39 research outputs found

    Structural studies of fragments of G-protein coupled receptors and their ligands by NMR

    Full text link
    In the course of my doctoral studies I characterized the structure and dynamics of G-protein coupled receptor (GPCRs) fragments and their ligands by high-resolution NMR. The receptors of the GPCR family are transmembrane proteins of prime biological importance. All members of this family possess similar architecture of seven membrane-spanning α-helices and are involved in various signal transduction processes. First part of my work is devoted to the investigation of the structural determinants of the GPCR ligand peptide YY and monitoring the folding process of this peptide in solution. PYY is a 36- residue C-terminally amidated polypeptide that belongs to the neuropeptide Y family of peptide hormones. These molecules are involved in the regulation of a variety of physiological processes, such as for example food uptake. In the second part of my thesis I directed my efforts towards elucidation of the structure and probing the dynamic properties of the transmembrane fragments of the GPCRs in native-like environments. The subject of my studies was the -factor G-protein coupled Ste2p receptor, which is involved in sensing pheromones in yeast. Two large polypeptide fragments including the first and the second (peptide TM1TM2) and the seventh (peptide TM7) transmembrane domains of the Ste2p receptor were structurally characterized in micellar solution. The obtained results provide important insights into the GPCR architecture in a membrane bilayer. In the first part of my work I focused on the structural determinants and the folding process of the peptide YY (PYY) in solution. Some of the peptides from neuropeptide Y family adopt a well-defined hairpin structure in water that was first shown for avian pancreatic peptide (aPP) using X-ray crystallography. This helical hairpin is commonly referred to as PP-fold and is characterized by a N-terminal polyproline helix, which is back-folded via a-turn onto a C-terminal -helix. The solution structure of the PYY displayed a highly similar helical hairpin, however in the highly homologous neuropeptide Y we were surprised by the absence of the tertiary structure. To investigate the significance of the tertiary contacts, Tyr and Pro residues at the hydrophobic interface of the hairpin- type structure of PYY were replaced by Ala residues, and the conformational and dynamical properties of the resulting peptides were analyzed by high-resolution NMR spectroscopy. Previously we established the 15N{1H}-NOE as a convenient method to quantify the extent of back-folding. A comparison of the data from different Ala mutant peptides to those of native PYY nicely reflected the differences in backbone rigidity of the N-terminus. Most of the Pro->Ala or the Tyr->Ala mutants possessed increased backbone dynamics, and the differences in N-terminal mobility among them reflected various degrees to which they sample conformations close to the PP-fold. By varying temperature or the methanol content of the aqueous solvent and monitoring chemical shifts we followed the residue-specific formation of tertiary contacts while changing the physical or chemical environment. The PYY peptide in methanol solution was characterized both by determining its solution structure as well as by its internal backbone dynamics as derived from 15N relaxation data. The latter is characterized by a complete loss of tertiary structure. Chemical shifts of Cα in the heat-denaturation experiments displayed sigmoidal curves with very similar points of inflection indicating that both secondary, as well as tertiary structure in the heat denaturation, was lost synchronously. The results suggest that helical hairpin formation in PYY peptide is both reversible and cooperative and that specific N- and C-terminal tertiary hydrophobic contacts between the polyproline and the -helix promote the folding process. In addition, structural analysis of substitutions in the turn region indicates that the loop does not constrain the hairpin structure. The results may also have implications for our understanding of the binding of these peptides to their receptors. In the second part of the thesis the structure and dynamics of two large fragments of Ste2p the G-protein coupled -factor receptor from yeast were investigated. Both GPCR fragments were expressed and purified by our colleagues from the group of Prof. Fred Naider (College of Staten Island, NY). At first I investigated the 73-residue (Ste2p(267-339)) peptide TM7 consisting of the 3rd extracellular loop, the 7th transmembrane helix and 40 residues from the cytosolic C-terminal domain in dodecylphosphocholine micelles using solution NMR spectroscopy. The structure revealed the presence of an -helix in the segment encompassing residues 10 to 30, which was perturbed around the internal Pro24 residue. 15N-relaxation and RDC data supported a rather stable fold for the transmembrane part of TM7, whereas the exposed segments were more flexible. Spin-label data indicated that the TM7 helix was integrated into dodecylphosphocholine micelles, but displayed flexibility around the internal Pro24 site, exposing residues 22 to 26 to solution and revealed a second site of interaction with the micelle within a region comprising residues 43-58, which formed part of a less well- defined nascent helix. Further I extended my work on a single membrane-spanning TM7 fragment to a longer 80-residue (Ste2p(31-110)) double membrane-spanning peptide TM1TM2, consisting of 19 residues from the N-terminal domain, the 1st transmembrane helix, the first cytoplasmic loop, the second transmembrane helix and 7 residues from the first extracellular loop of the Ste2p receptor. Because of the larger complexity of a double membrane-spanning fragment different isotope labeling patterns were utilized including [15N], [15N, 13C], [15N, 13C, 2H]-labeled and selectively [15N]-labeled at specific amino acid residues or protonated only at selected methyl groups peptides. The structure of TM1TM2 peptide in lyso-palmitoylphosphatidylglycerol micelles revealed the presence of three-helices encompassing residues 39-47, 49-72 and 80-103, with higher flexibility around the internal Arg58 site of the first transmembrane domain. Several long-range interhelical NOE connectivities supported the folding of TM1TM2 into a tertiary structure forming a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G56VRSG60 region. 15N-relaxation and hydrogen-deuterium exchange data support a stable fold for the transmembrane parts of TM1TM2, whereas the solvent-exposed segments were more flexible. Interestingly the NMR structure was consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor. The results obtained during my Ph.D. studies reveal important aspects of the GPCR ligand peptide PYY structure and folding in solution so as shed light on the structure of large fragments of yeast pheromone receptor Ste2p in native-like micellar environment. Zusammenfassung Im Laufe meiner Promotion habe ich die Struktur und Dynamik von G-Protein- gekoppelte Rezeptor-(GPCRs) Fragmenten und ihren Liganden mittels hochauflösender NMR charakterisiert. Die Rezeptoren der GPCR-Familie sind Transmembran-Proteine von zentraler biologischer Bedeutung. Alle Mitglieder dieser Familie besitzen eine Ă€hnliche Architektur mit sieben transmembranĂ€ren α-Helices, und nehmen in verschiedenen Signaltransduktionsprozessen teil. Der erste Teil meiner Arbeit widmet sich der Untersuchung der strukturellen Determinanten des GPCR Liganden Peptid YY und der Verfolgung des Faltungsprozesses dieses Peptids in Lösung. PYY ist ein Polypeptid mit 36 AminosĂ€uren und C-terminaler Amidierung, das zu der Neuropeptid Y-Familie von Peptid-Hormonen gehört. Diese MolekĂŒle sind in der Regulation einer Vielzahl physiologischer Prozesse involviert, wie zum Beispiel bei der Lebensmittelaufnahme. Im zweiten Teil meiner Arbeit richtete ich meine BemĂŒhungen auf die AufklĂ€rung der Struktur und die dynamischen Eigenschaften der Transmembran-Fragmente der GPCRs in nativen Bedingungen. Das Thema meiner Studien war der α-Faktor G-Protein-gekoppelter Rezeptor Ste2p, der involviert in der Pheromonerkennung in Hefe ist. Zwei große Polypeptid-Fragmente, bestehend aus der ersten und zweiten (Peptid TM1TM2) und der siebten Transmembran-DomĂ€n (Peptid TM7) des Ste2p-Rezeptors, wurden in micellĂ€rer Lösung strukturell charakterisiert. Die Ergebnisse liefern wichtige Einblicke in die GPCR-Architektur in einem Membran-Bilayer. Im ersten Teil meiner Arbeit konzentrierte ich mich auf die strukturellen Faktoren und den Faltungsprozess des Peptid YY (PYY) in Lösung. Einige der Peptide aus Neuropeptid Y-Familie haben eine klar definierte hairpin-Struktur in Wasser; diese wurde zum ersten Mal gezeigt fĂŒr das Avian Pankreas-Peptid mittels Röntgenstrahl-Kristallographie. Dieser helikale ‘hairpin’ wird gemeinhin als PP-fold bezeichnet und besteht aus einer N-terminalen Polyprolin-Helix, die zurĂŒckfaltet ĂŒber einen ÎČ-turn auf eine C-terminale α-Helix. Die Lösungsstruktur des PYY zeigt einen sehr Ă€hnlichen helikalen ‘hairpin’, jedoch im hoch-homologen Neuropeptid Y beobachteten wir zu unserem Erstauenen keine TertiĂ€rstruktur. Um die Bedeutung der tertiĂ€ren Kontakte zu untersuchen, wurden Tyr- und Pro-Reste an der hydrophoben OberflĂ€che der ‘hairpin’- Struktur von PYY ersetzt durch Alanin und die konformationellen und dynamischen Eigenschaften der resultierenden Peptide wurden analysiert mittels hochauflösender NMR-Spektroskopie. Zuvor haben wir die 15N{1H}-NOE als eine passende Methode zur Quantifizierung des Umfangs der RĂŒckfaltung etabliert. Ein Vergleich der Daten aus unterschiedlichen Ala-Peptid- Mutanten mit dem nativen PYY spiegelt schön die Unterschiede in der Steifheit des ‘backbones’ des N-Terminus wieder. Die meisten der Pro-> Ala oder der Tyr-> Ala Mutanten besaßen eine erhöhte ‘backbone’-Dynamik, und die Unterschiede in der N-terminalen MobilitĂ€t unter ihnen spiegelt verschiedene Grade wieder, zu dem sie Probe Konformationen annimmt, die dem ‘PP-fold’ Ă€hneln. Durch Variation der Temperatur oder des Methanolgehalts des wĂ€ssrigen Lösungsmittels und Verfolgung des ‘chemical shift’ konnten wir die aminosĂ€ure-spezifische Bildung der TertiĂ€rkontakte wĂ€hrend der Änderung der physikalischen oder chemischen Umgebung verfolgen. Das PYY Peptid in Methanollösung wurde charakterisiert sowohl durch die Bestimmung seiner Lösungsstruktur als auch durch ihre interne ‘backbone’-Dynamik mittels 15N-relaxation-Daten. Die ‘backbone’-Dynamik zeichnet sich durch einen vollstĂ€ndigen Verlust der tertiĂ€ren Struktur aus. Die ‘Chemical shifts’ der Cα in den Hitze-Denaturierungs-Experimenten zeigten sigmoidale Kurven mit sehr Ă€hnliche Wendepunkten, was darauf hinweist, dass sowohl SekundĂ€r- als auch TertiĂ€rstruktur in der Hitzedenaturierung synchron verloren werden. Die Ergebnisse deuten darauf hin, dass die Bildung des helikalen ‘hairpin’ im PYY Peptid reversibel und kooperativ ist und dass spezifische N-und C-terminale hydrophobe TertiĂ€rkontakte zwischen der Polyprolinhelix und der α-Helix den Faltungsprozess fördern. DarĂŒber hinaus deutet die Strukturanalyse von Substitutionen in der ‘turn’-Region darauf hin, dass der ‘loop’ die ‘hairpin’-Struktur nicht hemmt. Die Ergebnisse können auch Auswirkungen fĂŒr unser VerstĂ€ndnis der Bindung dieser Peptide auf ihren Rezeptoren haben. Im zweiten Teil der Dissertation wurde die Struktur und Dynamik von zwei großen Fragmenten von Ste2p, dem G-Protein-gekoppelten α-Faktor-Rezeptor von Hefe untersucht. Beide GPCR-Fragmente wurden exprimiert und aufgereinigt von unseren Kollegen aus der Arbeitsgruppe von Prof. Fred Naider (College of Staten Island, NY). Zuerst untersuchte ich das 73-aminosĂ€ure-Peptid TM7 (Ste2p (267-339)) bestehend aus dem dritten extrazellulĂ€ren ‘loop’, der siebten Transmembran-Helix und 40 AminosĂ€uren aus der zytosolische C-terminalen DomĂ€ne in Dodecylphosphocholin- Micellen mittels NMR-Spektroskopie. Die Struktur offenbarte die Anwesenheit einer α-Helix im Segment von AminosĂ€urerest 10 bis 30, die um das interne Pro24 gestört wird. 15N-relaxation und RDC-Daten unterstĂŒtzten einen recht stabilen ‘fold’ fĂŒr den Transmembran-Anteil des TM7, hingegen die ausgesetzten Segmente waren flexibler. Die Spin-Label-Daten weisten darauf hin, dass die TM7-Helix in die Dodecylphosphocholin-Micellen integriert wurde, aber zeigten FlexibilitĂ€t rund um das interne Pro24, da die AminosĂ€uren 22 bis 26 in die Lösung zeigen, desweiteren zeigten sie einen zweiten Interaktionsort mit der Micelle innerhalb der Region von AminosĂ€urerest 43 bis 58, die einen Teil einer weniger gut definierten im Entstehen begriffenen Helix bildet. Im weiteren verlĂ€ngerte ich meine Arbeit an einem einfachen Transmembran-Fragment TM7 zu einem lĂ€ngeren 80-AminosĂ€ure-Doppel-Transmembran-Peptid TM1TM2 (Ste2p (31-110)), bestehend vom 19 AminosĂ€uren aus der N-terminalen DomĂ€ne, die erste Transmembran-Helix, der erste zytoplasmatische ‘loop’, die zweite Transmembran-Helix und 7 AminosĂ€uren aus dem ersten extrazellulĂ€ren ‘loop’ des Ste2p-Rezeptors. Aufgrund der grĂ¶ĂŸeren KomplexitĂ€t des doppelten Transmembran-Fragments wurden verschiedene Isotopen-Labeling-Muster genutzt: [15N], [15N, 13C], [15N, 13C, 2H]-markiert und selektiv [15N]- markiert an bestimmten AminosĂ€uren oder protoniert nur an ausgewĂ€hlten Methyl-Gruppen-Peptiden. Die Struktur des TM 1 TM 2-Peptids in LYSO-palmitoylphosphatidylglycerol-Micellen zeigte das Vorhandensein von drei α-Helices, von AminosĂ€ure 39-47, 49-72 und 80-103, mit einer grĂ¶ĂŸeren FlexibilitĂ€t rund um das interne Arg58 der ersten Transmembran-DomĂ€ne. Mehrere ‘long range-interhelical NOE’ Verbindungen unterstĂŒtzen die Faltung von TM1TM2 in eine TertiĂ€rstruktur, die eine gekreuzte Helix bildet, die sich ausdehnt in Richtung der extrazellulĂ€ren Regionen und die erhebliche FlexibilitĂ€t in der G56VRSG60 Region enthĂ€lt. 15N-relaxation- und Wasserstoff-Deuterium-Austausch-Daten unterstĂŒtzten einen stabilen ‘fold’ fĂŒr die Transmembran-Teile von TM1TM2, wĂ€hrend die lösungsmittel-exponierten Segmente flexibler waren. Interessanterweise ist die NMR-Struktur im Einklang mit den Ergebnissen der biochemischen Experimente, die die Ligandenbindungsort in dieser Region des Rezeptors identifizierten. Die erzielten Ergebnisse wĂ€hrend meiner Promotionsstudien zeigen wichtige Aspekte der GPCR-Peptid-Liganden PYY-Struktur und seiner Faltung in der Lösung, sowie geben sie Aufschluss ĂŒber die Struktur der großen Fragmente des Hefe- Pheromon-Rezeptor Ste2p in nativer Micellenumgebung

    1H, 13C and 15N assignment of the GNA1946 outer membrane lipoprotein from Neisseria meningitidis

    Get PDF
    GNA1946 (Genome-derived Neisseria Antigen 1946) is a highly conserved exposed outer membrane lipoprotein from Neisseria meningitidis bacteria of 287 amino acid length (31 kDa). Although the structure of NMB1946 has been solved recently by X-Ray crystallography, understanding the behaviour of GNA1946 in aqueuos solution is highly relevant for the discovery of the antigenic determinants of the protein that will possibly lead to a more efficient vaccine development against virulent serogroup B strain of N.meningitidis. Here we report almost complete 1H, 13C and 15N resonance assignments of GNA1946 (residues 10–287) in aqueous buffer solution

    NMR Investigation of Structures of G-Protein Coupled Receptor Folding Intermediates

    Get PDF
    Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot et al., Biochemistry 29(1990), 4031) is postulated to proceed in 2 steps: Partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin et al., Biophys. J. 96(2009), 3187), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123 and TM127 adopt predominantly native-like topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior

    Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments

    Full text link
    Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 1 of the alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG) [1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol (TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information

    Moss-like Hierarchical Architecture Self-Assembled by Ultrathin Na2Ti3O7 Nanotubes: Synthesis, Electrical Conductivity, and Electrochemical Performance in Sodium-Ion Batteries

    No full text
    Nanocrystalline layer-structured monoclinic Na2Ti3O7 is currently under consideration for usage in solid state electrolyte applications or electrochemical devices, including sodium-ion batteries, fuel cells, and sensors. Herein, a facile one-pot hydrothermal synthetic procedure is developed to prepare self-assembled moss-like hierarchical porous structure constructed by ultrathin Na2Ti3O7 nanotubes with an outer diameter of 6–9 nm, a wall thickness of 2–3 nm, and a length of several hundred nanometers. The phase and chemical transformations, optoelectronic, conductive, and electrochemical properties of as-prepared hierarchically-organized Na2Ti3O7 nanotubes have been studied. It is established that the obtained substance possesses an electrical conductivity of 3.34 × 10−4 S/cm at room temperature allowing faster motion of charge carriers. Besides, the unique hierarchical Na2Ti3O7 architecture exhibits promising cycling and rate performance as an anode material for sodium-ion batteries. In particular, after 50 charge/discharge cycles at the current loads of 50, 150, 350, and 800 mA/g, the reversible capacities of about 145, 120, 100, and 80 mA∙h/g, respectively, were achieved. Upon prolonged cycling at 350 mA/g, the capacity of approximately 95 mA∙h/g at the 200th cycle was observed with a Coulombic efficiency of almost 100% showing the retention as high as 95.0% initial storage. At last, it is found that residual water in the un-annealed nanotubular Na2Ti3O7 affects its electrochemical properties
    corecore