Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling.

To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [(3)H]diazofluorene ([(3)H]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa alpha-subunit of the (Na(+)+K(+))-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [(3)H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the alpha-subunit of the (Na(+)+K(+))-ATPase and in a putative transmembrane beta-strand in VDAC. Collectively these results demonstrate that [(3)H]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by (3)H release.

Depth-dependent analysis of membranes using benzophenone-based phospholipids.

Any attempt to probe the membrane hydrophobic core with chemical reagents necessitates the use of reactive intermediates like carbenes and nitrenes, which can insert into C-H bonds. Several photoactivable reagents based on carbenes and nitrenes have been reported. However, the high reactivity of these reagents, often leads to very low insertion yields. We report here a high degree of cross-linking (35-40%) achieved with three benzophenone-based phospholipids and analyze the carbon functionalization data using a multiple Gaussian function. These phospholipids are so designed so as to permit depth-dependent labeling in membranes. Single bilayer vesicles were prepared from these phospholipids and dimyristoylphosphatidylcholine. The cross-linked product was isolated and characterized by mass spectroscopy. The results obtained indicated that the cross-linked product was dominated by dimeric product formed by intermolecular cross-linking. The Gaussian analysis used here provides insight into the relative depths of the probes inside the membrane.

Organization of diphtheria toxin in membranes. A hydrophobic photolabeling study.

Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a catalytic domain (C), a membrane-inserting domain (T), and a receptor-binding domain (R). It gains entry into cells by receptor-mediated endocytosis. The low pH ( approximately 5.5) inside the endosomes induces a conformational change in the toxin leading to insertion of the toxin in the membrane and subsequent translocation of the C domain into the cell, where it inactivates protein synthesis ultimately leading to cell death. We have used a highly reactive hydrophobic photoactivable reagent, DAF, to identify the segments of DT that interact with the membrane at pH 5.2. This reagent readily partitions into membranes and, on photolysis, indiscriminately inserts into lipids and membrane-inserted domains of proteins. Subsequent chemical and/or enzymatic fragmentation followed by peptide sequencing allows for identification of the modified residues. Using this approach it was observed that T domain helices, TH1, TH8, and TH9 insert into the membrane. Furthermore, the disulfide link was found on the trans side leaving part of the C domain on the trans side. This domain then comes out to the cis side via a highly hydrophobic patch corresponding to residues 134-141, originally corresponding to a beta-strand in the solution structure of DT. It appears that the three helices of the T domain could participate in the formation of a channel from a DT-oligomer, thus providing the transport route to the C domain after the disulfide reductase separates the two chains.

Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates.

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule (MG) state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent--2[3H]diazofluorene. This carbene-based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels alpha-lactalbumin as a function of pH (3-7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B (23-34) and C (86-98) helix of the native structure are extensively labeled. The small beta-domain (40-50) is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of alpha-lactalbumin, the alpha-domain has a greater degree of persistent structure than the beta-domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.

Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus.

The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.

Diagnosis and management of gastrointestinal angiodysplasia.

Eighteen patients with gastrointestinal angiodysplasia were seen in a single surgical unit over a period of 8 years. The mean age at onset of symptoms was 33 years. The average duration of symptoms was 54 months (range 2 days-16 years). Three patients had gastric angiodysplasia, two had colonic angiodysplasia, both diagnosed endoscopically preoperatively. The remaining patients required further investigation, which included small bowel enema (SBE), erythrocyte tagged scan (ETC), selective visceral angiography and intraoperative enteroscopy (IOE). SBE was useful but not diagnostic in 3, ETC in 3 and angiography in 5. Four patients required IOE for a diagnosis. Follow up of 17 months is available on all patients. Four had recurrence of symptoms. One required re-exploration and resection of 3 feet of small bowel and right hemicolectomy, another is on hormonal therapy and maintaining normal haemoglobin. Two others are asymptomatic on oral iron therapy.

Intraoperative enteroscopy in obscure gastrointestinal hemorrhage.

Forty three patients were diagnosed to have obscure gastrointestinal hemorrhage (OGH) between January 1987 and April 1996. Pre-operative diagnostic investigations were useful in localizing the site of bleeding in 28 patients (65.1%). These included small bowel enema (n = 12), erythrocyte tagged scan (n = 8), Meckel's scan (n = 2) and selective visceral angiography (n = 7). Following complete evaluation all patients underwent exploratory laparotomy. At laparotomy 31 patients were found to have gross lesions. Intraoperative enteroscopy (IOE) could detect lesions in 9 of the remaining 12 patients.

Unfolding of diphtheria toxin. Identification of hydrophobic sites exposed on lowering of pH by photolabeling.

We report here the use of a hydrophobic photoactivable reagent, 2-[3H]diazofluorene (DAF), to map the hydrophobic sites exposed when the pH is lowered in diphtheria toxin (DT). The reagent binds to DT, and on photolysis with light of wavelength >350 nm, it covalently attaches itself to DT. The labeling was observed to increase considerably when the pH was lowered from 7.4 to 5.2. Although both A- and B-chains were labeled to a similar degree at pH 7.4, at lower pH (5.2), B-chain was labeled to a much higher extent. Subsequent chemical and enzymatic fragmentation of DT followed by separation indicated that the putative transmembrane domain was labeled to its maximum extent at pH 5.2, with the bulk of labeling associated with residues 340-459. Protein sequencing analysis indicated that the two buried hydrophobic helices, identified in the crystal structure and suggested to insert and span the membrane bilayer, corresponding to residues 326-347 and 358-376, are strongly labeled. The Pro-345 residue was observed to be labeled maximally at lower pH values. Finally, the DAF labeling pattern indicated that the parent structural motifs are retained at low pH, suggesting that the low pH conformation of DT corresponds to an equilibrium molten globule state.

Probing the structure of the nicotinic acetylcholine receptor ion channel with the uncharged photoactivable compound -3H-diazofluorene.

The uncharged photoactivable probe 2-[3H]diazofluorene ([3H]DAF) was used to examine structural changes in the Torpedo californica nicotinic acetylcholine receptor (AChR) ion channel induced by agonists. Photoincorporation of [3H]DAF into the AChR consisted of the following two components: a nonspecific component consistent with incorporation into residues situated at the lipid-protein interface, and a specific component, inhibitable by noncompetitive antagonists and localized to the M2 hydrophobic segments of AChR subunits. The nonspecific [3H]DAF incorporation was characterized in the M4 segment of each AChR subunit. The observed distribution and periodicity of labeled residues reinforce the conclusion that the M4 segments are organized as transmembrane alpha-helices with a common "face" of each helix in contact with lipid. Within the M2 segments, in the absence of agonist [3H]DAF specifically labeled homologous residues betaVal-261 and deltaVal-269, with incorporation into deltaVal-269 at a 5-fold greater efficiency than into betaVal-261. This observation, coupled with the lack of detectable incorporation into alpha-M2 including the homologous alphaVal-255, indicates that within the resting channel [3H]DAF is bound with its photoreactive diazo group oriented toward deltaVal-269. In the presence of agonist, there is an approximately 90% reduction in the labeling of betaVal-261 and deltaVal-269 accompanied by specific incorporation into residues (betaLeu-257, betaAla-258, deltaSer-262, and deltaLeu-265) situated 1 or 2 turns of an alpha-helix closer to the cytoplasmic end of the M2 segments. The results provide a further characterization of agonist-induced rearrangements of the M2 (ion channel) domain of the AChR.

Membrane-protein interaction and the molten globule state: interaction of alpha-lactalbumin with membranes.

The insertion of soluble proteins into membranes has been a topic of considerable interest. We have studied the insertion of bovine alpha-lactalbumin into single-bilayer vesicles prepared from egg phosphatidylcholine (PC). Fluorescence studies indicated rapid and tight binding of apo-alpha-lactalbumin (apo-alpha-LA) to PC vesicles as a function of pH. The binding was maximal at pH values which favor the formation of the molten globule state. As an increase of hydrophobic surface is observed in the molten globule state, this conformational state can provide a molecular basis for insertion of soluble proteins into membranes. The membrane-bound complex formed at low pH (3.0) could be isolated and was found to be stable at neutral pH. The structural characterization of the apo-alpha-LA-PC complex was studied by fluorescence quenching using iodide, acrylamide, and 9,10-dibromostearic acid. The results obtained indicated that some of the tryptophans of apo-alpha-LA were buried in the membrane interior and some were exposed on the outer side. Fluorescence quenching and CD studies indicated the membrane-bound conformation of apo-alpha-LA was some conformational state that is between the soluble, fully folded conformation and the molten globule state.

Photolabeling of a pore-forming toxin with the hydrophobic probe 2-[3H]diazofluorene. Identification of membrane-inserted segments of Staphylococcus aureus alpha-toxin.

The identification of membrane-inserted segments of pore-forming soluble proteins is crucial to understanding the action of these proteins at the molecular level. A distinct member of this class of proteins is alpha-toxin, a 293-amino acid-long 33-kDa hemolytic toxin secreted by Staphylococcus aureus that can form pores in both artificial and natural membranes. We have studied the interaction of alpha-toxin with single bilayer vesicles prepared from asolectin using a hydrophobic photoactivable reagent, 2-[3H]diazofluorene ([3H]DAF) (Pradhan, D., and Lala, A. K. (1987) J. Biol. Chem. 262, 8242-8251). This reagent readily partitions into the membrane hydrophobic core and on photolysis labels the lipid and protein segments that penetrate the membrane. Current models on the mode of action of alpha-toxin indicate that, on interaction with membranes, alpha-toxin forms an oligomer, which represents the active pore. In keeping with these models, we observe that [3H]DAF photolabels the membrane-bound alpha-toxin oligomer. Cyanogen bromide fragmentation of [3H]DAF-labeled alpha-toxin gave several fragments, which were subjected to Edman degradation. We could thus sequence residues 1-19, 35-60, 114-139, 198-231, and 235-258. Radioactive analysis and phenylthiohydantoin-derivative analysis during sequencing permitted analysis of DAF insertion sites. The results obtained indicated that the N and C termini (residues 235-258) have been extensively labeled. The putative pore-forming glycine-rich central hinge region was poorly labeled, indicating that the apposing side of the lumen of the pore does not form the lipid-protein interface. The DAF labeling pattern indicated that the major structural motif in membrane-bound alpha-toxin was largely beta-sheet.

Gastric acid study in patients operated on for perforated duodenal ulcer.

OBJECTIVES: To determine the acid secretory status of patients operated on for perforated duodenal ulcer, with or without prior history suggestive of acid-peptic disease. METHOD: Basal and peak acid output were measured in 48 patients with perforated duodenal ulcer who were treated by simple closure alone, 10 patients with uncomplicated chronic duodenal ulcer and 30 normal controls. Patients operated on for perforated duodenal ulcer were analyzed according to presence (or absence) and duration of prior history of acid-peptic disease. RESULTS: Peak acid output was similar in controls and patients with perforated duodenal ulcer with no prior symptoms. However, patients with prior symptoms had significantly higher peak acid output, similar to those with uncomplicated duodenal ulcer. CONCLUSION: Patients with perforated duodenal ulcer with no antecedent history of acid-peptic disease have normal gastric acid output. These patients may therefore have other etiological factors for their ulcers.

Increased exposure of hydrophobic surface in molten globule state of alpha-lactalbumin. Fluorescence and hydrophobic photolabeling studies.

The involvement of molten globule state as a distinct intermediate in the denaturation process in proteins is well documented. However, the structural characterization of such an intermediate is far from complete. We have, using fluorescence and fluorescence quenching, studied the molten globule state of bovine alpha-lactalbumin. Unlike the native state, where all the 4 tryptophans are buried in the protein, 2 tryptophans are exposed in the molten globule state. Using the hydrophobic photoactivable reagent [3H]diazofluorene, we observe an increased hydrophobic exposure in the molten globule state. These structural characteristics conform to the current views on the molten globule state, i.e. it has similar secondary structure but a poorly defined tertiary structure. Our fluorescence studies indicate the involvement of a premolten globule state in the native to molten globule state transition. This premolten globule state exists at pH 5.0 and has a very compact structure involving increased hydrophobic interactions in the protein interior. These results are also supported by circular dichroism studies.

Fluorenyl fatty acids as fluorescent probes for depth-dependent analysis of artificial and natural membranes.

The main objective of depth-dependent fluorescent probes is to provide information at a distinct position in the membrane hydrophobic core. We report here a series of fluorenyl fatty acids which can probe both artificial and natural membranes at different depths. Long-chain acids (C4, C6, and C8) are attached to fluorene chromophore on one side, and a hydrophobic tail (C4) is attached on the other side, so that on incorporation in membranes the carboxyl end of the molecule is oriented toward the membrane-water interface and the hydrophobic tail points toward the membrane interior. These acids can be readily partitioned into membranes. The disposition of these fluorenyl fatty acids in membranes was studied by fluorescence quenching using iodide as a water-soluble and 9,10-dibromostearic acid as a lipid-soluble quencher. The results obtained indicate that attachment of a hydrophobic tail is essential for effective alignment of depth-dependent fluorescent probes. The length of the hydrophobic tail was varied and an n-butyl chain was found to be most effective. In all cases, the compounds with a hydrophobic tail were found to be probing the membrane deeper than their counterparts with no hydrophobic tail. Further, the compounds with hydrophobic tails were more strongly immobilized in the membrane as indicated by fluorescence polarization studies. However, the effect of such a tail varied with membrane type. Thus in artificial membranes an n-butyl chain was found to be extremely important for effective monitoring by shallow probes like 4-(2'-fluorenyl)butyric acid, whereas in erythrocyte ghost membranes the same n-butyl tail was found to be more desirable for deeper probes like 8-(2'-fluorenyl)octanoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrophobic photolabeling in membranes: the human erythrocyte glucose transporter.

Human erythrocyte membranes were labeled with a hydrophobic photoactivable reagent, 2-[3H]Diazofluorene. Electrophoretic analysis of the protein fraction showed that several membrane spanning proteins like Band 3 (the anion transporter), Band 4.5 (the glucose transporter), and the sialoglycoproteins PAS 1, 2, and 3 have been labeled. To isolate the diazofluorene-labeled glucose transporter, the membrane preparation was solubilized with Triton X-100 and passed through a DEAE-cellulose column. The flow-through fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive analysis of the gel indicated that besides the Band 4.5, two more proteins corresponding to the Band 3 and Band 6 regions also coelute with the glucose transporter in the flow-through fraction. On the other hand, use of n-octyl glucoside gave a relatively better preparation. The 2-[3H]DAF-labeled glucose transporter isolated by the latter method on tryptic digestion indicated that the Mr 18,000 fragment corresponding to the C-terminal transmembrane fragment is labeled.

Fluorescence studies on erythrocyte membrane isolated from Plasmodium berghei infected mice.

The erythrocyte host cell plays a key role in the well defined developmental stages of the malarial parasite growth and propogation in the erythrocyte cycle of malaria. The host cell serves the parasites by supplying metabolites and removing the catabolites produced by the obligatory parasites. It has been observed that the plasma membrane of the infected cells show a substantially higher fluidity due to the depletion of cholesterol content from the host cell. The protein component of the membrane is also modulated due to the insertion of new polypeptides of the parasitic origin, which confers upon it new antigenic properties. We have studied the membrane fraction isolated from mice erythrocytes infected with Plasmodium berghei using fluorescent probes like DPH, ANS and series of fluorenyl fatty acids, which permit depth dependent analysis of membrane. We have observed that there is a marked difference in the fluorescence emission wavelength maximum, the dissociation constant Kd of ANS when bound to normal and infected erythrocytes, though relatively small differences are observed in the fluorescence polarisation values of the two cell types. The fluorenyl fatty acids also show the differences when bound to normal and infected erythrocytes, indicating that either they are in a different environment or they have differing binding properties to the two cell types.

Transverse location of new fluorene based depth dependent fluorescent probes in membranes--quenching studies with 9,10-dibromostearic acid.

Fluorescence quenching technique has been used to determine the transverse location of the fluorescent fluorenyl fatty acids in single bilayer vesicles prepared from phosphatidylcholine. The fluorenyl fatty acids used here are 2-fluorenyl acetic, butyric, hexanoic and octanoic acid. In addition a new type of fluorescent probe, 7-n-butyl-fluorene-2-butyric acid, wherein a hydrophobic tail is attached to 2-fluorenyl-butyric acid has also been used to study its effect on alignment of these probes in the membrane. The association properties of the quencher 9,10-dibromostearic acid have been analysed. It is observed that the quencher association involves partitioning into the vesicles and does not involve any binding to the vesicles. The absolute partition coefficient of the 9,10-dibromostearic acid which partitions between the aqueous and the lipid phases of the phospholipid dispersion has been evaluated. Using this information the corrected Stern-Volmer plots were drawn and the bimolecular quenching constant evaluated.

Depth-dependent photolabelling of membrane hydrophobic core with 9-diazofluorene-2-butyric acid.

Hydrophobic photoactivable reagents, which readily partition into membranes, have proved very useful for studying membrane hydrophobic core. These reagents have been linked to fatty acids in order to obtain amphipathic photoactivable reagents which label membranes more effectively. By varying the length of these amphipathic reagents, an attempt to label membrane hydrophobic core at different depths can be made. We report here 9-diazofluorene-2-butyric acid as a new photoactivable reagent which labels the single bilayer vesicles prepared from egg phosphatidylcholine. The labelling site on the fatty acyl chains could be traced to be between the carbon atom 4 and 6. The new probe thus labels the membrane at a site which is proximal to what can be predicted from its length and transverse location in membranes.

Design, synthesis, and fluorescence studies of fluorenyl fatty acids as new depth-dependent fluorescent probes for membranes: getting over the looping-back problem.

Fluorescent fatty acids have proved very useful in studying the membrane hydrophobic core. They readily partition into membranes or can be converted to phospholipids, which form integral components of membranes. By attaching the fluorescent chromophore to different positions along the alkyl chain of fatty acids, e.g., an anthroyloxy group attached via an ester linkage to n-hydroxystearic acid, membranes have been probed at different depths. While this is an interesting approach and has been extensively used, relatively little attention has been paid to the molecular design of these probes in order to have minimal membrane perturbation. In the present study we have looked into the general problem of design of such depth-dependent membrane probes. We report here a series of fluorenyl fatty acids with varying fatty acid chain lengths, i.e., (2-fluorenyl)acetic acid, -butyric acid, -hexanoic acid, and -octanoic acid, in order to obtain information at different depths in the membrane hydrophobic core. To see the effect of attachment of a hydrophobic tail on the orientation of such fatty acids in membranes, an n-butyl group was linked to the C-7 position of fluorene in (2-fluorenyl)butyric acid to get 4-(7-n-butylfluoren-2-yl)butyric acid. Further, to assess their ability to act as depth-dependent fluorescent probes, these fatty acids were incorporated in vesicles prepared from egg phosphatidylcholine, and their fluorescence quenching was studied with potassium iodide, Cu(II), 9,10-dibromostearic acid, and 12-bromostearic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

A new carbene based heterobifunctional reagent. Photochemical crosslinking of aldolase.

The synthesis of a new photoactivatable heterobifunctional crosslinking reagent, the N-oxysuccinimide ester of 2-carboxy-9-diazofluorene, is described. The ability of the parent chromophore 2-carbomethoxy-9-diazofluorene to insert into cyclohexane and methanol has been established. The reagent has been linked to aldolase and the stoichiometry determined. Photolysis of the probe-linked aldolase indicated that photolysis was very rapid and that the photolysed product was constituted of crosslinked dimer, trimer and tetramer. Increase in concentration of probe linked to aldolase followed by photolysis gave rise to largely tetramer and higher oligomers of aldolase. The use of this carbene-based reagent vis a vis arylazide-based reagent for studying protein crosslinking is discussed.