fully transmembraneous state under the conditions examined in this report.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed1997

Use of Trp mutations to evaluate the conformational behavior and membrane insertion of A and B chains in whole diphtheria toxin.

The structure of diphtheria toxin was examined using its Trp fluorescence. To examine the interactions of the A and B chains of the toxin independently, mutants were constructed in which Trp residues were restricted to either the A or the B chain. The conformation and stability of the mutants were very similar to those of the wild-type protein. In addition, they underwent the low-pH conformational transition and membrane insertion at about the same pH as wild-type toxin. This shows Trp do not play a critical role in these processes which are necessary for the translocation of toxin across endosomal membranes in vivo. There was a shift in fluorescence of the Trp mutants which showed the low-pH-induced transition increases exposure of both the A and B Trp to a more polar environment. This supports a model in which the interdomain interactions present at neutral pH break down at low pH. To evaluate the location of the A and B chains in the membrane, the fluorescence quenching of model membrane inserted toxin was measured. Comparison of the amount of quenching by lipid labeled with nitroxides localized at shallow, medium, or deep depths within the bilayer demonstrated that both the A and B chains insert deeply, but the A chain Trp are somewhat less deeply inserted. Trp on the A chain are also less exposed to lipid than on the B chain, as judged by their weaker quenching by the nitroxide-labeled lipid. This conclusion was supported by the observation that the Trp of membrane-inserted isolated A chain is more lipid-exposed than when the A chain is part of the whole toxin. Both the A and B chain Trp become less exposed to lipid after neutralizing pH. However, both chains remain inserted, with at least part of the B chain remaining deeply inserted. These results support the "partial wrapper" model in which both the A and B chains are inserted but contacts between the two chains significantly reduce the exposure of the A chain to lipid.

Diphtheria Toxin↗
PubMed1997

Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes?

Detergent-resistant membrane domains (DRMs) can be isolated from a variety of eukaryotic cells. DRMs are of interest because of their potential importance in processes such as intracellular membrane sorting, and signal transduction at the cell surface. One type of DRM is also present in caveolae, non clathrin-coated plasma membrane pits with proposed roles in endocytosis, lipid transport, and signal transduction. Here we review recent advances in understanding the structure of these domains, and explore the possibility that DRMs are present in a phase separate from the surrounding bilayer. DRMs are rich in sphingolipids and cholesterol. The long saturated acyl chains and high acyl chain melting temperature of sphingolipids mediate their association in detergent resistant domains. These sphingolipid and cholesterol-rich domains have the properties of the liquid-ordered phase previously described in model membranes. Several lines of investigation support the idea that DRMs are not detergent-induced artifacts, but exist as domains in cell membranes. A striking feature of the proteins in DRMs is that many of them are linked to lipids. These include both GPI anchored proteins, and acylated proteins such as Src-family kinases. The linkage of these proteins to saturated acyl chains may help in targeting them to ordered membrane domains. Caveolin, the major structural protein of caveolae, is multiply palmitoylated. The presence of a high concentration of palmitate chains in DRMs in caveolae may help stabilize ordered domains.

Animals↗
PubMed1997

Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width.

The alpha-helix-rich, hydrophobic transmembrane (T) domain of diphtheria toxin is believed to play a central role in membrane insertion by the toxin and in the translocation of its catalytic domain across membranes. In this report, T domain structure was studied using site-directed single-Cys mutants. The residues chosen, 322 (near the amino-terminal end of helix TH8), 333 (within helix TH8), and 356 (within helix TH9) were substituted with Cys and labeled with the fluorescent probe bimane. (Residues 333 and 356 should be located within the bilayer in the transmembrane state, and residue 322 should not penetrate the bilayer.) After insertion of T domain into model membrane vesicles, the location of bimane label relative to the lipid bilayer was characterized by its fluorescence emission and by its quenching with nitroxide-labeled phospholipids. It was found that when the T domain is added to dioleoylphosphatidylcholine-containing vesicles, all three residues reside close to the outer surface. However, at high T domain concentration or in thinner dimyristoleoylphosphatidylcholine-containing vesicles, a large fraction of residues 333 and 356 penetrate deeply into the membrane. In contrast, residue 322 remains exposed to aqueous solution under these conditions. These conclusions were confirmed by a novel antibody binding method. Antibodies that quench the fluorescence of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene++ + (BODIPY) groups were used to evaluate the exposure of BODIPY-labeled 322, 333, and 356. Maximum exposure of residues 333 and 356 to externally added antibody was only observed under conditions in which bimane fluorescence showed that these residues do not penetrate the bilayer. In contrast, residue 322 remained exposed under all conditions. We propose that the deeply penetrating T domain conformation represents a transmembrane or near-transmembrane state. The regulation of the transmembrane/nontransmembrane equilibrium should be a key to understanding diphtheria toxin membrane insertion and translocation. Our results suggest that toxin-toxin interactions may play an important role in regulating this behavior.

Boron Compounds↗
PubMed1997

On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes.

Detergent-insoluble membrane fragments that are rich in sphingolipid and cholesterol can be isolated from both cell lysates and model membranes. We have proposed that these arise from membranes that are in the liquid-ordered phase both in vivo and in vitro [Schroeder et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130-12134]. In order to detect formation of the liquid-ordered phase while avoiding possible detergent artifacts, we have now used fluorescence quenching to examine the phase behavior of mixtures of phosphatidylcholines, sphingolipids, and cholesterol. Phase separation was found in binary mixtures of either dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) and a nitroxide-labeled phosphatidylcholine (12SLPC). A DPPC- or SM-enriched solidlike gel phase coexisted with a 12SLPC-enriched liquid-disordered fluid phase at 23 degrees C. As expected, phase separation was not seen at low concentrations of DPPC or SM. Instead, only a uniform fluid phase was present. Including 33 mol % cholesterol in model membranes greatly promoted phase separation. Phase separation was seen at higher temperatures and/or at lower concentrations of DPPC or SM in the presence of cholesterol than in its absence. Mixtures of DPPC or SM and cholesterol are known to form the liquid-ordered phase. Therefore, the fact that phase separation was observed in the cholesterol-containing membranes shows that liquid-ordered and liquid-disordered phase domains coexist. At 37 degrees C, the SM-enriched liquid-ordered phase was first seen at a SM/PC ratio of close to 0.25, when SM made up 17% of the total lipid including cholesterol. (This is similar to or less than the SM concentration of the plasma membranes of mammalian cells.) Furthermore, the detergent insolubility of cholesterol-containing model membranes correlated well with the amount of liquid-ordered phase as detected by fluorescence quenching. Thus, the detergent-insoluble membranes isolated from cells are likely to exist in the liquid-ordered phase prior to detergent extraction. The promotion of liquid-ordered phase formation may be an important function of cholesterol and sphingolipids in cells and may be a major distinction between the cholesterol- and sphingolipid-rich plasma membrane and most other cellular membranes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Transmembrane orientation of hydrophobic alpha-helices is regulated both by the relationship of helix length to bilayer thickness and by the cholesterol concentration.

A fluorescence-based approach to evaluate the regulation of transmembrane orientation of alpha-helices has been developed to examine the behavior of a membrane-inserted alpha-helical peptide with a 19 residue hydrophobic sequence. The emission lambdamax of a Trp residue in the helix was used to determine its location in the bilayer. To calibrate this method, Trp lambdamax and depth (determined by parallax analysis of fluorescence quenching) were measured for transmembrane peptides with Trp at different positions. Transmembrane orientation of the alpha-helix was found to be destabilized by differences between the width of the bilayer and the length of the hydrophobic sequence (i.e., hydrophobic mismatch). When bilayer width exceeded the length of the hydrophobic segment, mismatch induced formation of a nontransmembraneous orientation close to the polar/hydrocarbon interface. By manipulation of bilayer width in situ, it was found that the transmembrane and nontransmembrane orientations could interconvert. Cholesterol altered the transmembrane/nontransmembrane equilibrium to a degree consistent with its tendency to increase bilayer thickness. Evaluation of the energetics of transmembrane vs nontransmembrane insertion showed increased mismatch of a helix with bilayer width by the equivalent of just one hydrophobic residue can destabilize transmembrane orientation by roughly 0.5 kcal/mol. Inclusion of 30 mol % cholesterol in a bilayer can alter transmembrane insertion stability by 3-5 kcal/mol. Thus, physiologically relevant variations in both the hydrophobic helix length/membrane thickness ratio and the cholesterol levels influence transmembrane insertion significantly.

Amino Acid Sequence↗
PubMed1996

Comparison of the conformation, hydrophobicity, and model membrane interactions of diphtheria toxin to those of formaldehyde-treated toxin (diphtheria toxoid): formaldehyde stabilization of the native conformation inhibits changes that allow membrane insertion.

Toxoids are inactivated protein toxins that are used in vaccines. The behavior of diphtheria toxin reacted with formaldehyde (diphtheria toxoid) was compared to that of diphtheria toxin in order to understand the nature of the changes that occur in toxoids upon protein reaction with formaldehyde. Despite the intramolecular cross-links in the toxoid, the conformations of the toxoid and the toxin were very similar in both the native and low pH-induced membrane-penetrating states as judged by fluorescence and hydrophobicity properties. However, the toxoid underwent thermal-, low-pH-, and guanidinium chloride-induced conformational changes only at more extreme conditions than needed to induce such changes in the toxin. This implies that formaldehyde modification stabilizes the native conformation relative to several conformations that involve different degrees of unfolding. The stabilization to conformational changes induced by low pH is particularly interesting because low pH induces partial unfolding of the toxin to a molten globule-like state. It was found that the toxoid only gained the ability to interact with model membrane vesicles at a lower pH than the toxin. Because low-pH-induced unfolding and membrane interaction are critical steps in the entry of diphtheria toxin into cells, the resistance of the toxoid to these changes may be linked to its lack of toxicity. The implications of these results for the construction of toxoids are discussed.

Binding Sites↗
PubMed1995

Anchoring of tryptophan and tyrosine analogs at the hydrocarbon-polar boundary in model membrane vesicles: parallax analysis of fluorescence quenching induced by nitroxide-labeled phospholipids.

The role of Trp and Tyr residues in determining membrane protein structure is particularly interesting because indole and phenol structures combine hydrophobic and polar groups, and it is hard to predict the exact region of the membrane at which their energy would be at a minimum. To determine the depths intrinsically favored by these residues, the locations of membrane-associating Trp and Tyr analogs have been determined using a fluorescence quenching technique able to measure depth at high resolution. They are found to locate at the same depths as Trp and Tyr in membrane proteins, 14-15 A from the bilayer center, which implies an important role for these residues in aligning membrane proteins in precise relationship to the lipid bilayer.

Cell Membrane↗