RESUMEN
Conopeptides are a diverse array of small linear and reticulated peptides that interact with high potency and selectivity with a large diversity of receptors and ion channels. They are used by cone snails for prey capture or defense. Recent advances in venom gland transcriptomic and venom peptidomic/proteomic technologies combined with bioactivity screening approaches lead to the identification of new toxins with original pharmacological profiles. Here, from transcriptomic/proteomic analyses of the Conus consors cone snail, we identified a new conopeptide called τ-CnVA, which displays the typical cysteine framework V of the T1-conotoxin superfamily. This peptide was chemically synthesized and its three-dimensional structure was solved by NMR analysis and compared to that of TxVA belonging to the same family, revealing very few common structural features apart a common orientation of the intercysteine loop. Because of the lack of a clear biological function associated with the T-conotoxin family, τ-CnVA was screened against more than fifty different ion channels and receptors, highlighting its capacity to interact selectively with the somatostatine sst3 receptor. Pharmacological and functional studies show that τ-CnVA displays a micromolar (Ki of 1.5µM) antagonist property for the sst3 receptor, being currently the only known toxin to interact with this GPCR subfamily.
Asunto(s)
Conotoxinas/química , Conotoxinas/farmacología , Receptores de Somatostatina/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Células CHO , Calcio/metabolismo , Cricetinae , Cricetulus , Canales Iónicos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteómica , Transcriptoma , Xenopus laevisAsunto(s)
Acer , Ascomicetos/metabolismo , Enfermedades de los Caballos/diagnóstico , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/veterinaria , Acer/microbiología , Alimentación Animal/análisis , Animales , Ascomicetos/crecimiento & desarrollo , Dieta/veterinaria , Resultado Fatal , Enfermedades de los Caballos/sangre , Enfermedades de los Caballos/etiología , Caballos , Hipoglicinas/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/sangre , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/diagnóstico , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/etiología , Países Bajos , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Semillas/metabolismoRESUMEN
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 (1)H, (13)C and (15)N resonance assignments of GNA1946 (residues 10-287) in aqueous buffer solution.
Asunto(s)
Antígenos Bacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Lipoproteínas/química , Neisseria meningitidis , Secuencia de Aminoácidos , Isótopos de Carbono , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Proteínas Recombinantes/química , Alineación de SecuenciaRESUMEN
The C-terminal domain of the UvrC protein (UvrC CTD) is essential for 5' incision in the prokaryotic nucleotide excision repair process. We have determined the three-dimensional structure of the UvrC CTD using heteronuclear NMR techniques. The structure shows two helix-hairpin-helix (HhH) motifs connected by a small connector helix. The UvrC CTD is shown to mediate structure-specific DNA binding. The domain binds to a single-stranded-double-stranded junction DNA, with a strong specificity towards looped duplex DNA that contains at least six unpaired bases per loop ("bubble DNA"). Using chemical shift perturbation experiments, the DNA-binding surface is mapped to the first hairpin region encompassing the conserved glycine-valine-glycine residues followed by lysine-arginine-arginine, a positively charged surface patch and the second hairpin region consisting of glycine-isoleucine-serine. A model for the protein-DNA complex is proposed that accounts for this specificity.
Asunto(s)
ADN Bacteriano/metabolismo , Endodesoxirribonucleasas/química , Proteínas de Escherichia coli/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , ADN de Cadena Simple/metabolismo , Dimerización , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Soluciones , Relación Estructura-ActividadRESUMEN
Bacteria require three initiation factors, IF1, IF2 and IF3, to start protein synthesis. In the last few years the elucidation of both structural and mechanistic aspects pertaining to these proteins has made substantial progress. In this article we outline the translation initiation process in bacteria and review these recent developments giving a summary of the main features of the structure and function of the initiation factors.
Asunto(s)
Proteínas Bacterianas/química , Factores de Iniciación de Péptidos/química , Proteínas Bacterianas/fisiología , Factores de Iniciación de Péptidos/fisiología , Biosíntesis de Proteínas/fisiología , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
The combined structural and biochemical studies on Lac repressor bound to operator DNA have demonstrated the central role of the hinge helices in operator bending and the induction mechanism. We have constructed a covalently linked dimeric Lac-headpiece that binds DNA with four orders of magnitude higher affinity as compared with the monomeric form. This enabled a detailed biochemical and structural study of Lac binding to its cognate wild-type and selected DNA operators. The results indicate a profound contribution of hinge helices to the stability of the protein-DNA complex and highlight their central role in operator recognition. Furthermore, protein-DNA interactions in the minor groove appear to modulate hinge helix stability, thus accounting for affinity differences and protein-induced DNA bending among the various operator sites. Interestingly, the in vitro DNA-binding affinity of the reported dimeric Lac construct can de readily modulated by simple adjustment of redox conditions, thus rendering it a potential artificial gene regulator.
Asunto(s)
Proteínas Bacterianas/fisiología , ADN/metabolismo , Proteínas de Escherichia coli , Regiones Operadoras Genéticas , Proteínas Represoras/fisiología , Proteínas Bacterianas/metabolismo , Sitios de Unión , ADN/química , Dimerización , Represoras Lac , Oxidación-Reducción , Ingeniería de Proteínas , Estructura Secundaria de Proteína , Proteínas Represoras/metabolismoRESUMEN
The NOT4 protein is a component of the CCR4.NOT complex, a global regulator of RNA polymerase II transcription. Human NOT4 (hNOT4) contains a RING finger motif of the C(4)C(4) type. We expressed and purified the N-terminal region of hNOT4 (residues 1-78) encompassing the RING finger motif and determined the solution structure by heteronuclear NMR. NMR experiments using a (113)Cd-substituted hNOT4 RING finger showed that two metal ions are bound through cysteine residues in a cross-brace manner. The three-dimensional structure of the hNOT4 RING finger was refined with root mean square deviation values of 0.58 +/- 0.13 A for the backbone atoms and 1.08 +/- 0.12 A for heavy atoms. The hNOT4 RING finger consists of an alpha-helix and three long loops that are stabilized by zinc coordination. The overall folding of the hNOT4 RING finger is similar to that of the C(3)HC(4) RING fingers. The relative orientation of the two zinc-chelating loops and the alpha-helix is well conserved. However, for the other regions, the secondary structural elements are distinct.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Cisteína/química , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Transcripción Genética , Ubiquitina-Proteína Ligasas , Dedos de ZincRESUMEN
The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein chymotrypsin inhibitor 2. Dihedral angle restraints for the phi and psi angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond 3hbJNC' coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Péptidos/química , Pliegue de Proteína , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Proteínas de Plantas , Conformación ProteicaRESUMEN
The solution structure of the dimeric N-terminal domain of HIV-2 integrase (residues 1-55, named IN(1-55)) has been determined using NMR spectroscopy. The structure of the monomer, which was already reported previously [Eijkelenboom et al. (1997) Curr. Biol., 7, 739-746], consists of four alpha-helices and is well defined. Helices alpha1, alpha2 and alpha3 form a three-helix bundle that is stabilized by zinc binding to His12, His16, Cys40 and Cys43. The dimer interface is formed by the N-terminal tail and the first half of helix alpha3. The orientation of the two monomeric units with respect to each other shows considerable variation. 15N relaxation studies have been used to characterize the nature of the intermonomeric disorder. Comparison of the dimer interface with that of the well-defined dimer interface of HIV-1 IN(1-55) shows that the latter is stabilized by additional hydrophobic interactions and a potential salt bridge. Similar interactions cannot be formed in HIV-2 IN(1-55) [Cai et al. (1997) Nat. Struct. Biol., 4, 567-577], where the corresponding residues are positively charged and neutral ones.
Asunto(s)
Integrasa de VIH/química , VIH-2/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Dimerización , Integrasa de VIH/metabolismo , VIH-1/enzimología , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Soluciones , Zinc/metabolismoRESUMEN
The nature of the pB intermediate of photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been probed by NMR. pH-dependent changes in the NMR spectrum of the dark state of PYP are shown to closely mimic exchange broadening effects observed previously in the NMR spectrum of the pB intermediate in solution. Amide H-D exchange data show that while pB retains a solid protected core, two regions become significantly less protected than the dark state. The amide exchange data help to rationalize why the conformational exchange process affects the N-terminal 28-residue segment of the protein, which is not close to the site of chromophore rearrangement. At very low pH (pH 1.7), the dark state NMR spectrum displays approximately 30 very sharp signals, which are characteristic of a portion of the molecule becoming unfolded. Similarities between the dark state spectra at pH approximately 3.2 and the spectra of pB suggest a model for pB in solution where the protein exists in an equilibrium between a well-ordered state and a state in which a region is unfolded. Such a two-state model accounts for the exchange phenomena observed in the NMR spectra of pB, and the hydrophobic exposure and lability inferred from thermodynamic data. It is likely that in the crystalline environment the ordered form of pB is strongly favored.
Asunto(s)
Proteínas Bacterianas/química , Deuterio , Fotorreceptores Microbianos , Protones , Amidas , Halorhodospira halophila , Concentración de Iones de Hidrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Pliegue de Proteína , Soluciones , Termodinámica , VolumetríaRESUMEN
The interaction between fMet-tRNA(f)(Met) and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet-3'ACCAAC of the initiator tRNA and a fairly small area at the surface of the beta-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2). A weak but specific interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA(f)(Met) has been mapped using two independent approaches, site- directed mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA(f)(Met), and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3' to 5'.
Asunto(s)
Geobacillus stearothermophilus/química , Factores de Iniciación de Péptidos/química , Biosíntesis de Proteínas , ARN de Transferencia de Metionina/química , Sitios de Unión , Geobacillus stearothermophilus/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , N-Formilmetionina/química , N-Formilmetionina/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Factor 2 Procariótico de Iniciación , Conformación Proteica , ARN de Transferencia de Metionina/metabolismo , TermodinámicaRESUMEN
Two previously isolated mutations in the glucocorticoid receptor DNA-binding domain (DBD), S459A and P493R, have been postulated to mimic DNA-induced conformational changes in the glucocorticoid receptor DBD, thereby constitutively triggering an allosteric mechanism in which binding of specific DNA normally induces the exposure of otherwise silent glucocorticoid receptor transcriptional activation surfaces. Here we report the three-dimensional structure of the free S459A and P493R mutant DBDs as determined by NMR spectroscopy. The free S459A and P493R structures both display the conformational changes in the DBD dimerization interface that are characteristic of the DNA-bound wild-type DBD, confirming that these mutations mimic an allosteric effect of DNA. A transition between two packing arrangements of the DBD hydrophobic core provides a mechanism for long-range transmission of conformational changes, induced either by the mutations or by DNA binding, to protein-protein contact surfaces.
Asunto(s)
ADN/metabolismo , Mutación , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Regulación Alostérica , Sitio Alostérico , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Cisteína/genética , Cisteína/metabolismo , ADN/química , ADN/genética , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Receptores de Glucocorticoides/genética , Elementos de Respuesta/genéticaRESUMEN
The hydration of the collagen-like Ac-(Gly-Pro-Hyp)(6)-NH(2) triple-helical peptide in solution was investigated using an integrated set of high-resolution NMR hydration experiments, including different recently developed exchange-network editing methods. This approach was designed to explore the hydration dynamics in the proximity of labile groups, such as the hydroxyproline hydroxyl group, and revealed that the first shell of hydration in collagen-like triple helices is kinetically labile with upper limits for water molecule residence times in the nanosecond to sub-nanosecond range. This result is consistent with a "hopping" hydration model in which solvent molecules are exchanged in and out of solvation sites at a rate that is not directly correlated to the degree of site localization. The hopping model thus reconciles the dynamic view of hydration revealed by NMR with the previously suggested partially ordered semi-clathrate-like cylinder of hydration. In addition, the nanosecond to sub-nanosecond upper limits for water molecule residence times imply that hydration-dehydration events are not likely to be the rate-limiting step for triple helix self-recognition, complementing previous investigations on water dynamics in collagen fibers. This study has also revealed labile proton features expected to facilitate the characterization of the structure and folding of triple helices in collagen peptides.
Asunto(s)
Colágeno/química , Colágeno/metabolismo , Resonancia Magnética Nuclear Biomolecular , Agua/metabolismo , Animales , Biopolímeros/química , Biopolímeros/metabolismo , Hidroxiprolina/metabolismo , Cinética , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Protones , SolventesRESUMEN
The retinoid X receptor (RXR) is a prominent member of the nuclear receptor family of ligand-inducible transcription factors. Many proteins of this family exert their function as heterodimers with RXR as a common upstream partner. Studies of the DNA-binding domains of several nuclear receptors reveal differences in structure and dynamics, both between the different proteins and between the free- and DNA-bound receptor DBDs. We investigated the differences in dynamics between RXR free in solution and in complex with a 14 base-pair oligonucleotide, using (1)H and (15)N relaxation studies. Nano- to picosecond dynamics were probed on (15)N, employing Lipari-Szabo analysis with an axially symmetric tumbling model to estimate the exchange contributions to the transverse relaxation rates. Furthermore, milli- to microsecond dynamics were estimated qualitatively for (1)H and (15)N, using CPMG-HSQC and CPMG-T(2) measurements with differential pulse spacing. RXR shows hardly any nano- to picosecond time-scale internal motion. Upon DNA binding, the order parameters show a tiny increase. Dynamics in the milli- to microsecond time scale is more prevalent. It is localized in the first and second zinc fingers of the free RXR. Upon DNA-binding, exchange associated with specific/aspecific DNA-binding of RXR is observed throughout the sequence, whereas conformational flexibility of the D-box and the second zinc finger of RXR is greatly reduced. Since this DNA-binding induced folding transition occurs remote from the DNA in a region which is involved in protein-protein interactions, it may very well be related to the cooperativity of dimeric DNA binding.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/química , Oligodesoxirribonucleótidos/química , Receptores de Ácido Retinoico/química , Factores de Transcripción/química , Anisotropía , Emparejamiento Base , Sitios de Unión , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Difusión , Dimerización , Cinética , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/metabolismo , Estructura Terciaria de Proteína , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Soluciones , Termodinámica , Factores de Transcripción/metabolismoRESUMEN
To gain insight into intramolecular carbohydrate-protein interactions at the molecular level, the solution structure of differently deglycosylated variants of the alpha-subunit of human chorionic gonadotropin have been studied by NMR spectroscopy. Significant differences in chemical shifts and NOE intensities were observed for amino acid residues close to the carbohydrate chain at Asn78 upon deglycosylation beyond Asn78-bound GlcNAc. As no straightforward strategy is available for the calculation of the NMR structure of intact glycoproteins, a suitable computational protocol had to be developed. To this end, the X-PLOR carbohydrate force field designed for structure refinement was extended and modified. Furthermore, a computational strategy was devised to facilitate successful protein folding in the presence of extended glycans during the simulation. The values for phi and psi dihedral angles of the glycosidic linkages of the oligosaccharide core fragments GlcNAc2(beta1-4)GlcNAc1 and Man3(beta1-4)GlcNAc2 are restricted to a limited range of the broad conformational energy minima accessible for free glycans. This demonstrates that the protein core affects the dynamic behavior of the glycan at Asn78 by steric hindrance. Reciprocally, the NMR structures indicate that the glycan at Asn78 affects the stability of the protein core. The backbone angular order parameters and displacement data of the generated conformers display especially for the beta-turn 20-23 a decreased structural order upon splitting off the glycan beyond the Asn78-bound GlcNAc. In particular, the Asn-bound GlcNAc shields the protein surface from the hydrophilic environment through interaction with predominantly hydrophobic amino acid residues located in both twisted beta-hairpins consisting of residues 10-28 and 59-84.
Asunto(s)
Gonadotropina Coriónica/química , Polisacáridos/química , Acetilglucosamina/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicoproteínas/química , Glicosilación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Soluciones , Relación Estructura-ActividadRESUMEN
The three-dimensional structure of the fMet-tRNA(fMet) -binding domain of translation initiation factor IF2 from Bacillus stearothermophilus has been determined by heteronuclear NMR spectroscopy. Its structure consists of six antiparallel beta-strands, connected via loops, and forms a closed beta-barrel similar to domain II of elongation factors EF-Tu and EF-G, despite low sequence homology. Two structures of the ternary complexes of the EF-Tu small middle dotaminoacyl-tRNA small middle dot GDP analogue have been reported and were used to propose and discuss the possible fMet-tRNA(fMet)-binding site of IF2.
Asunto(s)
Geobacillus stearothermophilus/química , Factores de Iniciación de Péptidos/química , ARN de Transferencia de Metionina/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Bases de Datos Factuales , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Factor 2 Procariótico de Iniciación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Abstract The tetrameric Mnt repressor of bacteriophage P22 consists of two dimeric DNA-binding domains and a tetramerization domain. The NOE and chemical shift data demonstrate that the structures of the domains in the wild-type repressor protein are similar to those of the separate domains, the three-dimensional structures of which have been determined previously. (15)N relaxation measurements show that the linker that connects the anti-parallel four-helix bundle with the two ß-sheet DNA-binding dimers is highly flexible. No evidence was found for interactions between the distinct modules. The (15)N relaxation properties of the two domains differ substantially, confirming their structural independence. A model in which one two-stranded coiled coil of the four-helix bundle is attached to one N-terminal dimer is most consistent with the biochemical data and (15)N relaxation data. For the Mnt-DNA complex this geometry fits with a model in which the two ß-sheet DNA-binding domains are bound at two successive major grooves of the Mnt operator and the tetramerization domain is packed between these two DNA-bound dimers. In such a model the two-fold symmetry axis of the four-helix bundle coincides with that of the operator sequence and the two bound dimers. Bending of the Mnt operator of approximately 30° upon binding of the tetramer, as measured by gel-shift assays, is in agreement with this model of the Mnt-DNA complex.
Asunto(s)
Proteínas Represoras , Proteínas Reguladoras y Accesorias Virales , Secuencia de Aminoácidos , ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Represoras/química , Proteínas Virales/químicaRESUMEN
The hinge-region of the lac repressor plays an important role in the models for induction and DNA looping in the lac operon. When lac repressor is bound to a tight-binding symmetric operator, this region forms an alpha-helix that induces bending of the operator. The presence of the hinge-helices is questioned by previous data that suggest that the repressor does not bend the wild-type operator. We show that in the wild-type complex the hinge-helices are formed and the DNA is bent, similar to the symmetric complex. Furthermore, our data show differences in the binding of the DNA binding domains to the half-sites of the wild-type operator and reveal the role of the central base-pair of the wild-type operator in the repressor-operator interaction. The differences in binding to the operator half-sites are incorporated into a model that explains the relative affinities of the repressor for various lac operator sequences that contain left and right half-sites with different spacer lengths.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Operón Lac , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Secuencia de Bases , Clonación Molecular , Represoras Lac , Resonancia Magnética Nuclear Biomolecular , Conformación ProteicaRESUMEN
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.
Asunto(s)
Bacillus/enzimología , Serina Endopeptidasas/química , Subtilisinas/química , Sitios de Unión , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Protones , Serina Endopeptidasas/genética , Especificidad por Sustrato , Subtilisinas/genéticaRESUMEN
The structure of the C-terminal DNA-binding domain of human immunovirus-1 integrase has been refined using nuclear magnetic resonance spectroscopy. The protein is a dimer in solution and shows a well-defined dimer interface. The folding topology of the monomer consists of a five-stranded beta-barrel that resembles that of Src homology 3 domains. Compared with our previously reported structure, the structure is now defined far better. The final 42 structures display a back-bone root mean square deviation versus the average of 0.46 A. Correlation of the structure with recent mutagenesis studies suggests two possible models for DNA binding. Proteins 1999;36:556-564.