RESUMEN
Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli, had unprecedentedly high binding affinities (Kd â¼10-12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.
Asunto(s)
Anticuerpos contra la Hepatitis B/inmunología , Antígenos e de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Hepatitis B/sangre , Hepatitis B/tratamiento farmacológico , Anticuerpos contra la Hepatitis B/química , Anticuerpos contra la Hepatitis B/genética , Anticuerpos contra la Hepatitis B/uso terapéutico , Antígenos e de la Hepatitis B/sangre , Antígenos e de la Hepatitis B/química , Virus de la Hepatitis B/química , Humanos , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/uso terapéuticoRESUMEN
Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (Kd ≈ 10-8 to 10-9 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (Kd ≈ 10-7 M), but those retaining exon 10 (ABR+10) did so only weakly (Kd ≈ 10-5 M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg2+ ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.
Asunto(s)
Actinas/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas con Dominio LIM/química , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Empalme Alternativo , Sitios de Unión , Escherichia coli/genética , Escherichia coli/metabolismo , Exones , Expresión Génica , Humanos , Intrones , Proteínas con Dominio LIM/genética , Proteínas con Dominio LIM/metabolismo , Mutación , Concentración Osmolar , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcómeros/fisiología , Relación Estructura-ActividadRESUMEN
The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8-11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas con Dominio LIM/metabolismo , Mutación Missense , Miofibrillas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/patología , Actinina/genética , Actinina/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos , Animales , Línea Celular , Conectina/genética , Conectina/metabolismo , Humanos , Proteínas con Dominio LIM/genética , Ratones , Proteínas de Microfilamentos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miofibrillas/genética , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/metabolismo , Miopatías Estructurales Congénitas/patología , Estructura Terciaria de ProteínaRESUMEN
In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 µM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 µM) and tipranavir (TPV; EC50, 0.364 µM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRV(R)P20), with a 2.6-fold increase in its EC50 (0.097 µM) compared to its corresponding EC50 (0.038 µM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30' of HIVA02 protease (PRA02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30' due to an absence of polar groups. The P2' thiazolyl moiety of RTV showed two conformations in the crystal structure of the PRA02-RTV complex, one of which showed loss of contacts in the S2' binding pocket of PRA02, supporting RTV's compromised antiviral activity (EC50, >1 µM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30' most likely contributes to its persistently greater antiviral activity against HIVWT, HIVA02, and HIVDRV(R)P20.
Asunto(s)
Carbamatos/farmacología , Dominio Catalítico/efectos de los fármacos , Farmacorresistencia Viral Múltiple/efectos de los fármacos , Furanos/farmacología , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , Sulfonamidas/farmacología , Cristalización , Darunavir , Proteasa del VIH , Humanos , Enlace de Hidrógeno , Conformación Molecular , Datos de Secuencia Molecular , Pliegue de Proteína , Piridinas/farmacología , Pironas/farmacología , Difracción de Rayos XRESUMEN
GRL007 and GRL008, two structurally related nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) as the P2 moiety and a sulfonamide isostere consisting of benzene carboxylic acid and benzene carboxamide as the P2' moiety, respectively, were evaluated for their antiviral activity and interactions with wild-type protease (PR(WT)). Both GRL007 (Ki of 12.7 pM with PR(WT)) and GRL008 (Ki of 8.9 pM) inhibited PR(WT) with high potency in vitro. X-ray crystallographic analysis of PR(WT) in complex with GRL007 or GRL008 showed that the bis-THF moiety of both compounds has three direct polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30 of PR(WT). The P2' moiety of both compounds showed one direct contact with the backbone of Asp30' and a bridging polar contact with Gly48' through a water molecule. Cell-based antiviral assays showed that GRL007 was inactive (50% effective concentration [EC50] of >1 µM) while GRL008 was highly active (EC50 of 0.04 µM) against wild-type HIV-1. High-performance liquid chromatography (HPLC)/mass spectrometry-based cellular uptake assays showed 8.1- and 84-fold higher intracellular concentrations of GRL008 than GRL007 in human MT-2 and MT-4 cell extracts, respectively. Thus, GRL007, in spite of its favorable enzyme-inhibitory activity and protease binding profile, exhibited a lack of antiviral activity in cell-based assays, most likely due to its compromised cellular uptake associated with its P2' benzene carboxylic acid moiety. The anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2' moiety may improve its antiretroviral features.
Asunto(s)
Benceno/química , Benzoatos/química , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/química , Indoles/química , Dominio Catalítico , Línea Celular , Cromatografía Líquida de Alta Presión , Humanos , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Delta cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Regulación Fúngica de la Expresión Génica , Complejo Mediador , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Secuencia Conservada , Complejo Mediador/química , Complejo Mediador/genética , Complejo Mediador/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fenotipo , Dominios y Motivos de Interacción de Proteínas/fisiología , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Active-site inhibitors of HIV-1 PR (protease) block viral replication by preventing viral maturation. However, HIV-1 often develops resistance to active-site inhibitors through multiple mutations in PR and therefore recent efforts have focused on inhibiting PR dimerization as an alternative approach. Dimerization inhibitors have been identified using kinetic analysis, but additional characterization of the effect of these inhibitors on PR by physical methods has been difficult. In the present study, we identified a PR(MDR) (multi-drug-resistant HIV-1 PR) that was highly resistant to autoproteolysis. Using this PR and a novel size-exclusion chromatographic approach that incorporated fluorescence and MS detection, we were able to demonstrate inhibition of dimerization using P27 (peptide 27), a peptide dimerization inhibitor of PR previously identified on the basis of kinetic analysis. Incubation of PR(MDR) with P27, or other dimerization inhibitors, led to a dose- and time-dependent formation of PR monomers based on the change in elution time by size exclusion and its similar elution time to engineered forms of monomeric PR, namely PR(T26A) and glutathionylated PR. In contrast, incubation of PR(MDR) with a potent active-site inhibitor did not change the elution time for the PR(MDR) dimer. The monomeric PR induced by P27 had fluorescent characteristics which were consistent with unfolded PR. Structure-activity studies identified the active regions of P27 and experiments were performed to examine the effect of other dimerization inhibitors on PR. The present study is the first characterization of dimerization inhibition of PR(MDR), a prime target for these inhibitors, using a novel size-exclusion chromatographic approach.
Asunto(s)
Cromatografía en Gel/métodos , Proteasa del VIH/química , Proteasa del VIH/metabolismo , Multimerización de Proteína/efectos de los fármacos , Espectrometría de Masas , Péptidos/química , Péptidos/farmacología , Estabilidad Proteica/efectos de los fármacos , Relación Estructura-ActividadAsunto(s)
Proteína gp41 de Envoltorio del VIH/química , VIH-1/metabolismo , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Micelas , Simulación de Dinámica Molecular , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Dispersión del Ángulo Pequeño , Factores de Tiempo , Difracción de Rayos XRESUMEN
The HIV-1 envelope gp120/gp41 trimer mediates viral membrane fusion. After cluster of differentiation-4 recognition, gp120 detaches from the virus, exposing gp41 which triggers fusion. During the fusion process, gp41 may not remain trimeric, which could have functional importance. Here, we probe the reversible association of full length gp41 (minus the cytoplasmic domain) in detergent micelles (with probes attached to transmembrane domain) by fluorescence resonance energy transfer (FRET) with a µm dissociation constant. This is compared with other methods. A gp41-targeted fusion inhibitor must interfere with this transition, and monomeric, partially monomeric or trimeric states all present potential binding epitopes. The gp41 self-association is a valid drug target model and FRET, a potential high-throughput assay system, could be used to screen drug libraries.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Multimerización de Proteína , Transferencia Resonante de Energía de Fluorescencia , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , MicelasRESUMEN
Hepatitis B virus (HBV) is the leading cause of liver disease worldwide. While an adequate vaccine is available, current treatment options are limited, not highly effective, and associated with adverse effects, encouraging the development of alternative therapeutics. The HBV core gene encodes two different proteins: core, which forms the viral nucleocapsid, and pre-core, which serves as an immune modulator with multiple points of action. The two proteins mostly have the same sequence, although they differ at their N and C termini and in their dimeric arrangements. Previously, we engineered two human-framework antibody fragments (Fab/scFv) with nano- to picomolar affinities for both proteins. Here, by means of X-ray crystallography, analytical ultracentrifugation, and electron microscopy, we demonstrate that the antibodies have non-overlapping epitopes and effectively block biologically important assemblies of both proteins. These properties, together with the anticipated high tolerability and long half-lives of the antibodies, make them promising therapeutics.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Antígenos del Núcleo de la Hepatitis B/química , Antígenos e de la Hepatitis B/química , Virus de la Hepatitis B/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Antivirales , Sitios de Unión , Cristalografía por Rayos X , Antígenos del Núcleo de la Hepatitis B/metabolismo , Antígenos e de la Hepatitis B/metabolismo , Virus de la Hepatitis B/química , Humanos , Microscopía Electrónica , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , UltracentrifugaciónRESUMEN
HIV-1 Rev protein mediates nuclear export of unspliced and partially spliced viral RNAs for production of viral genomes and structural proteins. Rev assembles on a 351-nt Rev response element (RRE) within viral transcripts and recruits host export machinery. Small (<40-nt) RNA aptamers that compete with the RRE for Rev binding inhibit HIV-1 viral replication. We determined the X-ray crystal structure of a potential anti-HIV-1 aptamer that binds Rev with high affinity (Kd = 5.9 nM). The aptamer is structurally similar to the RRE high-affinity site but forms additional contacts with Rev unique to its sequence. Exposed bases of the aptamer interleave with the guanidinium groups of two arginines of Rev, forming stacking interactions and hydrogen bonds. The aptamer also obstructs an oligomerization interface of Rev, blocking Rev self-assembly. We propose that this aptamer can inhibit HIV-1 replication by interfering with Rev-RRE, Rev-Rev, and possibly Rev-host protein interactions.
Asunto(s)
Aptámeros de Nucleótidos/química , VIH-1/fisiología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Aptámeros de Nucleótidos/farmacología , Arginina/metabolismo , Sitios de Unión , Cristalografía por Rayos X , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Elementos de Respuesta , Replicación Viral/efectos de los fármacos , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Human immunodeficiency viral (HIV-1) fusion is mediated by the viral envelope gp120/gp41 complex (ENVelope glycoprotein). After gp120 shedding, gp41 is exposed and elicits membrane fusion via a cascade of conformational changes. In contrast to prefusion and postfusion conformation, little is known about any intermediate conformation. We report on a solution NMR investigation of homotrimeric HIV-1 gp41(27-194), comprising the transmembrane region and reconstituted in dodecyl phosphocholine (DPC) micelles. The protein is mainly α-helical, but experiences internal dynamics on the nanosecond and micro to millisecond time scale and transient α-helical behavior for certain residues in the N-terminal heptad repeat (NHR). Strong lipid interactions are observed, in particular for C-terminal residues of the NHR and imunodominant loop region connecting NHR and C-terminal heptad repeat (CHR). Our data indicate an extended conformation with features anticipated for a prefusion intermediate, presumably in exchange with a lowly populated postfusion six-helical bundle conformation.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/química , Fosforilcolina/análogos & derivados , Detergentes/química , VIH-1/química , Micelas , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fosforilcolina/química , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Internalización del VirusRESUMEN
Rev is a key regulatory protein of human immunodeficiency virus type 1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA, thereby allowing the synthesis of structural proteins. Despite its evident importance, the structure of Rev has remained unknown, primarily because Rev's proclivity for polymerization and aggregation is an impediment to crystallization. Monoclonal antibody antigen-binding domains (Fabs) have proven useful for the co-crystallization of other refractory proteins. In the present study, a chimeric rabbit/human anti-Rev Fab was selected by phage display, expressed in a bacterial secretion system, and purified from the media. The Fab readily solubilized polymeric Rev. The resulting Fab/Rev complex was purified by metal ion affinity chromatography and characterized by analytical ultracentrifugation, which demonstrated monodispersity and indicated a 1:1 molar stoichiometry. The Fab binds with very high affinity, as determined by surface plasmon resonance, to a conformational epitope in the N-terminal half of Rev. The complex forms crystals suitable for structure determination. The ability to serve as a crystallization aid is a new application of broad utility for chimeric rabbit/human Fab. The corresponding single-chain antibody (scFv) was also prepared, offering the potential of intracellular antibody therapeutics against human immunodeficiency virus type 1.
Asunto(s)
VIH-1/fisiología , Fragmentos Fab de Inmunoglobulinas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Cristalización , Cristalografía por Rayos X , Mapeo Epitopo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conformación Proteica , ARN Viral/inmunología , ARN Viral/metabolismo , Conejos , Proteínas Recombinantes de Fusión/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de Superficie , Replicación ViralRESUMEN
The N-terminal fusion domain of the HIV-1 gp41 envelope glycoprotein is responsible for initiating the fusion of viral and cellular membranes, leading to the subsequent infection of the host cell by HIV-1. We have investigated the backbone structure and dynamics of the 30 N-terminal residues of HIV-1 gp41 in membrane-mimicking environments using NMR spectroscopy and (15)N- and (15)N,(13)C,(2)H-labeled peptides. Similar (15)N-(1)H HSQC spectra were obtained in a variety of detergents, including SDS, DPC, mixed DPC/SDS, and LPPG micelles, indicating that the peptide structure is not strongly influenced by the type of detergent used. Detailed characterization was carried out in SDS micelles, where the long-term sample stability was found to be optimal. In addition to J-coupling and NOE restraints, a nearly complete set of backbone residual dipolar coupling restraints was recorded for the fusion domain-micelle complex aligned with respect to the magnetic field using a stretched polyacrylamide gel. Backbone amide (15)N spin relaxation and amide hydrogen exchange rates with the solvent were also measured. The ensemble of NMR structures reveals an uninterrupted alpha-helix for the least mobile residues (S(2) > 0.65), Ile-4 to Met-19, with transient helical character extending up to Ala-22. A 12-residue (Ile-4 to Ala-15) segment is fully shielded from solvent, with Gly-3 and Gly-16 found at micelle-solvent interfaces. Residues external to the micelle exhibit enhanced picosecond to nanosecond time scale dynamics relative to the residues buried in the micelle, and their mobility increases with the distance from the micelle.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/química , Micelas , Conformación Proteica , Secuencia de Aminoácidos , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/química , VIH-1/metabolismo , Humanos , Fusión de Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The structure of a water-insoluble fragment encompassing residues 282-304 of the HIV envelope protein gp41 is studied when solubilized by dihexanoyl phosphatidylcholine (DHPC) and by small bicelles, consisting of a 4:1 molar ratio of DHPC and dimyristoyl phosphatidylcholine (DMPC). Weak alignment with the magnetic field was accomplished in a stretched polyacrylamide gel, permitting measurement of one-bond (1)H-(15)N, (13)Ca-(13)C', and (13)C'-(15)N dipolar couplings, which formed the basis for determining the peptide structure. In both detergent systems, the peptide adopts an alpha-helical conformation from residue 4 through 18. In the presence of the DHPC micelles the helix is strongly curved towards the hydrophobic surface, whereas in the presence of bicelles a much weaker curvature in the opposite direction is observed.