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1.
We have previously described the functional activity of a human TCR specific for an HLA-A2-presented peptide derived from the Wilms tumor Ag 1 (WT1). Recent studies showed that the expression and function of human TCR was improved by the introduction of an additional disulfide bond between the alpha- and beta-chains or by the exchange of the human constant region for murine sequences. In this study, we analyzed the functional activity of WT1-TCR variants expressed in Jurkat cells and in primary T cells. The introduction of cysteine residues or murine constant sequences into the WT1-TCR did not result in a global reduction of mispairing with wild-type TCR chains. Instead, the level of mispairing was affected by the variable region sequences of the wild-type TCR chains. The analysis of freshly transduced peripheral blood T cells showed that the transfer of modified TCR constructs generated a higher frequency of Ag-responsive T cells than the transfer of the wild-type TCR. After several rounds of peptide stimulation this difference was no longer observed, as all transduced T cell populations accumulated approximately 90% of Ag-responsive T cells. Although the Ag-responsive T cells expressing the modified TCR bound the HLA-A2/WT1 tetramer more efficiently than T cells expressing the wild-type TCR, this did not improve the avidity of transduced T cells nor did it result in a measurable enhancement in IFN-gamma production and cytotoxic activity. This indicated that the enhanced tetramer binding of modified WT1-TCR variants was not associated with improved WT1-specific T cell function.  相似文献   

2.
The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8-10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the beta-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3 beta loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.  相似文献   

3.
The second transmembrane domain (TM2) of neurotransmitter transporters has been invoked to control oligomerization and surface expression. This transmembrane domain lies between TM1 and TM3, which have both been proposed to contain residues that contribute to the substrate binding site. Rat serotonin transporter (SERT) TM2 was investigated by cysteine scanning mutagenesis. Six mutants in which cysteine replaced an endogenous TM2 residue had low transport activity, and two were inactive. Most of the reduction in transport activity was due to decreased surface expression. In contrast, M124C and G128C showed increased activity and surface expression. Random mutagenesis at positions 124 and 128 revealed that hydrophobic residues at these positions also increased activity. When modeled as an alpha-helix, positions where mutation to cysteine strongly affects expression levels clustered on the face of TM2 surrounding the leucine heptad repeat conserved within this transporter family. 2-(Aminoethyl)-methanethiosulfonate hydrobromide (MTSEA)-biotin labeled A116C and Y136C but not F117C, M135C, or Y134C, suggesting that these residues may delimit the transmembrane domain. None of the cysteine substitution mutants from 117 through 135 were sensitive to [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) or MTSEA. However, treatment with MTSEA increased 5-hydroxytryptamine transport by A116C. Activation of A116C by MTSEA was observed only in mutants containing Cys to Ile mutation at position 357, suggesting that modification of Cys-116 activated transport by compensating for a disruption in transport in response to Cys-357 replacement. The reactivity of A116C toward MTSEA was substantially increased in the presence of substrates but not inhibitors. This increase required Na+ and Cl-, and was likely to result from conformational changes during the transport process.  相似文献   

4.
The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.  相似文献   

5.
Role of CD3 gamma in T cell receptor assembly   总被引:3,自引:0,他引:3       下载免费PDF全文
The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression.  相似文献   

6.
7.
One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.  相似文献   

8.
High-alkaline serine proteases have been successfully applied as protein degrading components of detergent formulations and are subject to extensive protein engineering efforts to improve their stability and performance. Dynamics has been suggested to play an important role in determining enzyme activity and specificity and it is therefore of interest to establish how local changes in internal mobility affect protein stability, specificity and performance. Here we present the dynamic properties of the 269 residue serine proteases subtilisin PB92 (Maxacal(TM)) and subtilisin BLS (Savinase(TM)), secreted by Bacillus lentus, and an engineered quadruple variant, DSAI, that has improved washing performance. T1, T2 and heteronuclear NOE measurements of the 15N nuclei indicate that for all three proteins the majority of the backbone is very rigid, with only a limited number of residues being involved in local mobility. Many of the residues that constitute the S1 and S4 pockets, determining substrate specificity, are flexible in solution. In contrast, the backbone amides of the residues that constitute the catalytic triad do not exhibit any motion. Subtilisins PB92, BLS and DSAI demonstrate similar but not identical NMR relaxation rates. A detailed analysis of local flexibility indicates that the motion of residues Thr143 and Ala194 becomes more restricted in subtilisin BLS and DSAI. Noteworthy, the loop regions involved in substrate binding become more structured in the engineered variant as compared with the two native proteases, suggesting a relation between altered dynamics and performance. Similar conclusions have been established by X-ray crystallograpic methods, as shown in the accompanying paper.  相似文献   

9.
The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.  相似文献   

10.
Karim CB  Marquardt CG  Stamm JD  Barany G  Thomas DD 《Biochemistry》2000,39(35):10892-10897
Chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) have been used to analyze the structure and function of phospholamban (PLB), a 52-residue integral membrane protein that regulates the calcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-PAGE, EPR, and fluorescence. It has been proposed that inhibition of the pump is due primarily to the monomeric form, with both pentameric stability and inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-PAGE, and compared it with the synthetic null-cysteine TM domain (residues 26-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isolated TM domain is more oligomeric than the full-length parent molecule. These results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic TM domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic domains (residues 1-25). When reconstituted into liposomes containing the Ca-ATPase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule. We conclude that the TM domain of PLB is sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.  相似文献   

11.
Peripheral blood lymphocytes (PBL) genetically modified to express T cell receptors (TCR) specific to known melanoma antigens, such as melanoma antigen recognized by T cells-1 (MART-1), and gp100 can elicit objective tumor regression when administered to patients with metastatic melanoma. It has also been demonstrated that modifications within the constant regions of a fully human TCR can enhance surface expression and stability without altering antigen specificity. In this study, we evaluated the substitution of murine constant regions for their human counterpart within the DMF5 MART-1-specific TCR. Unlike previous studies, all modified TCRs were inserted into retroviral vectors and analyzed for expression and function following a clinical transduction protocol. PBL were transduced with retroviral supernatant generated from stable packaging lines encoding melanoma-specific TCRs. This protocol resulted in high levels of antigen-specific T cells without the need for additional peptide stimulation and selection. Both the human and murinized TCR efficiently transduced PBL; however, the murinized TCR exhibited significantly higher tetramer binding, mean fluorescence intensity, as well as, increased in vitro effector function following our clinical transduction and expansion protocol. Additional TCR modifications including insertion of a second disulfide bond or the linker modifications evaluated herein did not significantly enhance TCR expression or subsequent in vitro effector function. We conclude that the substitution of a human constant region with a murine constant region was sufficient to increase receptor expression and tetramer binding as well as antitumor activity of the DMF5 TCR and could be a tool to augment other antigen-specific TCR.  相似文献   

12.
YscD is an essential component of the plasmid pCD1-encoded type III secretion system (T3SS) of Yersinia pestis. YscD has a single transmembrane (TM) domain that connects a small N-terminal cytoplasmic region (residues 1 to 121) to a larger periplasmic region (residues 143 to 419). Deletion analyses established that both the N-terminal cytoplasmic region and the C-terminal periplasmic region are required for YscD function. Smaller targeted deletions demonstrated that a predicted cytoplasmic forkhead-associated (FHA) domain is also required to assemble a functional T3SS; in contrast, a predicted periplasmic phospholipid binding (BON) domain and a putative periplasmic "ring-building motif" domain of YscD could be deleted with no significant effect on the T3S process. Although deletion of the putative "ring-building motif" domain did not disrupt T3S activity per se, the calcium-dependent regulation of the T3S apparatus was affected. The extreme C-terminal region of YscD (residues 354 to 419) was essential for secretion activity and had a strong dominant-negative effect on the T3S process when exported to the periplasm of the wild-type parent strain. Coimmunoprecipitation studies demonstrated that this region of YscD mediates the interaction of YscD with the outer membrane YscC secretin complex. Finally, replacement of the YscD TM domain with a TM domain of dissimilar sequence had no effect on the T3S process, indicating that the TM domain has no sequence-specific function in the assembly or function of the T3SS.  相似文献   

13.
Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.  相似文献   

14.
Overcoming the problems associated with the expression, purification and in vitro handling of membrane proteins requires an understanding of the factors governing the folding and stability of such proteins in detergent solutions. As a sequel to our earlier report (Biochim. Biophys. Acta 1747(2005), 133-140), we describe an improved purification procedure and a detailed structural analysis of a fragment of the μ-opioid receptor (‘TM2-3’) that comprises the second and third transmembrane segments and the extracellular loop that connects them. Circular dichroism (CD) spectroscopy of TM2-3 in 2,2,2-trifluoroethanol gave a helical content similar to that predicted by published homology models, while spectra acquired in several detergents showed significantly lower helical contents. This indicates that this part of the μ-opioid receptor has an intrinsic propensity to be highly helical in membrane-like environments, but that in detergent solutions, this helical structure is not fully formed. Proteolysis of TM2-3 with trypsin showed that the helical portions of TM2 and TM3 are both shorter than their predicted lengths, indicating that helix-helix interactions in the full-length receptor are apparently important for stabilizing their conformation. Lengthening the alkyl chain of the detergent led to a small but significant increase in the helicity of TM2-3, suggesting that hydrophobic mismatch could play an important role in the stabilization of transmembrane helices by detergents. Protonation of aspartic acid residues in detergent-solubilized TM2-3 also caused a significant increase in helicity. Our results thus suggest that detergent alkyl chain-length and pH may influence membrane protein stability by modulating the stability of individual transmembrane segments.  相似文献   

15.
Although transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, beta-sheet promoters (Val, Ile, Thr) often account for approximately 40% of TM residue composition. We are examining the conformational role(s) of these residues, using as a model system the major coat protein of the filamentous bacteriophage M13. This 50-residue protein, which is located at the Escherichia coli host membrane during phage reproduction, contains a prototypic 19-residue hydrophobic midregion (residues 21-39: YIGYAWAMVVVIVGATIGI). Using "Eckstein" site-directed mutagenesis, we have generated several viable M13 coat protein mutants with beta-branched amino acid substitutions within their TM region. Mutant coat proteins, including Ile32----Val (I32V) and Ala27----Thr (A27T), were obtained in milligram quantities by growing M13 mutant phages in liter preparations, confirming that these coat proteins are capable of assuming their normal biological function(s) in phage reproduction. Circular dichroism spectroscopy performed in the membrane-mimetic medium of deoxycholate micelles indicated comparable alpha-helical contents of mutants I32V and A27T to wild-type protein. 13C nuclear magnetic resonance experiments with mutant A27T demonstrated that the combination of additional beta-branched content and introduction of an -OH substituent induced chemical shift and temperature-dependent changes and influenced the local protein environment at sites up to 12 residues remote from the mutation site. In contrast, mutant I32V (of which a salient feature is a mid-TM pentavaline segment) behaved very similarly to wild-type coat. These findings are interpreted in terms of the range of TM secondary structure and stability which can be accommodated by viable M13 coat protein mutants.  相似文献   

16.
The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta.  相似文献   

17.
Overcoming the problems associated with the expression, purification and in vitro handling of membrane proteins requires an understanding of the factors governing the folding and stability of such proteins in detergent solutions. As a sequel to our earlier report (Biochim. Biophys. Acta 1747(2005), 133-140), we describe an improved purification procedure and a detailed structural analysis of a fragment of the mu-opioid receptor ('TM2-3') that comprises the second and third transmembrane segments and the extracellular loop that connects them. Circular dichroism (CD) spectroscopy of TM2-3 in 2,2,2-trifluoroethanol gave a helical content similar to that predicted by published homology models, while spectra acquired in several detergents showed significantly lower helical contents. This indicates that this part of the mu-opioid receptor has an intrinsic propensity to be highly helical in membrane-like environments, but that in detergent solutions, this helical structure is not fully formed. Proteolysis of TM2-3 with trypsin showed that the helical portions of TM2 and TM3 are both shorter than their predicted lengths, indicating that helix-helix interactions in the full-length receptor are apparently important for stabilizing their conformation. Lengthening the alkyl chain of the detergent led to a small but significant increase in the helicity of TM2-3, suggesting that hydrophobic mismatch could play an important role in the stabilization of transmembrane helices by detergents. Protonation of aspartic acid residues in detergent-solubilized TM2-3 also caused a significant increase in helicity. Our results thus suggest that detergent alkyl chain-length and pH may influence membrane protein stability by modulating the stability of individual transmembrane segments.  相似文献   

18.
19.
The alphabeta T cell receptor (TCR) can be triggered by a class of ligands called superantigens. Enterotoxins secreted by bacteria act as superantigens by simultaneously binding to an MHC class II molecule on an antigen- presenting cell and to a TCR beta-chain, thereby causing activation of the T cell. The cross-reactivity of enterotoxins with different Vbeta regions can lead to stimulation of a large fraction of T cells. To understand the molecular details of TCR-enterotoxin interactions and to generate potential antagonists of these serious hyperimmune reactions, we engineered soluble TCR mutants with improved affinity for staphylococcal enterotoxin C3 (SEC3). A library of randomly mutated, single-chain TCRs (Vbeta-linker-Valpha) were expressed as fusions to the Aga2p protein on the surface of yeast cells. Mutants were selected by flow cytometric cell sorting with a fluorescent-labeled SEC3. Various mutations were identified, primarily in Vbeta residues that are located at the TCR:SEC3 interface. The combined mutations created a remodeled SEC3-binding surface and yielded a Vbeta domain with an affinity that was increased by 1000-fold (K(D)=7 nM). A soluble form of this Vbeta mutant was a potent inhibitor of SEC3-mediated T cell activity, suggesting that these engineered proteins may be useful as antagonists.  相似文献   

20.
Jittikoon J  East JM  Lee AG 《Biochemistry》2007,46(38):10950-10959
Hydropathy plots have problems in identifying the sequences of transmembrane (TM) alpha-helices when they contain charged residues. Here we show that fluorescence spectroscopy can be used to define the ends of TM alpha-helices. Diacylglycerol kinase (DGK) from Escherichia coli contains three transmembrane (TM) alpha-helices per monomer. We have used fluorescence techniques to define the region of the putative first TM helix (TM1) that spans the hydrophobic core of the lipid bilayer surrounding DGK in reconstituted membranes. Single Cys mutants were introduced into TM1 and flanking sites, in a mutant of DGK lacking the two native Cys residues. Introduction of Cys residues into the region between residues 28 and 34 resulted in mutants with low activities, due to a combination of reduced affinities for ATP and diacylglycerol and a reduced maximum rate. Cross-linking experiments showed that the low-activity mutants were present largely in the normal, trimeric form after reconstitution. Fluorescence emission maxima for the Cys mutants labeled with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD) reconstituted into bilayers of dioleoylphosphatidylcholine varied with position, suggesting that the region of TM1 spanning the hydrophobic core of the bilayer runs from Glu-28 on the cytoplasmic side to Asp-49 or Val-50 on the periplasmic side. This locates the charged/polar cluster 32RQE34 within the hydrophobic core of the bilayer. Fluorescence quenching experiments agree with this assignment for TM1, the results showing a periodicity consistent with distinct stripes of amino acid residues along the length of the helix, the stripes facing the lipid bilayer and facing the rest of the protein, respectively. The residues located close to the glycerol backbone region of the bilayer remained the same when the lipid fatty acyl chain length was changed in the range C14 to C22, showing that hydrophobic matching between the protein and the surrounding lipid bilayer is highly efficient.  相似文献   

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