共查询到20条相似文献,搜索用时 31 毫秒
1.
J Sopkova M Vincent M Takahashi A Lewit-Bentley J Gallay 《Canadian Metallurgical Quarterly》1998,37(34):11962-11970
The conformation and dynamics of domain III of annexin V was studied by steady-state and time-resolved fluorescence of its single tryptophan residue (Trp187) as a function of pH in the absence of calcium. At neutral pH, the maximum of emission occurs at 326 nm, in agreement with the hydrophobic location of the tryptophan residue seen in the three-dimensional structure. Upon decreasing the pH, a progressive red-shift by about 12 nm of the fluorescence emission spectrum is observed. The effect is complete between pH 6 and 4.5, and most likely involves at least one and maybe two carboxylic group(s). Circular dichroism mesurements give evidence for a preservation of the native folding of the protein in these mild acidic conditions. A fluorescence red-shift of smaller amplitude is also observed at high pH (approximately 11). The aggregation state of the protein is affected by pH: while at neutral pH, the protein is monomeric (rotational correlation time = 14 ns); it forms aggregates larger than a dimer (rotational correlation time > 40 ns) in acidic pH conditions. These results suggest that electrostatic interactions are probably important for the stabilization of the folding of domain III without calcium. The conformational change may be related to the aggregation state of the molecule. Examination of the protein crystal structures with and without calcium ion in domain III shows an interplay of salt bridges implying charged amino acid side chains at the molecule surface of domain III. These observations may provide a further clue to the mechanism of the conformational change of domain III of annexin V induced by high calcium concentrations and interaction at the membrane/water interface. 相似文献
2.
The tryptophan residues in pig kidney aminoacylase (N-acylamino acid amido hydrolase, EC 3.5.1.14) have been modified by N-bromosuccinimide (NBS) at low pH. The modification of eight tryptophan residues as measured by spectrophotometric and spectrofluorimetric methods leads to complete loss of enzymatic activity. The decreases in absorption at 280 nm and fluorescence emission at 337 nm indicate the modification of tryptophan residues. Both the inactivation and tryptophan residual modification are monophasic, first-order reactions. Quantitative treatment of the data (Tsou, C. L., Sci. Sin., 1962, 11, 1535-1558) shows that among the tryptophan residues modified, two are essential for aminoacylase catalytic activity. K?rdel and Schneider (Hoppe-Seyler's Physiol. Chem. 1976, 357, 1109-1115) reported that the modification of tryptophan residues led to inactivation of aminoacylase, and suggested that tryptophan residues are essential for enzymatic activity. We have now shown that eight tryptophan residues can be modified by N-bromosuccinimide and that two of them are essential for the catalytic activity of this enzyme. 相似文献
3.
The relation between serum lipids, membrane fluidity, insulin, and the activity of the sodium-hydrogen exchanger was investigated in human lymphocytes from 83 subjects. Subjects had a wide range of serum lipids and no concurrent disease. Lymphocyte membrane anisotropy (inversely related to membrane fluidity) was measured with a fluorescence polarization method. Sodium-hydrogen exchange maximal proton efflux rate, affinity for external sodium, and resting pH were determined with the intracellular pH-sensitive fluorochrome 2',5'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Sodium-hydrogen exchange maximal proton efflux rate was negatively correlated with the age of the subject (p = 0.03). Membrane anisotropy correlated with serum triglyceride (p = 0.04). Multiple regression analysis demonstrated that the maximal proton efflux rate in human lymphocytes was significantly related to age (p = 0.005), systolic blood pressure (p = 0.04), membrane anisotropy (p = 0.03), and serum cholesterol (p = 0.03). Incubation of lymphocytes with insulin failed to affect sodium-hydrogen exchange kinetics, intracellular buffering power, or resting intracellular pH. These results suggest that membrane-bound transport proteins may be influenced by serum lipids and the fluidity of the lipid membrane in which they are bound, but they are unlikely to be affected by insulin. 相似文献
4.
One of the ubiquitous features of membrane proteins is the preference of tryptophan and tyrosine residues for membrane surfaces that presumably arises from enhanced stability due to distinct interfacial interactions. The physical basis for this preference is widely believed to arise from amphipathic interactions related to imino group hydrogen bonding and/or dipole interactions. We have examined these and other possibilities for tryptophan's interfacial preference by using 1H magic angle spinning (MAS) chemical shift measurements, two-dimensional (2D) nuclear Overhauser effect spectroscopy (2D-NOESY) 1H MAS NMR, and solid state 2H NMR to study the interactions of four tryptophan analogues with phosphatidylcholine membranes. We find that the analogues reside in the vicinity of the glycerol group where they all cause similar modest changes in acyl chain organization and that hydrocarbon penetration was not increased by reduction of hydrogen bonding or electric dipole interaction ability. These observations rule out simple amphipathic or dipolar interactions as the physical basis for the interfacial preference. More likely, the preference is dominated by tryptophan's flat rigid shape that limits access to the hydrocarbon core and its pi electronic structure and associated quadrupolar moment (aromaticity) that favor residing in the electrostatically complex interface environment. 相似文献
5.
E Breukink C van Kraaij A van Dalen RA Demel RJ Siezen B de Kruijff OP Kuipers 《Canadian Metallurgical Quarterly》1998,37(22):8153-8162
Nisin is a 34 residue long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The antimicrobial activity is based on pore formation in the cytoplasmic membrane of target organisms. The mechanism which leads to pore formation remains to be clarified. We studied the orientation of nisin via site-directed tryptophan fluorescence spectroscopy. Therefore, we engineered three nisin Z variants with unique tryptophan residues at positions 1, 17, and 32, respectively. The activity of the tryptophan mutants against Gram-positive bacteria and in model membrane systems composed of DOPC or DOPG was established to be similar to that of wild type nisin Z. The tryptophan fluorescence emission maximum showed an increasing blue-shift upon interaction with vesicles containing increased amounts of DOPG, with the largest effect for the 1W peptide. Studies with the aqueous quencher acrylamide showed that all tryptophans became inaccessible from the aqueous phase in the presence of negatively charged lipids in the vesicles. From these results it is concluded that anionic lipids mediate insertion of the tryptophan residues in at least three positions of the molecule into the lipid bilayer. The depth of insertion of the tryptophan residues was determined via quenching of the tryptophan fluorescence by spin-labeled lipids. The results showed that the depth of insertion was dependent on the amount of negatively charged lipids. In membranes containing 50% DOPG, the distances from the bilayer center were determined to be 15.7, 15.0, and 18.4 A for the tryptophan at position 1, 17, and 32, respectively. In membranes containing 90% DOPG, these distances were calculated to be 10.8, 11.5, and 13.1 A, respectively. These results suggest an overall parallel average orientation of nisin in the membrane, with respect to the membrane surface, with the N-terminus more deeply inserted than the C-terminus. These data were used to model the orientation of nisin in the membrane. 相似文献
6.
To elucidate the dependence of protein intrinsic fluorescence characteristics on the microenvironment of their tryptophan residue localization, more than a hundred tryptophan residues of a number of proteins were analysed and compared with experimental data on their intrinsic fluorescence. Some factors were revealed, which determine the fluorescence spectrum position of certain tryptophan residues and their contribution to the total protein fluorescence. Specifically, the role of aromatic residues and proline, as well as of tryptophan residue side chain conformation, in the formation of the unique blue fluorescence spectrum of a number of proteins was demonstrated. It was shown that the quenching effect of sulphur atoms of cysteine and methionine, imidazole rings of histidine, guanyl groups of arginine, etc. depends not only on their distance from the indole ring of the tryptophan residue but, to a great extent, on their orientation to indole ring. 相似文献
7.
Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing groups with pK values of 5.4, that must be unprotonated, and 8.4, that must be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modification with (DEP). The histidine specific reagent inactivated the enzyme following a pseudo first-order kinetics with a second-order rate constant of 8.9 10(-3) (+/- 1.8 10(-3)) M-1 min-1. The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern-Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are buried in the native molecule. Tryptophan residues also showed a high heterogeneity that was increased after binding of ground- and transition-state analogs to the enzyme. 相似文献
8.
1. Cataract is the single major cause of blindness worldwide; however, the reasons for the development of this condition remain unknown. It has been suggested that the essential amino acid tryptophan may be implicated in the aetiology but definitive evidence has been lacking. 2. The serum levels of tryptophan and seven of its metabolites have been measured in both cataract patients and control subjects, after administration of tryptophan, in order to determine the typical response profile and to discover whether differences could be found in tryptophan metabolism in the two groups. 3. Tryptophan, kynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, 5-hydroxyanthranilic acid, 5-hydroxytryptophan and anthranilic acid were measured by HPLC with dual electrochemical and programmable wavelength fluorescence detection. Fasting cataract patients (n = 42) and control subjects (n = 37) were given an oral dose of L-tryptophan and sera were sampled at 0, 1, 2, 4 and 6 h. 4. Statistically significant differences in the distribution of data between the two groups were observed. The responses of kynurenine and 5-hydroxyanthranilic acid were higher in cataract patients, but those of kynurenic acid and total tryptophan were lower than in control subjects. No statistically significant differences in free tryptophan, anthranilic acid, 3-hydroxyanthranilic acid, xanthurenic acid or 5-hydroxytryptophan levels were noted. 5. We conclude that there is a major subgroup of age-related cataract patients with a dysfunction in the metabolism of tryptophan. This may be related to the onset of cataract. The mechanism remains to be established but may operate via the action of tryptophan metabolites, such as 5-hydroxyanthranilic acid, which become reactive towards protein upon oxidation. 相似文献
9.
Influenza virus hemagglutinin (HA) has served as a paradigm for both pH-dependent and -independent viral membrane fusion. Although large conformational changes were observed by X-ray crystallography when soluble fragments of HA were subjected to fusion-pH conditions, it is not clear whether the same changes occur in membrane-bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH-induced conformational change of the membrane-bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane-bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of beta-sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time-resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane-bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6:1993-2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined studies. 相似文献
10.
AM Evangelou MP Malamas P Vezyraki SC Karkabounas 《Canadian Metallurgical Quarterly》1998,12(3):321-325
Fluorescence spectroscopy revealed a dramatic difference between the intracellular distributions of doxorubicin (DOX) and its paramagnetic analogue, ruboxyl (Rb). Modification of the anthracyclin structure by introduction of a paramagnetic label at position 14 in the DOX molecule resulted in reduced DNA binding and enhanced partitioning into phospholipid membranes, as evidenced by the fluorescence-quenching experiments. Higher partitioning into cell membranes resulted in stronger intracellular fluorescence of Rb. In addition, a paramagnetic label on the Rb molecule provided a unique opportunity for an EPR investigation of the drug's intracellular distribution and dynamics. The observed changes in the EPR spectra of drug-containing cells during their life cycle suggested either a progressive release of the intercalated drug, cell membrane fluidization, or both. 相似文献
11.
A general approach to dissecting the complex photophysics of tryptophan is presented and used to elucidate the effects of amino acid functional groups on tryptophan fluorescence. We have definitively identified the amino acid side chains that quench tryptophan fluorescence and delineated the respective quenching mechanisms in a simple model system. Steady-state and time-resolved fluorescence techniques, photochemical H-D exchange experiments, and transient absorption techniques were used to measure individual contributions to the total nonradiative rate for deactivation of the excited state, including intersystem crossing, solvent quenching, and excited-state proton and electron transfer rates. Eight amino acid side chains representing six functional groups quench 3-methylindole fluorescence with a 100-fold range in quenching rate constant. Lysine and tyrosine side chains quench by excited-state proton transfer; glutamine, asparagine, glutamic and aspartic acid, cysteine, and histidine side chains quench by excited-state electron transfer. These studies provide a framework for deriving detailed structural and dynamical information from tryptophan fluorescence intensity and lifetime data in peptides and proteins. 相似文献
12.
M Coles W Bicknell AA Watson DP Fairlie DJ Craik 《Canadian Metallurgical Quarterly》1998,37(31):11064-11077
The three-dimensional solution structure of the 40 residue amyloid beta-peptide, Abeta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles. In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of Abeta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6. 0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for Abeta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of Abeta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease. 相似文献
13.
In order to assess the importance of a variety of environmental factors on the structure of bovine prothrombin fragment 1, we have examined acrylamide quenching of fragment 1 intrinsic fluorescence. Tryptophan exposure, determined from Stern-Volmer plots, is heterogeneous with one or more of the three fragment 1 tryptophans being exposed to solvent. In the presence of Ca2+ or Mg2+ even the most accessible tryptophan(s) are relatively buried. Only small differences in tryptophan exposure may exist between fragment 1-Ca2+ and fragment 1-Mg2+ complexes. Lowering pH, on the other hand, results in increased tryptophan exposure. Finally, structural isomers of fragment 1 which exist in the absence of metal ions have identical tryptophan exposure as determined by acrylamide quenching and fluorescence intensity. 相似文献
14.
PF Augustijns 《Canadian Metallurgical Quarterly》1996,48(3):277-280
The transepithelial transport and uptake of chloroquine were studied in cultured human intestinal Caco-2 cell layers, to investigate whether a specific mechanism facilitates the flux of chloroquine. Due to ionization of chloroquine at the pH of the intestinal lumen, the fraction of the neutral form, which is required for partitioning into biological membranes, is very low, while oral bioavailability has been reported to be nearly complete. Several observations, such as concentration-dependent uptake and temperature-dependent transepithelial flux, suggest the presence of carrier mediated transport. However, alternative mechanisms may be invoked to explain these observations. It is suggested that concentration dependence can originate from ion-trapping in acidic compartments of the cell or non-specific binding to cell components, while temperature-dependent transport can, at least partly, be explained by the temperature dependence of the acid dissociation constants of chloroquine. No differences were observed in the transepithelial flux of the enantiomers of chloroquine. pH-dependent uptake as well as pH-dependent transepithelial transport suggest that the translocation of chloroquine occurs according to the fraction of neutral molecules. From the data obtained in this study, it is concluded that chloroquine crosses the gastrointestinal barrier by passive diffusion. The extensive area of the gastrointestinal tract probably compensates for the low fraction of the neutral molecule. An interesting finding of this study was the concentration-dependent increase in transepithelial electrical resistance across monolayers incubated with chloroquine at the apical side. 相似文献
15.
M Opekarová P Urbanová I Konopásek P Kvasnicka K Strzalka K Sigler E Amler 《Canadian Metallurgical Quarterly》1996,386(2-3):181-184
The F?rster energy transfer from tryptophan residues of membrane proteins to nystatin was measured in reconstituted yeast plasma membrane vesicles free of, or doped with, ergosterol. We wanted to elucidate whether the functional change of membrane transport proteins from H+ symporters to facilitators, observed in ergosterol-containing plasma membrane vesicles on addition of nystatin [Opekarová and Tanner (1994) FEBS Lett. 350, 46-50], is reflected in altered protein-nystatin relations within the membrane. Both frequency-domain and time-domain time-resolved fluorescence spectroscopy showed that in the presence of ergosterol nystatin is located much closer to membrane proteins than in its absence. 相似文献
16.
F Bobanovi? MD Bootman MJ Berridge NA Parkinson P Lipp 《Canadian Metallurgical Quarterly》1999,13(2):365-376
Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp28, Trp101, and Trp227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature (Tm) of the first transition phase by about 7 degrees C, and the double mutation by about 11 degrees C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing Tm and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein. 相似文献
17.
SD Swain SL Helgerson AR Davis LK Nelson MT Quinn 《Canadian Metallurgical Quarterly》1997,272(47):29502-29510
Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47(phox), p67(phox), and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47(phox) undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes in p47(phox) using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47(phox) with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose-dependent quenching in the intrinsic tryptophan fluorescence of p47(phox), whereas treatment with a number of other amphiphilic agents that failed to activate the oxidase had no effect on p47(phox) fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell-free assay system. We next determined if activation by phosphorylation caused the same type of conformational changes in p47(phox). Protein kinase C phosphorylation of p47(phox) in vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity. Finally, the circular dichroism (CD) spectrum of p47(phox) was significantly changed by the addition of SDS, whereas treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47(phox). Thus, our studies provide direct evidence linking conformational changes in p47(phox) to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47(phox) that are similar to those mediated by phosphorylation in vivo. 相似文献
18.
A quantitative estimation of surface accessibility of aromatic residues in alpha-crystallin from goat lens has been accomplished by chemical modifications using different specific reagents having varying sizes. Results of modification of tyrosine residues with N-acetylimidazole and tetranitromethane when combined with those of ionization studies carried out with hydroxyl ions having the smallest size reveal different classes of tyrosine residues in the native protein: 78 +/- 2 residues have been found to be easily available for modification; among the rest, 94 +/- 2 residues appear to be comparatively less exposed to the reagents while 28 +/- 2 residues are found to be completely unavailable for modification in the native protein and are modified only when the protein is denatured. Modification of tryptophan residues with H2O2 also indicates different classes of these residues available for oxidation at different concentrations of the oxidant. 34 +/- 2 residues of tryptophan are found to be easily oxidized at a lower concentration of H2O2 during the first phase of the reaction. The remaining tryptophan residues appear to be less exposed to the reagent. This is also corroborated from the studies of reactivities of these residues towards another specific but bulkier reagent, 2-hydroxy-5-nitrobenzyl bromide. These surface exposed aromatic residues in alpha-crystallin may be considered to be vulnerable to in vivo oxidative modifications forming insoluble aggregates which may finally contribute to the formation of cataract. 相似文献
19.
Because tryptophans are found as part of the phosphate binding sites in a number of proteins, human prostatic acid phosphatase (hPAP) was examined for the presence and the role of essential tryptophan residues. The pH dependence of the intrinsic fluorescence of hPAP resembled the kinetic pH dependence. Chemical modification by N-bromosuccinimide (NBS) resulted in an inactivation of the enzyme and produced a characteristic reduction of the protein absorbance at 280 nm. Two tryptophans per subunit were modified, and this was accompanied by an apparently complete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantly reduced as compared to the rate of tryptophan modification. After labeling the protein with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), two tryptic peptides containing DNPS-labeled tryptophans were isolated and the sequences were identified by amino acid sequence analysis and mass spectroscopy. One peptide corresponded to residues 172-176, and included Trp174. The other corresponded to the C-terminal sequence, including Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibitor tartrate in the DNPS-Cl modification studies. This is also consistent with the location of a homologous residue in the structure of the rat enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lower Km values with several aromatic substrates, and also exhibited reduced stability towards urea denaturation. 相似文献
20.
The membrane insertion and translocation of diphtheria toxin, which is induced in vivo by low pH, is thought to be directed by the hydrophobic alpha-helices of its transmembrane (T) domain. In this study the structure of membrane-associated T domain was examined. Site-directed mutants of the T domain with single Trp residues were prepared at the two naturally occurring positions, 206 (near the N-terminal end of helix TH1) and 281 (within helix TH5), as well as at three residues in helix TH9, in which the substitutions F355W (near the N-terminal end of TH9), I364W (close to the center of TH9), and Y375W (near the C-terminal end of TH9) were made. All these mutants were found to undergo the low-pH-induced conformational change observed with wild-type T domain and insert into model membranes at low pH. The location of Trp residues relative to the lipid bilayer was characterized in model membrane vesicles by fluorescence emission and by quenching with nitroxide-labeled phospholipids. In TH9, residue 375 was shallowly inserted, residue 364 deeply inserted, and residue 355 located at an intermediate depth. Residues 206 and 281 exhibited moderately deep insertion. It was also found, in agreement with our previous study using bimane-labeled protein (Wang et al. (1997) J. Biol. Chem. 272, 25091-25098), that TH9 switches from a relatively shallowly inserted state to a more deeply inserted state when the concentration of the T domain in the membrane is increased or the thickness of the membrane bilayer is decreased. In particular, the depth of residue 355 was found to increase under the conditions giving deeper insertion. In contrast, residue 375 remained shallowly located in both states, as predicted from its location on the polar C-terminus of TH9. It is concluded that TH1 and TH5 insert into the lipid bilayer in both T domain conformations. In addition, Trp depths suggest that even in the shallowly inserted state there is a significant degree of insertion of TH9. These results suggest regions of the T domain in addition to the hydrophobic TH8/TH9 hairpin insert into membranes. Models for the structure of the membrane-inserted T domain are discussed. 相似文献