Structure and topology of a peptide segment of the 6th transmembrane domain of theSaccharomyces cerevisae ?-factor receptor in phospholipid bilayers

Valentine, Kathleen G.; Liu, Shi Feng; Marassi, Francesca M.; Veglia, Gianluigi; Opella, Stanley J.; Ding, Fa Xiang; Wang, Shu Hua; Arshava, Boris; Becker, Jeffrey M.; Naider, Fred

doi:10.1002/1097-0282(20011005)59:4<243::aid-bip1021>3.0.co;2-h

Cited by 50 publications

(40 citation statements)

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“…Recent developments in sample preparation, recombinant bacterial expression systems for the preparation of isotopically labeled membrane proteins, pulse sequences for high-resolution spectroscopy, and structural indices that guide the structure assembly process, have greatly extended the capabilities of the technique. The structures of a variety of membrane peptides and proteins have been examined using this approach, and several atomic-resolution structures have been determined and deposited in the PDB (Ketchem et al 1993, Valentine et al 2001, Park et al 2003. In this chapter, the methods are illustrated with examples from the Bcl-2 family proteins, which regulate a major mechanism for commitment to programmed cell death (apoptosis), and play critical roles in tissue development, differentiation, and homeostasis.…”

Section: Solid-state Nmr Spectroscopysupporting

confidence: 88%

Structural Basis for Bc12-Regulated Mitochondrion-Dependent Apoptosis

Marassi¹

2005

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Public reporting burden for this collection of information is estimated to average 1 hour per response, induding the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and The Bcl-2 family proteins are key regulators of programmed cell death, in health and major human diseases, including cancer. Their pro-or anti-apoptotic functions are regulated by subcellular location, as the proteins cycle between soluble and membrane-bound forms; by dimerization with other Bcl-2 family members; by binding to other non-homologous proteins; and by formation of membrane pores that are believed to regulate apoptosis by perturbing mitochondrial physiology. The solution structures of several Bcl-2 family proteins are very similar despite their antagonistic activities, however, the structures of the membrane-associated proteins are not known and may be key to their opposing functions. The goals of this project are: (1) to determine the structures of the meribrane-asaociated Bd-2 proteins; and (2) to determine their mechanism of apoptosis regulation. The research stategy combines NMR structure determination in lipid environments with biological assays carried out in parallel with structure determination. INTRODUCTIONThe goal of this project was to determine the structures of the Bcl-2 family proteins in their membraneassociated forms. The Bcl-2 family proteins play major regulatory roles in programmed cell death, or apoptosis, and exert their activities through dimerization with other Bcl-2 family members, binding to nonhomologous proteins, and the formation of membrane pores, believed to regulate apoptosis by perturbing mitochondrial physiology. Their functions are also regulated by subcellular location as the proteins cycle between soluble and membrane-bound forms. The soluble structures of several pro-and anti-apoptotic Bcl-2 family proteins are very similar, despite their antagonistic activities, however, the structures of their membrane-associated forms have not been previously examined, and may be important for understanding their opposing functions. Moreover, most of the previous structural and functional studies have focused on soluble truncated proteins, lacking the C-terminal 20-residue hydrophobic domain, which is present in many of the family members and is important for membrane targeting.The support of this grant has enabled us to initiate a major research effort to determine the structures of membrane-bound and full-length Bcl-2 proteins. The results from these studies serve as a platform for additional structural and biological experiments aimed at examining the structural consequences of modificat...

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Section: Solid-state Nmr Spectroscopysupporting

confidence: 88%

Structural Basis for Bc12-Regulated Mitochondrion-Dependent Apoptosis

Marassi¹

2005

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“…This conclusion is consistent with the findings on the WALP peptides that reported small tilt angles (ϳ10 -15°) for peptides shorter than the bilayer and large tilt angles (ϳ30°) for peptides expected to be significantly longer than the bilayer (58). It is also consistent with results on synthetic peptide segments of bacteriorhodopsin that contained 35-45 residues and also exhibited tilt angles between 30 and 40°(59) and with findings on an 18-residue peptide fragment of the sixth TMD of Ste2p that had a very small tilt angle (22).…”

Section: Discussionmentioning

confidence: 99%

“…Ultimately, the peptide was elongated to 35 residues. In this strategy, seven portions of resin with peptide chain lengths of 10,14,18,22,26,30, and 35 were obtained during one batch synthesis. The synthesis of M2-30B was carried out using a Wang resin (0.4 mmol of OH/g) where the first Fmoc-Leu-OH was loaded manually as described above.…”

Section: Methodsmentioning

confidence: 99%

“…This G protein-coupled receptor recognizes the tridecapeptide phero-mone (WHWLQLKPGQPMY-␣-factor) and triggers a cascade of intracellular events ultimately resulting in sexual conjugation and diploid formation (16,17). Studies on fragments of the receptor using CD (18,19), IR (20), and NMR spectroscopies (21,22) have revealed the conformational tendencies of the seven transmembrane domains and, in the case of IR analysis, gave information on the orientation of the peptides in lipid bilayers.…”

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confidence: 99%

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The Chain Length Dependence of Helix Formation of the Second Transmembrane Domain of a G Protein-coupled Receptor ofSaccharomyces cerevisiae

Ding¹,

Schreiber²,

VerBerkmoes³

et al. 2002

Journal of Biological Chemistry

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The chain length dependence of helix formation of transmembrane peptides in lipids was investigated using fragments corresponding to the second transmembrane domain of the ␣-factor receptor from Saccharomyces cerevisiae. Seven peptides with chain lengths of 10 (M2-10; FKYLLSNYSS), 14 (M2-14), 18 (M2-18), 22 (M2-22), 26 (M2-26), 30 (M2-30) and 35 (M2-35; RSRKTPIFIINQVSLFLIILH-SALYFKYLLSNYSS) residues, respectively, were synthesized. CD spectra revealed that M2-10 was disor-dered, and all of the other peptides assumed partially ␣-helical secondary structures in 99% trifluoroethanol (TFE)/H 2 O. In 50% TFE/H 2 O, M2-30 assumed a ␤-like structure. The other six peptides exhibited the same CD patterns as those found in 99% TFE/H 2 O. In 1,2-dimyristoyl-snglycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1 ratio) vesicles, M2-22, M2-26, and M2-35 formed ␣-helical structures, whereas the other peptides formed ␤-like structures. Fourier transform infrared spectroscopy in 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphorac-(1-glycerol) (4:1) multilayers showed that M2-10, M2-14, M2-18, and M2-30 assumed ␤-structures in this environment. Another homologous 30-residue pep-tide (M2-30B), missing residues SNYSS from the N terminus and extending to RSRKT on the C terminus, was helical in lipid bilayers, suggesting that residues at the termini of transmembrane domains influence their biophysical properties. Attenuated total reflection Fourier transform infrared spectroscopy revealed that M2-22, M2-26, M2-30B, and M2-35 were ␣-helical and oriented at angles of 12°, 13°, 36°, and 34°, respectively, with respect to the multilayer normal. This study showed that chain length must be taken into consideration when using peptides representing single transmembrane domains as surrogates for regions of an intact receptor. Furthermore, this work indicates that the tilt angle and conformation of transmembrane portions of G protein-coupled receptors may be estimated by detailed spectroscopic measurements of single transmembrane peptides.The folding and structure of integral membrane proteins is driven by entropic factors that cause the apolar side chains of many amino acid residues to seek the interior of lipid bilayers and enthalpic factors that require that the hydrogen bond potential of the peptide group be satisfied in the low dielectric membrane interior (1). Thermodynamic analyses indicate that the ␣-helix is the most favored conformation of membranespanning regions of proteins. Indeed, it is believed that preassembled ␣-helices form in the aqueous cytoplasm of the cell and then insert into the bilayer (2). The thickness of the hydrocarbon milieu of the bilayer depends on the fatty acid composition and is often assumed to be about 30 Å. On this basis, the minimum number of residues that can form an ␣-helix and span the bilayer is predicted to be 20. However, this prediction assumes that the peptide inserts into the membrane parallel to the bilayer normal.Recent x-ray stud...

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“…O mesmo grupo também observou por espectroscopia de fluorescência que esse peptídeo era capaz de atravessar a membrana (63), O grupo de Naider et aI. (64)(65)(66)(67) também têm tido uma contribuição relevante na literatura ao estudar fragmentos sintéticos correspondentes a TMs de um GPCR de Saccharomyces cerevisiae. Esse grupo observou que com exceção das TMs 3 e 6 na presença de modelos de membrana, todos os outros peptídeos adotaram conformação ahelicoidal na presença de TFE, misturas de TFE-água e na presença de micelas de detergentes e de vesículas fosfolipídicas (64).…”

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Estudos conformacionais de seqüências hidrofóbicas de domínios de receptores acoplados a proteínas G (GPCR) e da proteína G. Um estudo de CD e espectroscopia de fluorescência

Casallanovo¹

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AgradecimentosAo pessoal do laboratório, Salay, Felipe, Myriam, Fábio Dyszy, Tatiana e Marcelo, obrigado pela paciência e pela ajuda. À Aninha, obrigado pelos papos, discussões e pelo apoio.Ao Carlos Àlvares, que me ajudou muito nos momentos finais.Ao pessoal que passou pelo LBE, Raquel, Berê, Noboru, Roberto Salinas, Goretti, Thelma e Mercedes.Ã Renata Fischer, pela amizade.Ao Claúdio S. Shida, que me ajudou muito em vários momentos, obrigado pelos conselhos e pela amizade.Aos meus amigos Eroc, Paulo, Luís Mayer e Luiz, obrigado pelo apoio e por me agüentar nos momentos mais dificeis.À Lua, que é uma pessoa que eu gosto muito e com a qual tive uma ótima amizade desde o primeiro dia em que entrei no laboratório como aluno de iniciação científica. Obrigado pelos conselhos e pelos momentos de alegria. À Dra. Shirley, que é uma pessoa muito especial. Poucas pessoas sabem contar estórias e atrair a admiração dos outros como você. Com você aprendi muito, sempre admirei sua postura e inteligência. Você também soube ser muito compreensiva nos momentos mais dificeis.À Fapesp e ao CNPq, obrigado pelo apoio finaceiro.o. Índice 1 Índice Lista de abreviações .................................................................................................................................... iv Resumo ........................................................................................................................................................ v O peptídeo estudado mostrou-se muito sensível à força iônica, pH e TFE, observando-se mudança de estrutura ao acaso para a-hélice em alguns pHs e na presença de TFE e para folha ~ com o aumento da força iônica.Na presença de detergentes, observou-se que o peptídeo era capaz de ligar-se tanto a interfaces com carga líquida zero (HPS e LPC) quanto com carga líquida negativa. Observou-se também que o CG tem maior afinidade por LPC do que por HPS.O estudo do CG em diferentes condições forneceu subsídios para investigannos a interação deste fragmento com regiões do receptor A TIA de angiotensina 11 que estão envolvidas com a ativação da proteína G. analog displayed the highest propensity to acquire a-helical conformation in the presence of TFE and in aqueous solution.In the presence of detergents we observed that the peptides were able to bind to different interfaces, especially negatively charged ones. In the presence of phospholipid membranes, all peptides displayed p-sheet conformation.In order to study the effect of aggregation, we performed experiments in the presence of denaturing agents such as urea and as a function of temperature. Even in the presence of high urea concentrations and at higher temperatures, the fragments remained aggregated. This result led us to another approach, where peptide films were hydrated in the presence of detergents. Using this methodology it was observed that the fragments were less aggregated, indicating that under adequate conditions the peptides tend to adopt a-helical conformation. In summary, it was possible to observe experimentall...

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Structure and topology of a peptide segment of the 6th transmembrane domain of theSaccharomyces cerevisae ?-factor receptor in phospholipid bilayers

Cited by 50 publications

References 66 publications

Structural Basis for Bc12-Regulated Mitochondrion-Dependent Apoptosis

Structural Basis for Bc12-Regulated Mitochondrion-Dependent Apoptosis

The Chain Length Dependence of Helix Formation of the Second Transmembrane Domain of a G Protein-coupled Receptor ofSaccharomyces cerevisiae

Estudos conformacionais de seqüências hidrofóbicas de domínios de receptores acoplados a proteínas G (GPCR) e da proteína G. Um estudo de CD e espectroscopia de fluorescência

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