RESUMEN
Scalar couplings of the form JI(1) x I(2) between nuclei impart valuable information about molecular structure to nuclear magnetic-resonance spectra. Here we demonstrate direct detection of J-spectra due to both heteronuclear and homonuclear J-coupling in a zero-field environment where the Zeeman interaction is completely absent. We show that characteristic functional groups exhibit distinct spectra with straightforward interpretation for chemical identification. Detection is performed with a microfabricated optical atomic magnetometer, providing high sensitivity to samples of microliter volumes. We obtain 0.1 Hz linewidths and measure scalar-coupling parameters with 4-mHz statistical uncertainty. We anticipate that the technique described here will provide a new modality for high-precision "J spectroscopy" using small samples on microchip devices for multiplexed screening, assaying, and sample identification in chemistry and biomedicine.
Asunto(s)
Espectroscopía de Resonancia Magnética/instrumentación , Magnetismo/instrumentación , Dispositivos Ópticos , Refractometría/instrumentación , Análisis Espectral/instrumentación , Diseño de Equipo , Análisis de Falla de EquipoRESUMEN
Bacteria use a strategy referred to as two-component signal transduction to sense a variety of stimuli and initiate an appropriate response. Signal processing begins with proteins referred to as histidine kinases. In most cases, these are membrane-bound receptors that respond to environmental cues. Histidine kinases use ATP as a phosphodonor to phosphorylate a conserved histidine residue. Subsequent transfer of the phosphoryl group to a conserved aspartyl residue in the cognate response regulator results in an appropriate output. Recent structural studies of activated (phosphorylated) response regulators and their aspartate-bearing regulatory domains have provided insight into the links between the chemistry and biology of these ubiquitous regulatory proteins. Chemical aspects of their function appear to generalize broadly to enzymes that adopt a phosphoaspartate intermediate.
Asunto(s)
Ácido Aspártico/metabolismo , Fenómenos Fisiológicos Bacterianos , Transducción de Señal/fisiología , Proteínas Bacterianas/fisiología , Sitios de Unión , Fosforilación , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Nicking of duplex DNA by the iron-mediated Fenton reaction occurs preferentially at a limited number of sequences. Of these, purine-T-G-purine (RTGR) is of particular interest because it is a required element in the upstream regulatory regions of many genes involved in iron and oxidative-stress responses. In order to study the basis of this preferential nicking, NMR studies were undertaken on the RTGR-containing duplex oligonucleotide, d(CGCGATATGACACTAG)/d(CTAGTGTCATATCGCG). One-dimensional and two-dimensional 1H NMR measurements show that Fe(2+) interacts preferentially and reversibly at the ATGA site within the duplex at a rate that is rapid relative to the chemical-shift timescale, while selective paramagnetic NMR line-broadening of the ATGA guanine H8 suggests that Fe(2+) interacts with the guanine N7 moiety. Localization at this site is supported by Fe(2+) titrations of a duplex containing a 7-deazaguanine substitution in place of the guanine in the ATGA sequence. The addition of a 100-fold excess of Mg(2+) over Fe(2+) does not affect the Fe(2+)-dependent broadening. When the ATGA site in the duplex is replaced by ATGT, an RTGR site (GTGA) is created on the opposite strand. Preferential iron localization then takes place at the 3' guanine in GTGA but no longer at the guanine in ATGT, consistent with the lack of preferential cleavage of ATGT sites relative to ATGA sites.
Asunto(s)
Cationes Bivalentes/metabolismo , Daño del ADN , ADN/genética , ADN/metabolismo , Peróxido de Hidrógeno/metabolismo , Hierro/metabolismo , Emparejamiento Base , Secuencia de Bases , Cationes Bivalentes/farmacología , Cobalto/metabolismo , ADN/química , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Peróxido de Hidrógeno/farmacología , Hierro/farmacología , Espectroscopía de Resonancia Magnética , Manganeso/metabolismo , Modelos Moleculares , Estrés Oxidativo/efectos de los fármacos , Especificidad por Sustrato , TermodinámicaRESUMEN
We have carried out a solid-state magic-angle sample-spinning (MAS) nuclear magnetic resonance (NMR) spectroscopic investigation of the (13)C(alpha) chemical shielding tensors of alanine, valine, and leucine residues in a series of crystalline peptides of known structure. For alanine and leucine, which are not branched at the beta-carbon, the experimental chemical shift anisotropy (CSA) spans (Omega) are large, about 30 ppm, independent of whether the residues adopt helical or sheet geometries, and are in generally good accord with Omega values calculated by using ab initio Hartree-Fock quantum chemical methods. The experimental Omegas for valine C(alpha) in two peptides (in sheet geometries) are also large and in good agreement with theoretical predictions. In contrast, the "CSAs" (Deltasigma) obtained from solution NMR data for alanine, valine, and leucine residues in proteins show major differences, with helical residues having Deltasigma values of approximately 6 ppm while sheet residues have Deltasigma approximately 27 ppm. The origins of these differences are shown to be due to the different definitions of the CSA. When defined in terms of the solution NMR CSA, the solid-state results also show small helical but large sheet CSA values. These results are of interest since they lead to the idea that only the beta-branched amino acids threonine, valine, and isoleucine can have small (static) tensor spans, Omega (in helical geometries), and that the small helical "CSAs" seen in solution NMR are overwhelmingly dominated by changes in tensor orientation, from sheet to helix. These results have important implications for solid-state NMR structural studies which utilize the CSA span, Omega, to differentiate between helical and sheet residues. Specifically, there will be only a small degree of spectral editing possible in solid proteins since the spans, Omega, for the dominant nonbranched amino acids are quite similar. Editing on the basis of Omega will, however, be very effective for many Thr, Val, and Ileu residues, which frequently have small ( approximately 15-20 ppm) helical CSA (Omega) spans.
Asunto(s)
Aminoácidos/química , Resonancia Magnética Nuclear Biomolecular/métodos , Oligopéptidos/química , Alanina/química , Isótopos de Carbono , Leucina/química , Modelos Químicos , Modelos Moleculares , Estructura Secundaria de Proteína , Teoría Cuántica , Soluciones , Valina/químicaRESUMEN
The detection of biological molecules and their interactions is a significant component of modern biomedical research. In current biosensor technologies, simultaneous detection is limited to a small number of analytes by the spectral overlap of their signals. We have developed an NMR-based xenon biosensor that capitalizes on the enhanced signal-to-noise, spectral simplicity, and chemical-shift sensitivity of laser-polarized xenon to detect specific biomolecules at the level of tens of nanomoles. We present results using xenon "functionalized" by a biotin-modified supramolecular cage to detect biotin-avidin binding. This biosensor methodology can be extended to a multiplexing assay for multiple analytes.
Asunto(s)
Técnicas Biosensibles/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Xenón , Avidina/química , Biotina/químicaRESUMEN
The sensitivity of (129)Xe chemical shifts to weak nonspecific xenon-protein interactions has suggested the use of xenon to probe biomolecular structure and interactions. The realization of this potential necessitates a further understanding of how different macromolecular properties influence the (129)Xe chemical shift in aqueous solution. Toward this goal, we have acquired (129)Xe NMR spectra of xenon dissolved in amino acid, peptide, and protein solutions under both native and denaturing conditions. In general, these cosolutes induce (129)Xe chemical shifts that are downfield relative to the shift in water, as they deshield the xenon nucleus through weak, diffusion-mediated interactions. Correlations between the extent of deshielding and molecular properties including chemical identity, structure, and charge are reported. Xenon deshielding was found to depend linearly on protein size under denaturing solution conditions; the denaturant itself has a characteristic effect on the (129)Xe chemical shift that likely results from a change in the xenon solvation shell structure. In native protein solutions, contributions to the overall (129)Xe chemical shift arise from the presence of weak xenon binding either in cavities or at the protein surface. Potential applications of xenon as a probe of biological systems including the detection of conformational changes and the possible quantification of buried surface area at protein-protein interfaces are discussed.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/farmacología , Xenón/farmacología , Aminoácidos/farmacología , Interacciones Farmacológicas , Agua , Isótopos de XenónRESUMEN
The secondary structure of a 55-residue fragment of the mouse prion protein, MoPrP(89-143), was studied in randomly aggregated (dried from water) and fibrillar (precipitated from water/acetonitrile) forms by (13)C solid-state NMR. Recent studies have shown that the fibrillar form of the P101L mutant of MoPrP(89-143) is capable of inducing prion disease in transgenic mice, whereas unaggregated or randomly aggregated samples do not provoke disease. Through analysis of (13)C chemical shifts, we have determined that both wild-type and mutant sequence MoPrP(89-143) form a mixture of beta-sheet and alpha-helical conformations in the randomly aggregated state although the beta-sheet content in MoPrP(89-143, P101L) is significantly higher than in the wild-type peptide. In a fibrillar state, MoPrP(89-143, P101L) is completely converted into beta-sheet, suggesting that the formation of a specific beta-sheet structure may be required for the peptide to induce disease. Studies of an analogous peptide from Syrian hamster PrP verify that sequence alterations in residues 101-117 affect the conformation of aggregated forms of the peptides.
Asunto(s)
Fragmentos de Péptidos/química , Proteínas PrPC/química , Priones/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Isótopos de Carbono , Marcaje Isotópico/métodos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Enfermedades por Prión , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de SecuenciaAsunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Portadoras/química , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Unión a Maltosa , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Isótopos de XenónRESUMEN
INTRODUCTION: The RSG-1.2 peptide was selected for specific binding to the Rev response element RNA, as the natural Rev peptide does. The RSG-1.2 sequence has features incompatible with the helical structure of the bound Rev peptide, indicating that it must bind in a different conformation. RESULTS: The binding of the RSG-1.2 peptide to the Rev response element RNA was characterized using multinuclear, multidimensional NMR. The RSG-1.2 peptide is shown to bind with the N-terminal segment of the peptide along the major groove in an extended conformation and turn preceding a C-terminal helical segment, which crosses the RNA groove in the region widened by the presence of purine-purine base pairs. These features make the details of the bound state rather different than that of the Rev peptide which targets the same RNA sequence binding as a single helix along the groove axis. CONCLUSIONS: These studies further demonstrate the versatility of arginine-rich peptides in recognition of specific RNA elements and the lack of conserved structural features in the bound state.
Asunto(s)
Arginina/química , Genes env/genética , Péptidos/química , Purinas/química , ARN/química , Sitios de Unión/fisiología , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , ARN/genéticaRESUMEN
Protein actions are usually discussed in terms of static structures, but function requires motion. We find a strong correlation between phosphorylation-driven activation of the signaling protein NtrC and microsecond time-scale backbone dynamics. Using nuclear magnetic resonance relaxation, we characterized the motions of NtrC in three functional states: unphosphorylated (inactive), phosphorylated (active), and a partially active mutant. These dynamics are indicative of exchange between inactive and active conformations. Both states are populated in unphosphorylated NtrC, and phosphorylation shifts the equilibrium toward the active species. These results support a dynamic population shift between two preexisting conformations as the underlying mechanism of activation.
Asunto(s)
Proteínas Bacterianas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Conformación Proteica , Transactivadores , Factores de Transcripción , Regulación Alostérica , Sitios de Unión , Proteínas de Unión al ADN/genética , Modelos Moleculares , Movimiento (Física) , Mutación , Resonancia Magnética Nuclear Biomolecular , Proteínas PII Reguladoras del Nitrógeno , Fosforilación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transducción de Señal , TiempoRESUMEN
The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Quimiotaxis , Cristalografía por Rayos X/métodos , Escherichia coli/genética , Proteínas de Escherichia coli , Magnesio/metabolismo , Manganeso/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , Modelos Moleculares , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The solution structure of the protein disulfide oxidoreductase Mj0307 in the reduced form has been solved by nuclear magnetic resonance. The secondary and tertiary structure of this protein from the archaebacterium Methanococcus jannaschii is similar to the structures that have been solved for the glutaredoxin proteins from Escherichia coli, although Mj0307 also shows features that are characteristic of thioredoxin proteins. Some aspects of Mj0307's unique behavior can be explained by comparing structure-based sequence alignments with mesophilic bacterial and eukaryotic glutaredoxin and thioredoxin proteins. It is proposed that Mj0307, and similar archaebacterial proteins, may be most closely related to the mesophilic bacterial NrdH proteins. Together these proteins may form a unique subgroup within the family of protein disulfide oxidoreductases.
Asunto(s)
Methanococcus/enzimología , NADH NADPH Oxidorreductasas/química , Oxidorreductasas , Secuencia de Aminoácidos , Clonación Molecular , Disulfuros , Escherichia coli/química , Glutarredoxinas , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Homología de Secuencia de Aminoácido , Temperatura , Tiorredoxinas/químicaRESUMEN
Two-dimensional homonuclear NMR was used to characterize synthetic DNA minor groove-binding ligands in complexes with oligonucleotides containing three different A-T binding sites. The three ligands studied have a C(2) axis of symmetry and have the same general structural motif of a central para-substituted benzene ring flanked by two meta-substituted rings, giving the molecules a crescent shape. As with other ligands of this shape, specificity seems to arise from a tight fit in the narrow minor groove of the preferred A-T-rich sequences. We found that these ligands slide between binding subsites, behavior attributed to the fact that all of the amide protons in the ligand backbone cannot hydrogen bond to the minor groove simultaneously.
Asunto(s)
Benceno/química , Benceno/metabolismo , ADN/química , ADN/metabolismo , Secuencia Rica en At/genética , Secuencia de Bases , Sitios de Unión , ADN/genética , Diseño de Fármacos , Enlace de Hidrógeno , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismoRESUMEN
Pancreatic ribonuclease A may be cleaved to produce two fragments: the S-peptide (residues 1-20) and the S-protein (residues 21-124). The S-peptide, or a truncated version designated as the S15 peptide (residues 1-15), combines with the S-protein to produce catalytically active complexes. The conformation of these peptides and many of their analogues is predominantly random coil at room temperature; however, they populate a significant fraction of helical form at low temperature under certain solution conditions. Moreover, they adopt a helical conformation when bound to the S-protein. A hybrid sequence, disulfide-stabilized peptide (ApaS-25), designed to stabilize the helical structure of the S-peptide in solution, also combines with the S-protein to yield a catalytically active complex. We have performed high-precision titration microcalorimetric measurements to determine the free energy, enthalpy, entropy, and heat capacity changes for the binding of ApaS-25 to S-protein within the temperature range 5-25 degrees C. The thermodynamic parameters for both the complex formation reactions and the helix-to-coil transition also were calculated, using a structure-based approach, by calculating changes in accessible surface area and using published empirical parameters. A simple thermodynamic model is presented in an attempt to account for the differences between the binding of ApaS-25 and the S-peptide. From this model, the thermodynamic parameters of the helix-to-coil transition of S15 can be calculated.
Asunto(s)
Fragmentos de Péptidos/química , Conformación Proteica , Ribonucleasa Pancreática/química , Ribonucleasas/química , Aminoácidos , Calorimetría/métodos , Fenómenos Químicos , Química Física , Metionina , TermodinámicaRESUMEN
The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3--activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the beta4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-beta5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Berilio/farmacología , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/fisiología , Proteínas de Escherichia coli , Flagelos/fisiología , Fluoruros/farmacología , Proteínas Quimiotácticas Aceptoras de Metilo , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Estructura Secundaria de Proteína , Rotación , Alineación de Secuencia , Electricidad EstáticaRESUMEN
alpha-Conotoxins are small disulfide-constrained peptide toxins which act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nACh receptors). In this study, we analyzed the structures and activities of three mutants of alpha-conotoxin ImI, a 12 amino acid peptide active at alpha7 nACh receptors, in order to gain insight into the primary and tertiary structural requirements of neuronal alpha-conotoxin specificity. NMR solution structures were determined for mutants R11E, R7L, and D5N, resulting in representative ensembles of 20 conformers with average pairwise RMSD values of 0.46, 0.52, and 0.62 A from their mean structures, respectively, for the backbone atoms N, C(alpha), and C' of residues 2-11. The R11E mutant was found to have activity near that of wild-type ImI, while R7L and D5N demonstrated activities reduced by at least two orders of magnitude. Comparison of the structures reveals a common two-loop architecture, with variations observed in backbone and side-chain dihedral angles as well as surface electrostatic potentials upon mutation. Correlation of these structures and activities with those from previously published studies emphasizes that existing hypotheses regarding the molecular determinants of alpha-conotoxin specificity are not adequate for explaining peptide activity, and suggests that more subtle features, visualized here at the atomic level, are important for receptor binding. These data, in conjunction with reported characterizations of the acetylcholine binding site, support a model of toxin activity in which a single solvent-accessible toxin side-chain anchors the complex, with supporting weak interactions determining both the efficacy and the subtype specificity of the inhibitory activity.
Asunto(s)
Conotoxinas/química , Conotoxinas/farmacología , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Conotoxinas/genética , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Subunidades de Proteína , Receptores Nicotínicos/química , Alineación de Secuencia , Electricidad Estática , Relación Estructura-Actividad , Especificidad por Sustrato , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Transthyretin is a human protein capable of amyloid formation that is believed to cause several types of amyloid disease, depending on the sequence deposited. Previous studies have demonstrated that wild-type transthyretin (TTR), although quite stable, forms amyloid upon dissociation from its native tetrameric form into monomers with an altered conformation. Many naturally occurring single-site variants of TTR display decreased stability in vitro, manifested by the early onset familial amyloid diseases in vivo. Only subtle structural changes were observed in X-ray crystallographic structures of these disease associated variants. In this study, the stability of the wild-type TTR tetramer was investigated at the residue-resolution level by monitoring (2)H-H exchange via NMR spectroscopy. The measured protection factors for slowly-exchanging amide hydrogen atoms reveal a stable core consisting of strands A, B, E, F, and interestingly, the loop between strands A and B. In addition, the faster exchange of amide groups from residues at the subunit interfaces suggests unexpected mobility in these regions. This information is crucial for future comparisons between disease-associated and wild-type tetramers. Such studies can directly address the regions of TTR that become destabilized as a consequence of single amino acid substitutions, providing clues to aspects of TTR amyloidogenesis.
Asunto(s)
Deuterio/metabolismo , Prealbúmina/química , Prealbúmina/metabolismo , Amidas/metabolismo , Sitios de Unión , Dimerización , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína , ProtonesRESUMEN
The high sensitivity of the magnetic resonance properties of xenon to its local chemical environment and the large (129)Xe NMR signals attainable through optical pumping have motivated the use of xenon as a probe of macromolecular structure and dynamics. In the present work, we report evidence for nonspecific interactions between xenon and the exterior of myoglobin in aqueous solution, in addition to a previously reported internal binding interaction. (129)Xe chemical shift measurements in denatured myoglobin solutions and under native conditions with varying xenon concentrations confirm the presence of nonspecific interactions. Titration data are modeled quantitatively with treatment of the nonspecific interactions as weak binding sites. Using laser-polarized xenon to measure (129)Xe spin-lattice relaxation times (T(1)), we observed a shorter T(1) in the presence of 1 mM denatured apomyoglobin in 6 M deuterated urea (T(1) = 59 +/- 1 s) compared with that in 6 M deuterated urea alone (T(1) = 291 +/- 2 s), suggesting that nonspecific xenon-protein interactions can enhance (129)Xe relaxation. An even shorter T(1) was measured in 1 mM apomyoglobin in D(2)O (T(1) = 15 +/- 0.3 s), compared with that in D(2)O alone (T(1) = 506 +/- 5 s). This difference in relaxation efficiency likely results from couplings between laser-polarized xenon and protons in the binding cavity of apomyoglobin that may permit the transfer of polarization between these nuclei via the nuclear Overhauser effect.
Asunto(s)
Mioglobina/metabolismo , Xenón/química , Xenón/metabolismo , Animales , Apoproteínas/química , Apoproteínas/metabolismo , Sitios de Unión , Deuterio/metabolismo , Caballos , Rayos Láser , Espectroscopía de Resonancia Magnética , Metamioglobina/química , Metamioglobina/metabolismo , Músculo Esquelético , Mioglobina/química , Conformación Proteica , Desnaturalización Proteica , Protones , Soluciones , Solventes , Propiedades de Superficie , Factores de Tiempo , Volumetría , Isótopos de XenónRESUMEN
Studies have indicated that partially unfolded states occur under conditions that favor amyloid formation by transthyretin (TTR), as well as other amyloidogenic proteins. In this study, we used hydrogen exchange measurements to show that there is selective destabilization of one half of the beta-sandwich structure of TTR under such conditions. This provides more direct information about conformational fluctuations than previously available, and will facilitate design of future experiments to probe the intermediates critical to amyloid formation.
Asunto(s)
Placa Amiloide/química , Placa Amiloide/metabolismo , Prealbúmina/química , Prealbúmina/metabolismo , Amidas/metabolismo , Deuterio/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Desnaturalización Proteica , Estructura Secundaria de Proteína , ProtonesRESUMEN
In the past decade, a general design for sequence-specific minor groove ligands has evolved, based on the natural products distamycin and netropsin. By utilizing a basic set of design rules for connecting pyrrole, imidazole, and hydroxypyrrole modules, new ligands can be prepared to target almost any sequence of interest with both high affinity and specificity. In this review we present the design rules with a brief history of how they evolved. The structural basis for sequence-specific recognition is explained, together with developments that allow linking of recognition modules that enable targeting of long DNA sequences. Examples of the affinity and specificity that can be achieved with a number of variations on the basic design are given. Recently these molecules have been used to compete with proteins both in vitro and in vivo, and a brief description of the experimental results are given.