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1.
BMC Biotechnol ; 14: 111, 2014 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-25540031

RESUMEN

BACKGROUND: Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form. RESULTS: The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25 °C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach. CONCLUSION: rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.


Asunto(s)
Cisteína/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/química , Vacunas contra la Influenza/inmunología , Gripe Humana/virología , Animales , Cisteína/genética , Cisteína/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Gripe Humana/inmunología , Gripe Humana/prevención & control , Ratones , Ratones Endogámicos BALB C , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología
2.
FASEB J ; 25(7): 2234-44, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21402716

RESUMEN

We report how rotational variations in transmembrane (TM) helix interactions participate in the activity states of the thrombopoietin receptor (TpoR), a type 1 cytokine receptor that controls the production of blood platelets. We also explore the mechanism of small-molecule agonists that do not mimic the natural ligand. We show, by a combination of cysteine cross-linking, alanine-scanning mutagenesis, and computational simulations, that the TpoR TM dimerizes strongly and can adopt 3 different stable, rotationally related conformations, which may correspond to specific states of the full-length receptor (active, inactive, and partially active). Thus, our data suggest that signaling and inactive states of the receptor are related by receptor subunit rotations, rather than a simple monomer-dimer transition. Moreover, results from experiments with and without agonists in vitro and in cells allow us to propose a novel allosteric mechanism of action for a class of small molecules, in which they activate TpoR by binding to the TM region and by exploiting the rotational states of the dimeric receptor. Overall, our results support the emerging view of the participation of mutual rotations of the TM domains in cytokine receptor activation.


Asunto(s)
Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Trombopoyetina/química , Regulación Alostérica , Secuencia de Aminoácidos , Simulación por Computador , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Piperidinas/química , Piperidinas/farmacología , Conformación Proteica , Receptores de Trombopoyetina/agonistas , Receptores de Trombopoyetina/genética , Rotación
3.
Cell ; 127(3): 447-50, 2006 Nov 03.
Artículo en Inglés | MEDLINE | ID: mdl-17081964

RESUMEN

Studying how protein transmembrane domains transmit signals across membranes is beset by unique challenges. Here, we discuss the circumstances that have led to success and reflect on what has been learned from these examples. Such efforts suggest that some of the most interesting properties of transmembrane helix interactions may be the least amenable to study by current techniques.


Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Transducción de Señal , Membrana Celular/química , Membrana Celular/genética , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/genética , Micelas , Modelos Moleculares , Modelos Estructurales , Mutación , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo
4.
J Am Chem Soc ; 128(35): 11338-9, 2006 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-16939241

RESUMEN

Folded polymers in nature are assembled from simple monomers and adopt complex folded structures through networks of stabilizing noncovalent interactions. These interactions define secondary and tertiary structure and in most cases specify a unique three-dimensional architecture. Individual secondary or tertiary structures can also associate with one another to form multi-subunit quaternary structures. Nonnatural folded polymers have potential for similar structural versatility. Here we describe a pair of beta3-peptides whose sequences were designed to promote a 14-helix structure in water, favor hetero-oligomer formation, and disfavor nonspecific aggregation. These beta3-peptides assemble noncovalently into a well-defined hetero-oligomer characterized by a defined stoichiometry, a highly stabilized secondary structure, and a cooperative melting transition (TM > 55 degrees C). This work demonstrates that beta3-peptides can assemble into defined, cooperatively folded quaternary structures and constitutes an important step toward designing protein-like assemblies from nonnatural polymers.


Asunto(s)
Aminoácidos/química , Péptidos/química , Proteínas/química , Dicroismo Circular , Conformación Proteica , Pliegue de Proteína , Estructura Cuaternaria de Proteína
5.
Biochemistry ; 45(16): 5228-34, 2006 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-16618111

RESUMEN

The transmembrane (TM) domain of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) has long been implicated in both correct folding and function of the MHC class II complex. To function correctly, Ii must form a trimer, and the TM domain is one of the domains thought to stabilize the trimeric state. Specific mutations in the TM domain have been shown previously to disrupt MHC class II functions such as mature complex formation and antigen presentation, possibly due to disruption of Ii TM helix-helix interactions. Although this hypothesis has been reported several times in the literature, thus far no experimental measurements have been made to explore the relationship between TM domain structure and TM mutations that affect Ii function. We have applied biophysical and computational methods to study the folding and assembly of the Ii TM domain in isolation and find that the TM domain strongly self-associates. According to analytical ultracentrifugation analyses, the primary oligomeric state for this TM domain is a strongly associated trimer with a dissociation constant of approximately 120 nM in DPC micelles. We have also examined the effect of functionally important mutations of glutamine and threonine residues in the TM domain on its structure, providing results that now link the disruption of TM helix interactions to previously reported losses of Ii function.


Asunto(s)
Antígenos de Diferenciación de Linfocitos B/química , Antígenos de Diferenciación de Linfocitos B/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Detergentes/farmacología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido
6.
J Mol Biol ; 355(4): 697-707, 2006 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-16325852

RESUMEN

The Escherichia coli DEAD-box protein A (DbpA) belongs to the highly conserved superfamily-II of nucleic acid helicases that play key roles in RNA metabolism. A central question regarding helicase activity is whether the process of coupling ATP hydrolysis to nucleic acid unwinding requires an oligomeric form of the enzyme. We have investigated the structural and functional properties of DbpA by multi-angle laser light-scattering, size-exclusion chromatography, analytical ultracentrifugation, chemical cross-linking and hydrodynamic modeling. DbpA is monomeric in solution up to a concentration of 25 microM and over the temperature range of 4 degrees C to 22 degrees C. Binding of neither nucleotide (ATP or ADP) nor peptidyl transferase center (PTC) RNA, the presumed physiological RNA substrate, favor oligomerization. The hydrodynamic parameters were used together with hydrodynamic bead modeling and structural homology in conjunction with ab initio structure prediction methods to define plausible shapes of DbpA. Collectively, the results favor models where DbpA functions as an active monomer that possesses two distinct RNA binding sites, one in the helicase core domain and the other in the carboxyl-terminal domain that recognizes 23S rRNA and interacts specifically with hairpin 92 of the PTC.


Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , ARN Helicasas/química , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Cromatografía en Gel , Computadores , Reactivos de Enlaces Cruzados/química , ARN Helicasas DEAD-box , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Biológicos , Modelos Moleculares , Estructura Terciaria de Proteína , ARN/metabolismo , ARN Helicasas/genética , Proteínas de Unión al ARN/genética , Homología Estructural de Proteína
7.
FEBS Lett ; 555(1): 122-5, 2003 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-14630331

RESUMEN

The folding of alpha-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable alpha-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.


Asunto(s)
Proteínas de la Membrana/química , Acuaporinas/química , Proteínas Bacterianas/química , Bacteriorodopsinas/química , Sitios de Unión , Proteínas de Escherichia coli/química , Ligandos , Membrana Dobles de Lípidos/química , Modelos Moleculares , Canales de Potasio/química , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Termodinámica
8.
J Am Chem Soc ; 125(14): 4022-3, 2003 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-12670203

RESUMEN

beta-Peptides have attracted considerable attention by virtue of their ability to populate helical secondary structures in methanol, even in the absence of stabilizing tertiary interactions. Recent efforts in beta-peptide design have produced few beta3-peptides that form stable 14-helices in water; those that do require stabilizing intramolecular salt bridges on two of three helical faces and therefore possess limited utility as tools in biological research. Here we show that favorable interactions with the 14-helix macrodipole significantly stabilize the 14-helix in water, alleviating the need for multiple salt bridges on two of three helical faces. We also report the previously unrecognized stabilization of 14-helix structure by gamma-branched beta3-amino acids. The most structured molecules we describe are highly heterogeneous at the primary sequence level, containing seven different beta3-amino acids within an 11-residue sequence. These results represent the essential first step toward the design of well-folded 14-helices that explore the interactions between beta3-peptides and biological macromolecules in vitro and in vivo.


Asunto(s)
Aminoácidos/química , Péptidos/química , Estructura Secundaria de Proteína , Agua/química , Dicroismo Circular , Péptidos/síntesis química
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