RESUMEN
Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the (15)N-{(1)H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the kon values and koff values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model ([Formula: see text]), (ii) there is a strong electrostatic component in the determined kon, and (iii) the binding is not diffusion-limited.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Glutamato-Amoníaco Ligasa/metabolismo , Synechocystis/metabolismo , Dicroismo Circular , Cinética , Espectroscopía de Resonancia Magnética , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
NMR assignment of intrinsically disordered proteins (IDPs) by conventional HN-detected methods is hampered by the small dispersion of the amide protons chemical shifts and exchange broadening of amide proton signals. Therefore several alternative assignment strategies have been proposed in the last years. Attempting to seize that dispersion of (13)C' and (15)N chemical shifts holds even in IDPs, we recently proposed two (13)C-detected experiments to directly correlate the chemical shifts of two consecutive (13)C'-(15)N groups in proteins, i.e. without mediation of other nuclei. Main drawback of these experiments is the interruption of the connection at prolines. Here we present new (13)C-detected experiments to correlate consecutive (13)C'-(15)N groups in IDPs, hacacoNcaNCO and hacaCOncaNCO, that overcome this limitation. Moreover, the experiments provide recognition of glycine residues, thereby facilitating the assignment process.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular , Isótopos de Carbono/química , Isótopos de Nitrógeno/química , Resonancia Magnética Nuclear Biomolecular/métodosRESUMEN
The stress protein Nupr1 is a highly basic, multifunctional, intrinsically disordered protein (IDP). MSL1 is a histone acetyl transferase-associated protein, known to intervene in the dosage compensation complex (DCC). In this work, we show that both Nupr1 and MSL1 proteins were recruited and formed a complex into the nucleus in response to DNA-damage, which was essential for cell survival in reply to cisplatin damage. We studied the interaction of Nupr1 and MSL1, and their binding affinities to DNA by spectroscopic and biophysical methods. The MSL1 bound to Nupr1, with a moderate affinity (2.8 µM) in an entropically-driven process. MSL1 did not bind to non-damaged DNA, but it bound to chemically-damaged-DNA with a moderate affinity (1.2 µM) also in an entropically-driven process. The Nupr1 protein bound to chemically-damaged-DNA with a slightly larger affinity (0.4 µM), but in an enthalpically-driven process. Nupr1 showed different interacting regions in the formed complexes with Nupr1 or DNA; however, they were always disordered ("fuzzy"), as shown by NMR. These results underline a stochastic description of the functionality of the Nupr1 and its other interacting partners.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Histona Acetiltransferasas/metabolismo , Proteínas de Neoplasias/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Cisplatino/toxicidad , Daño del ADN/genética , Daño del ADN/fisiología , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Técnica del Anticuerpo Fluorescente , Histona Acetiltransferasas/genética , Humanos , Espectroscopía de Resonancia Magnética , Proteínas de Neoplasias/genética , Unión ProteicaRESUMEN
Two novel 3D (13)C-detected experiments, hNcocaNCO and hnCOcaNCO, are proposed to facilitate the resonance assignment of intrinsically disordered proteins. The experiments correlate the (15)N and (13)C' chemical shifts of two consecutive amide moieties without involving other nuclei, thus taking advantage of the good dispersion shown by the (15)N-(13)C' correlations, even for proteins that lack a well defined tertiary structure. The new pulse sequences were successfully tested using Nupr1, an intrinsically disordered protein of 93 residues.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Proteínas Intrínsecamente Desordenadas/química , Proteínas de Neoplasias/química , Resonancia Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono/química , Isótopos de Nitrógeno/químicaRESUMEN
A suite of (13)C-detected NMR pulse sequences to edit the correlation peaks of the CACO and CON spectra according to the amino acid residue type is presented. The pulse sequences exploit the topology of the C(ß) carbon and led to the sorting of the CACO or CON signals into several classes depending on the nature of the generating residue. A set of four or eight correlation spectra is recorded where the sign of the cross peaks changes from one spectrum to another according to the amino acid type of the corresponding residue in the protein sequence. Linear combination of these spectra produces subspectra showing signals from residues having similar C(ß) topology. The presence of weak breakthrough peaks does not prevent the proper classification, since this is obtained from the subspectrum in which the correlation peak is more intense. The experiments were tested on a globular protonated protein ((13)C, (15)N labeled Ubiquitin), on a globular deuterated protein ((2)H, (13)C, (15)N labeled Ubiquitin), and on an intrinsically disordered protein ((13)C, (15)N labeled Nupr1).
Asunto(s)
Aminoácidos/análisis , Espectroscopía de Resonancia Magnética/métodos , Isótopos de Carbono , Proteínas de Unión al ADN/química , Deuterio , Marcaje Isotópico , Proteínas de Neoplasias/química , Proteínas/química , Protones , Ubiquitina/químicaRESUMEN
Two experiments are presented that yield amino acid type identification of individual residues in a protein by editing the (1)H-(15)N correlations into four different 2D subspectra, each corresponding to a different amino acid type class, and that can be applied to deuterated proteins. One experiment provides information on the amino acid type of the residue preceding the detected amide (1)H-(15)N correlation, while the other gives information on the type of its own residue. Versions for protonated proteins are also presented, and in this case it is possible to classify the residues into six different classes. Both sequential and intraresidue experiments provide highly complementary information, greatly facilitating the assignment of protein resonances. The experiments will also assist in transferring the assignment of a protein to the spectra obtained under different experimental conditions (e.g. temperature, pH, presence of ligands, cofactors, etc.).
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Aminoácidos/química , Deuterio , Conformación Proteica , ProtonesRESUMEN
Tick-derived protease inhibitor (TdPI) is a tight-binding Kunitz-related inhibitor of human tryptase ß with a unique structure and disulfide-bond pattern. Here we analyzed its oxidative folding and reductive unfolding by chromatographic and disulfide analyses of acid-trapped intermediates. TdPI folds through a stepwise generation of heterogeneous populations of one-disulfide, two-disulfide, and three-disulfide intermediates, with a major accumulation of the nonnative three-disulfide species IIIa. The rate-limiting step of the process is disulfide reshuffling within the three-disulfide population towards a productive intermediate that oxidizes directly into the native four-disulfide protein. TdPI unfolds through a major accumulation of the native three-disulfide species IIIb and the subsequent formation of two-disulfide and one-disulfide intermediates. NMR characterization of the acid-trapped and further isolated IIIa intermediate revealed a highly disordered conformation that is maintained by the presence of the disulfide bonds. Conversely, the NMR structure of IIIb showed a native-like conformation, with three native disulfide bonds and increased flexibility only around the two free cysteines, thus providing a molecular basis for its role as a productive intermediate. Comparison of TdPI with a shortened variant lacking the flexible prehead and posthead segments revealed that these regions do not contribute to the protein conformational stability or the inhibition of trypsin but are important for both the initial steps of the folding reaction and the inhibition of tryptase ß. Taken together, the results provide insights into the mechanism of oxidative folding of Kunitz inhibitors and pave the way for the design of TdPI variants with improved properties for biomedical applications.
Asunto(s)
Inhibidores de Proteasas/química , Animales , Cisteína/química , Disulfuros/química , Relación Dosis-Respuesta a Droga , Glutatión/química , Humanos , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Estrés Oxidativo , Oxígeno/química , Pliegue de Proteína , Garrapatas , Triptasas/antagonistas & inhibidores , Triptasas/químicaRESUMEN
We present a filter element to observe exclusively the resonances of Asp and Glu residues in several (13)C detected experiments, CACO, CBCACO, and CCCO. As in these experiments the carboxylate resonances appear in the directly detected dimension, it is possible to measure their chemical shifts with a high precision. Therefore, the experiments can be of great utility in the accurate determination of the pKa 's of these residues. Furthermore, the experiments can be applied in the study of deuterated proteins, where the usual experiments for pKa determination cannot be used. Finally, the good resolution with which the carboxylate spectrum is obtained allows observing the coupling between the carboxyl carbon and the backbone CO in Asp residues, what provides information on their side chain conformation.
Asunto(s)
Ácido Aspártico/química , Ácido Glutámico/química , Resonancia Magnética Nuclear Biomolecular/métodos , Ubiquitina/química , Isótopos de Carbono , Modelos Moleculares , Conformación ProteicaRESUMEN
Protein self-recognition is essential in many biochemical processes and its study is of fundamental interest to understand the molecular mechanism of amyloid formation. Ribonuclease A (RNase A) is a monomeric protein that may form several oligomers by 3D domain swapping of its N-terminal alpha-helix, C-terminal beta-strand, or both. RNase A oligomerization is induced by 40% acetic acid, which has been assumed to mildly unfold the protein by detaching the terminal segments and consequently facilitating intersubunit swapping, once the acetic acid is removed by lyophilization and the protein is redissolved in a benign buffer. Using UV difference, near UV circular dichroism, folding kinetics, and multidimensional heteronuclear NMR spectroscopy, the conformation of RNase A in 40% acetic acid and in 8 M urea has been characterized. These studies demonstrate that RNase A is chiefly unfolded in 40% acetic acid; it partially retains the native helices, whereas the beta-sheet is fully denatured and all X-Pro peptide bonds are predominantly in the trans conformation. Refolding occurs via an intermediate, I(N), with non-native X-Pro peptide bonds. I(N) is known to be populated during RNase A refolding following denaturation in concentrated solutions of urea or guanidinium chloride, and we find that urea- or GdmCl-denatured RNase A can oligomerize during refolding. By revealing the importance of a chiefly denaturated state and a refolding intermediate with non-native X-Pro peptide bonds, these findings revise the model for RNase A oligomerization via 3D domain swapping and have general implications for amyloid formation.
Asunto(s)
Ribonucleasa Pancreática/química , Ácido Acético/química , Animales , Bovinos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Desnaturalización Proteica , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
We present a pulse sequence for the simultaneous measurement of N-H and Calpha-Halpha couplings in double-labeled proteins from 2D spectra. The proposed sequence, a modification of the HN(CO)CA experiment, combines the J-modulation method and the IPAP scheme. The couplings can be readily retrieved from a series of 2D (15)N-(1)H correlation spectra, differing in the time point at which a (1)H 180 degrees pulse is applied. This induces an intensity modulation of the (15)N-(1)H correlation peaks with the Calpha-Halpha coupling. The Calpha-Halpha coupling is then obtained by fitting the observed intensities to the modulation equation. The N-H coupling is measured in each member of the set from peak-to-peak separations in the IPAP subspectra. The pulse sequence is experimentally verified with a sample of (15)N/(13)C-enriched ubiquitin.
Asunto(s)
Carbono/química , Hidrógeno/química , Nitrógeno/química , Proteínas/química , Espectroscopía de Resonancia Magnética/métodos , Ubiquitina/químicaRESUMEN
Protein folding mechanisms have remained elusive mainly because of the transient nature of intermediates. Leech-derived tryptase inhibitor (LDTI) is a Kazal-type serine proteinase inhibitor that is emerging as an attractive model for folding studies. It comprises 46 amino acid residues with three disulfide bonds, with one located inside a small triple-stranded antiparallel beta-sheet and with two involved in a cystine-stabilized alpha-helix, a motif that is widely distributed in bioactive peptides. Here, we analyzed the oxidative folding and reductive unfolding of LDTI by chromatographic and disulfide analyses of acid-trapped intermediates. It folds and unfolds, respectively, via sequential oxidation and reduction of the cysteine residues that give rise to a few 1- and 2-disulfide intermediates. Species containing two native disulfide bonds predominate during LDTI folding (IIa and IIc) and unfolding (IIa and IIb). Stop/go folding experiments demonstrate that only intermediate IIa is productive and oxidizes directly into the native form. The NMR structures of acid-trapped and further isolated IIa, IIb, and IIc reveal global folds similar to that of the native protein, including a native-like canonical inhibitory loop. Enzyme kinetics shows that both IIa and IIc are inhibitory-active, which may substantially reduce proteolysis of LDTI during its folding process. The results reported show that the kinetics of the folding reaction is modulated by the specific structural properties of the intermediates and together provide insights into the interdependence of conformational folding and the assembly of native disulfides during oxidative folding.
Asunto(s)
Hirudo medicinalis/química , Pliegue de Proteína , Proteínas/química , Secuencias de Aminoácidos/fisiología , Animales , Disulfuros/química , Resonancia Magnética Nuclear Biomolecular , Oxidación-ReducciónRESUMEN
Reduced dimensionality NMR spectra usually require very large spectral widths in the shared dimension. In this paper we show that aliasing can be introduced in reduced dimensionality NMR spectra either to decrease the acquisition time or increase the resolution of the experiments without losing information. The gains of introducing aliasing are illustrated with two examples, the (3,2)D HNHA and the (4,2)D HN(COCA)NH experiments. In both cases a reduction of the spectral width of more than 50% was possible.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/normas , Modelos Teóricos , UbiquitinaRESUMEN
Sticholysin I is an actinoporin, a pore forming toxin, of 176 aminoacids produced by the sea anemone Stichodactyla heliantus. Isotopically labelled (13)C/(15)N recombinant protein was produced in E. coli. Here we report the complete NMR (15)N, (13)C and (1)H chemical shifts assignments of Stn I at pH 4.0 and 25 degrees C (BMRB No. 15927).
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteínas Citotóxicas Formadoras de Poros/química , Anémonas de Mar/química , Animales , Isótopos de Carbono/química , Isótopos de Nitrógeno/química , Compuestos Orgánicos/química , ProtonesRESUMEN
CdnL, a 164-residue protein essential for Myxococcus xanthus viability, is a member of a large family of bacterial proteins of unknown structure and function. Here, we report the (1)H, (13)C and (15)N backbone and side chain assignments for the stable C-terminal domain of CdnL identified by limited proteolysis.
Asunto(s)
Proteínas Bacterianas/química , Espectroscopía de Resonancia Magnética/métodos , Myxococcus xanthus/química , Secuencia de Aminoácidos , Isótopos de Carbono/química , Datos de Secuencia Molecular , Isótopos de Nitrógeno/química , Estructura Terciaria de Proteína , ProtonesRESUMEN
Eosinophil cationic protein (ECP)/human RNase 3, a member of the RNase A family, is a remarkably cytotoxic protein implicated in asthma and allergies. These activities are probably due to ECP's ability to interact with and disrupt membranes and depend on two Trp, 19 Arg, and possibly an extremely high conformational stability. Here, we have used NMR spectroscopy to assign essentially all (1)H, (15)N, and backbone (13)C resonances, to solve the 3D structure in aqueous solution and to quantify its residue-level stability. The NMR solution structure was determined on the basis of 2316 distance constraints and is well-defined (backbone RMSD = 0.81 A). The N-terminus and the loop composed of residues 114-123 are relatively well-ordered; in contrast, conformational diversity is observed for the loop segments 17-22, 65-68, and 92-95 and most exposed sidechains. The side chain NH groups of the two Trp and 19 Arg showed no significant protection against hydrogen/deuterium exchange. The most protected NH groups belong to the first and last two beta-strands, and curiously, the first alpha-helix. Analysis of their exchange rates reveals a strikingly high global stability of 11.8 kcal/mol. This value and other stability measurements are used to better quantify ECP's unfolding thermodynamics.
Asunto(s)
Proteína Catiónica del Eosinófilo/química , Espectroscopía de Resonancia Magnética/métodos , Secuencia de Aminoácidos , Sitios de Unión , Isótopos de Carbono , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Estabilidad de Enzimas , Proteína Catiónica del Eosinófilo/genética , Proteína Catiónica del Eosinófilo/metabolismo , Humanos , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Protones , Soluciones , TermodinámicaRESUMEN
In this work, we introduce a set of pulse sequences that provide amino acid type identification of the NH correlation signals of proteins. The first pulse sequence is a modification of the CBCA(CO)NH experiment that exploits spin-coupling topologies to differentiate between amino acid types. A set of eight 2D (1)H-(15)N correlation spectra is recorded where the sign of the cross-peaks change from one spectrum to another according to the amino acid type of the preceding residue in the protein sequence. Linear combination of these eight data sets produces four subspectra. Taking also into account the sign of the correlation signals, this method allows the classification of the NH signals into six different groups, depending on the character of the preceding residue. This sequence is complemented with a (CGCBCACO)NH experiment that allows the subdivision of the largest of these groups into two smaller ones. Finally, a modification of the CBCANH experiment led to a similar classification of NH signals into six different groups, but now depending on the type of its own amino acid. The set of pulse sequences is demonstrated with two proteins of small to moderate size.
Asunto(s)
Algoritmos , Aminoácidos/análisis , Aminoácidos/química , Carbono/análisis , Carbono/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas/análisis , Proteínas/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The effects of the inactivating peptide from the eukaryotic Shaker BK(+) channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr(8), which exhibit the strongest STD effects; the C4H in the imidazole ring of His(16); the methyl protons of Val(4), Leu(7), and Leu(10) and the side chain amine protons of one, if not both, the Lys(18) and Lys(19) residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val(4) and Leu(10), whereas His(16) seems similarly affected as before. Conversely, STD effects on Tyr(8) are strongly diminished, and those on Lys(18) and/or Lys(19) are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of (13)C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a beta-hairpin structure when bound to the KcsA channel. Indeed, docking such a beta-hairpin structure into an open pore model for K(+) channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.
Asunto(s)
Proteínas Bacterianas/química , Epítopos/química , Canales de Potasio/química , Secuencia de Aminoácidos , Aminoácidos/química , Electrofisiología , Proteínas de Escherichia coli/química , Lisina/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Mutación , Péptidos/química , Canales de Potasio con Entrada de Voltaje , Unión Proteica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Ole e 9 is an olive pollen allergen belonging to group 2 of pathogenesis-related proteins. The protein is composed of two immunological independent domains: an N-terminal domain (NtD) with 1,3-beta-glucanase activity, and a C-terminal domain (CtD) that binds 1,3-beta-glucans. We have determined the three-dimensional structure of CtD-Ole e 9 (101 amino acids), which consists of two parallel alpha-helices forming an angle of approximately 55 degrees , a small antiparallel beta-sheet with two short strands, and a 3-10 helix turn, all connected by long coil segments, resembling a novel type of folding among allergens. Two regions surrounded by aromatic residues (F49, Y60, F96, Y91 and Y31, H68, Y65, F78) have been localized on the protein surface, and a role for sugar binding is suggested. The epitope mapping of CtD-Ole e 9 shows that B-cell epitopes are mainly located on loops, although some of them are contained in secondary structural elements. Interestingly, the IgG and IgE epitopes are contiguous or overlapped, rather than coincident. The three-dimensional structure of CtD-Ole e 9 might help to understand the underlying mechanism of its biochemical function and to determine possible structure-allergenicity relationships.
Asunto(s)
Alérgenos/química , Epítopos/química , Proteínas de Plantas/química , Polen/química , beta-Glucosidasa/química , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Plantas , Epítopos/inmunología , Humanos , Inmunoglobulina E/química , Inmunoglobulina E/inmunología , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Olea , Proteínas de Plantas/inmunología , Polen/inmunología , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Conejos , beta-Glucosidasa/inmunologíaRESUMEN
TA0095 is a 96-residue hypothetical protein from Thermoplasma acidophilum that exhibits no sequence similarity to any protein of known structure. Also, TA0095 is a member of the COG4004 orthologous group of unknown function found in Archaea bacteria. We determined its three-dimensional structure by NMR methods. The structure displays an alpha/beta two-layer sandwich architecture formed by three alpha-helices and five beta-strands following the order beta1-alpha1-beta2-beta3-beta4-beta5-alpha2-alpha3. Searches for structural homologs indicate that the TA0095 structure belongs to the TBP-like fold, constituting a novel superfamily characterized by an additional C-terminal helix. The TA0095 structure provides a fold common to the COG4004 proteins that will obviously belong to this new superfamily. Most hydrophobic residues conserved in the COG4004 proteins are buried in the structure determined herein, thus underlying their importance for structure stability. Considering that the TA0095 surface shows a large positively charged patch with a high degree of residue conservation within the COG4004 domain, the biological function of TA0095 and the rest of COG4004 proteins might occur through binding a negatively charged molecule. Like other TBP-like fold proteins, the COG4004 proteins might be DNA-binding proteins. The fact that TA0095 is shown to interact with large DNA fragments is in favor of this hypothesis, although nonspecific DNA binding cannot be ruled out.
Asunto(s)
Proteínas Arqueales/química , Proteínas de Unión al ADN/química , Modelos Moleculares , Thermoplasma , Secuencia de Aminoácidos , Proteínas Arqueales/clasificación , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
Alpha-sarcin is an exquisitely specific ribonuclease that binds and cleaves a single phosphodiester bond in the large rRNA of the eukaryotic ribosome, inactivating it. To better understand this remarkable activity, the contributions of the active site residues (His 50, Glu 96, and His 137) to the conformational stability have been determined as a function of pH using variant proteins containing uncharged substitutes. Wild-type alpha-sarcin and the variants are maximally stable near pH 5.5, coinciding with the pH of optimal activity. A comparison of the stability vs pH profiles determined by thermal denaturation experiments to those calculated on the basis of pKa values shows that the charged forms of Glu 96 and His 137 compromise the enzyme's stability, lowering it. In contrast to barnase, there is little evidence for significant electrostatic interactions in the denatured states of alpha-sarcin or its active site variants between pH 3.5 and pH 8.5. Alpha-sarcin contains a long beta-hairpin and surface loops which are highly positively charged and which play key roles in membrane translocation and in ribosome binding. These positive charges decrease the stability of alpha-sarcin, particularly below pH 5. Hydrogen exchange measurements have been performed at pH 5.5 and reveal that the catalytic residues are firmly anchored in highly stable elements of secondary structure. Significant, though lower, levels of protection are observed for many amide protons in the positively charged beta-hairpin and long loops.