RESUMEN
In this paper we describe solid-state NMR experiments that provide information on the structures of surface-immobilized peptides. The peptides are covalently bound to alkanethiolates that are self-assembled as monolayers on colloidal gold nanoparticles. The secondary structure of the immobilized peptides was characterized by quantifying the Ramachandran angles phi and psi. These angles were determined in turn from distances between backbone carbonyl 13C spins, measured with the double-quantum filtered dipolar recoupling with a windowless sequence experiment, and by determination of the mutual orientation of chemical shift anisotropy tensors of 13C carbonyl spins on adjacent peptide planes, obtained from the double-quantum cross-polarization magic-angle spinning spectrum. It was found that peptides composed of periodic sequences of leucines and lysines were bound along the length of the peptide sequence and displayed a tight alpha-helical secondary structure on the gold nanoparticles. These results are compared to similar studies of peptides immobilized on hydrophobic surfaces.
Asunto(s)
Oro/química , Nanoestructuras/química , Péptidos/química , Análisis de Fourier , Espectroscopía de Resonancia Magnética , Estructura Molecular , Tiopronina/químicaRESUMEN
Dipolar recoupling pulse sequences are of great importance in magic angle spinning solid-state NMR. Recoupling sequences are used for excitation of double-quantum coherence, which, in turn, is employed in experiments to estimate internuclear distances and molecular torsion angles. Much effort is spent on the design of recoupling sequences that are able to produce double-quantum coherence with high efficiency in demanding spin systems, i.e., spin systems with small dipole-dipole couplings and large chemical-shift anisotropies (CSAs). The sequence should perform robustly under a variety of experimental conditions. This paper presents experiments and computer calculations that extend the theory of double-quantum coherence preparation from the strong coupling/small CSA limit to the weak coupling limit. The performance of several popular dipole-dipole recoupling sequences-DRAWS, POST-C7, SPC-5, R1, and R2-are compared. It is found that the optimum performance for several of these sequences, in the weak coupling/large CSA limit, varies dramatically, with respect to the sample spinning speed, the magnitude and orientation of the CSAs, and the magnitude of dipole-dipole couplings. It is found that the efficiency of double-quantum coherence preparation by gamma-encoded sequences departs from the predictions of first-order theory. The discussion is supported by density-matrix calculations.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Modelos Químicos , Alanina/química , Isótopos de Carbono , Simulación por Computador , Malonatos/química , Ácido Oxálico/química , Teoría Cuántica , Ácido Succínico/químicaRESUMEN
Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composites technologies. Here, we describe both the theory and practice of double-quantum solid-state NMR (ssNMR) structure-determination techniques, as they are used to determine the secondary structures of surface-adsorbed peptides and proteins. In particular, we have used ssNMR dipolar techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Here, we also review NMR data on peptides designed to adsorb from aqueous solutions onto highly porous hydrophobic surfaces with specific helical secondary structures. The adsorption or covalent attachment of biological macromolecules onto polymer materials to improve their biocompatibility has been pursued using a variety of approaches, but key to understanding their efficacy is the verification of the structure and dynamics of the immobilized biomolecules using double-quantum ssNMR spectroscopy.
Asunto(s)
Materiales Biocompatibles/química , Durapatita/química , Proteínas y Péptidos Salivales/química , Secuencia de Aminoácidos , Animales , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Propiedades de SuperficieRESUMEN
Proteins directly control the nucleation and growth of biominerals, but the details of molecular recognition at the protein-biomineral interface remain poorly understood. The elucidation of recognition mechanisms at this interface may provide design principles for advanced materials development in medical and ceramic composite technologies. Here, we have used solid-state NMR techniques to provide the first high-resolution structural and dynamic characterization of a hydrated biomineralization protein, salivary statherin, adsorbed to its biologically relevant hydroxyapatite (HAP) surface. Backbone secondary structure for the N-terminal dodecyl region was determined using a combination of homonuclear and heteronuclear dipolar recoupling techniques. Both sets of experiments indicate the N-terminus is alpha-helical in character with the residues directly binding to the HAP being stabilized in the alpha-helical conformation by the presence of water. Dynamic NMR studies demonstrate that the highly anionic N-terminus is strongly adsorbed and immobilized on the HAP surface, while the middle and C-terminal regions of this domain are mobile and thus weakly interacting with the mineral surface. The direct binding footprint of statherin is thus localized to the negatively charged N-terminal pentapeptide sequence. Study of a site-directed mutant demonstrated that alteration of the only anionic side chain outside of this domain did not affect the dynamics of statherin on the HAP surface, suggesting that it does not play an important role in HAP binding.
Asunto(s)
Durapatita , Proteínas y Péptidos Salivales/química , Secuencia de Aminoácidos , Animales , Durapatita/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Termodinámica , AguaRESUMEN
Determination of the conformational flexibility of the furanose ring is of vital importance in understanding the structure of DNA. In this work we have applied a model of furanose ring motion to the analysis of deuterium line shape data obtained from sugar rings in solid hydrated DNA. The model describes the angular trajectories of the atoms in the furanose ring in terms of pseudorotation puckering amplitude (q) and the pseudorotation puckering phase phi. Fixing q, the motion is thus treated as Brownian diffusion through an angular-dependent potential U(phi). We have simulated numerous line shapes varying the adjustable parameters, including the diffusion coefficient D, pseudorotation puckering amplitude q, and the form of the potential U(phi). We have used several forms of the potential, including equal double-well potentials, unequal double-well potentials, and a potential truncated to "second order" in the Fourier series. To date, we have obtained best simulations for both equilibrium and nonequilibrium (partially relaxed) solid-state deuterium NMR line shapes for the sample [2' '-2H]-2'-deoxycytidine at the position C3 (underlined) in the DNA sequence [d(CGCGAATTCGCG)]2, using a double-well potential with an equal barrier height of U(0) = 5.5k(B)T ( approximately 3.3 kcal/mol), a puckering amplitude of q = 0.4 A, and a diffusion coefficient characterizing the underlying stochastic jump rate D = 9.9 x 10(8) Hz. Then the rate of flux for the C-D bond over the barrier, i.e., the escape velocity or the overall rate of puckering between modes, was found to be 0.7 x 10(7) Hz.
Asunto(s)
ADN/química , Desoxicitidina/química , Modelos Químicos , Conformación de Carbohidratos , Simulación por Computador , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , TermodinámicaRESUMEN
Base methylation plays an important role in numerous biological functions of DNA, from inhibition of cleavage by endonucleases to inhibition of transcription factor binding. Studies of nucleic acid structure have shown little differences in unmethylated DNAs and the identical sequence containing methylated analogues. We have investigated changes in the local dynamics of DNA upon substitution of a methylated cytosine analogue for cytosine using solid-state deuterium NMR. In particular, we have observed changes in the local dynamics at the target site of the M. HhaI restriction system. These studies observe changes in the amplitudes of the local backbone dynamics at the actual target site of the HhaI methyltransferase. This conclusion is another indication that the significant result of base methylation is to perturb the local dynamics, and therefore the local conformational flexibility, of the DNA helix, inhibiting or restricting the protein's ability to manipulate the DNA helix in order to perform its chemical alterations.
Asunto(s)
Metilación de ADN , ADN-Citosina Metilasas/metabolismo , ADN/química , ADN/metabolismo , Secuencia de Bases , Sitios de Unión , Islas de CpG , Deuterio , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Especificidad por SustratoRESUMEN
Solid-state deuterium NMR is used to investigate perturbations of the local, internal dynamics in the EcoRI restriction binding site, -GAATTC- induced by cytidine methylation. Methylation of the cytidine base in this sequence is known to suppress hydrolysis by the EcoRI restriction enzyme. Previous solid-state deuterium NMR studies have detected large amplitude motions of the phosphate-sugar backbone at the AT-CG junction of the unmethylated DNA sequence. This study shows that methylation of the cytidine base in a CpG dinucleotide reduces the amplitudes of motions of the phosphate-sugar backbone. These observations suggest a direct link between suppression of the amplitudes of localized, internal motions of the sugar-phosphate backbone of the DNA and inhibition of restriction enzyme cleavage.
Asunto(s)
Islas de CpG/genética , Metilación de ADN , ADN/química , ADN/metabolismo , Secuencia de Bases , Simulación por Computador , Citidina/química , Citidina/genética , Citidina/metabolismo , ADN/genética , Desoxirribonucleasa EcoRI/metabolismo , Deuterio/metabolismo , Difusión , Cinética , Espectroscopía de Resonancia Magnética , Movimiento (Física) , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Extracellular matrix proteins play key roles in controlling the activities of osteoblasts and osteoclasts in bone remodeling. These bone-specific extracellular matrix proteins contain amino acid sequences that mediate cell adhesion, and many of the bone-specific matrix proteins also contain acidic domains that interact with the mineral surface and may orient the signaling domains. Here we report a fusion peptide design that is based on this natural approach for the display of signaling peptide sequences at biomineral surfaces. Salivary statherin contains a 15-amino acid hydroxyapatite binding domain (N15) that is loosely helical in solution. To test whether N15 can serve to orient active peptide sequences on hydroxyapatite, the RGD and flanking residues from osteopontin were fused to the C terminus. The fusion peptides bound tightly to hydroxyapatite, and the N15-PGRGDS peptide mediated the dose-dependent adhesion of Moalpha(v) melanoma cells when immobilized on the hydroxyapatite surface. Experiments with an integrin-sorted Moalpha(v) subpopulation demonstrated that the alpha(v)beta(3) integrin was the primary receptor target for the fusion peptide. Solid state NMR experiments showed that the RGD portion of the hydrated fusion peptide is highly dynamic on the hydroxyapatite surface. This fusion peptide framework may thus provide a straightforward design for immobilizing bioactive sequences on hydroxyapatite for biomaterials, tissue engineering, and vaccine applications.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Durapatita/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas y Péptidos Salivales/metabolismo , Sialoglicoproteínas/metabolismo , Adsorción , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Dicroismo Circular , Humanos , Espectroscopía de Resonancia Magnética , Melanoma/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Osteopontina , Fragmentos de Péptidos/farmacología , Unión Proteica , Estructura Secundaria de Proteína , Receptores de Vitronectina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas y Péptidos Salivales/química , Sialoglicoproteínas/química , Células Tumorales CultivadasRESUMEN
Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 +/- 0.1 A. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete alpha-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an alpha-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.
Asunto(s)
Durapatita/química , Péptidos/química , Secuencia de Aminoácidos , Cristalización , Cinética , Espectroscopía de Resonancia Magnética , Conformación Proteica , Proteínas y Péptidos Salivales/química , Propiedades de SuperficieRESUMEN
A windowless, homonuclear dipolar recoupling pulse sequence (DRAWS) is described and a theoretical basis for describing its recoupling performance is developed using numerical techniques. It is demonstrated that DRAWS recouples weak dipolar interactions over a broad range of experimental and molecular conditions. We discuss two spectroscopic control experiments, which help to take into account effects due to insufficient proton decoupling, relaxation, and static dipolar couplings to nearby 13C spins at natural abundance. Finally DRAWS is used in combination with selective 13C labeling to measure 13C-13C distances in five doubly labeled DNA dodecamers, [d(CGCGAAT*T*CGCG)]2, which contain the binding site for the restriction enzyme EcoRI. The longest distance reported is 4.8 A. In most cases the distances agree well with those derived from X-ray crystallographic data, although small changes in hydration level can result in relatively large changes in internuclear distances.
Asunto(s)
ADN/química , Espectroscopía de Resonancia Magnética/métodos , Ácidos Nucleicos/química , Isótopos de Carbono , Simulación por Computador , Cristalografía por Rayos X , Cómputos Matemáticos , Conformación de Ácido NucleicoRESUMEN
At TpA steps in DNA, the adenine base experiences exceptionally large amplitude (20 degrees-50 degrees) and slow (10 ms-1 microsecond) motion [Kennedy et al. (1993) Biochemistry 32, 8022-8035] which has been correlated with transitions between multiple conformational states [Lefevre et al. (1985) FEBS Lett. 190, 37-40]. The base dynamics can be detected in one-dimensional 1H NMR spectra as excess line width of the aromatic proton resonances. The magnitude of the excess line width is temperature dependent and reaches a maximum at some temperature. In order to better understand the origin of the dynamics, we have studied the effect of N6-methylation of the TpA adenine on both the line widths and its local structure. Here, solution-state 500 and 750 MHz 1H NMR data collected on [d(CGAGGTTTAAACCTCG)]2 show that the excess line width of the TpA adenine-H2 is diminished when the TpA adenine is N6-methylated and that the line width no longer experiences a maximum as the temperature is varied. The resonances sharpen upon methylation because the amplitude of base motion is restricted due to steric effects and due to other structural changes at the TpA site. Additionally, both the TpA adenine-H8 and the exchangeable imino resonance of thymine at the TpA step were also found to have excess line width that is diminished upon N6-methylation. In order to elucidate the structural features responsible for TpA base dynamics, solution-state NMR structures of [d(CGAGGTTTAAACCTCG)]2 and its A9 N6-methylated derivative were determined at 750 MHz. Comparison of the structures shows that poor base stacking at the TpA step may contribute to, or be the origin of, its base dynamics.
Asunto(s)
Adenina/metabolismo , ADN/química , Secuencia de Bases , Espectroscopía de Resonancia Magnética , Metilación , Modelos Estructurales , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
The widely used antitumor drug cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) reacts with DNA, cross-linking two purine residues through the N7 atoms, which reside in the major groove in B-form DNA. The solution structure of the short duplex [d(CAT-AGCTATG)]2 cross-linked at the GC:GC site was determined by nuclear magnetic resonance (NMR). The deoxyguanosine-bridging cis-diammineplatinum(II) lies in the minor groove, and the complementary deoxycytidines are extrahelical. The double helix is locally reversed to a left-handed form, and the helix is unwound and bent toward the minor groove. These findings were independently confirmed by results from a phase-sensitive gel electrophoresis bending assay. The NMR structure differs markedly from previously proposed models but accounts for the chemical reactivity, the unwinding, and the bending of cis-DDP interstrand cross-linked DNA and may be important in the formation and repair of these cross-links in chromatin.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , ADN/química , Conformación de Ácido Nucleico , Secuencia de Bases , ADN/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , SolucionesAsunto(s)
ADN/química , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , Composición de Base , Secuencia de Bases , Carbohidratos/química , ADN/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Matemática , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/químicaRESUMEN
This chapter reviews recent progress in understanding duplex DNA dynamics. The weakly bending rod model of Schurr and coworkers is described and compared to a model-free formulation of DNA dynamics. Numerical trajectory methods for obtaining dynamic information are also discussed. The general principles of magnetic resonance relaxation are the reviewed, and the methods by which molecular motions are incorporated into the calculation of relaxation rates or the simulation of experimental NMR and EPR data are described. The impact that the time scale of the dynamics exerts on various computational methods is considered, and in particular, the implementation of (a) the stochastic Liouville equation, (b) the Redfield relaxation matrix, and (c) tensorial preaveraging is described. Expressions for direct and cross-relaxation processes are developed by expanding the density matrix in terms of an irreducible tensor basis set.
Asunto(s)
Simulación por Computador , ADN/química , Modelos Químicos , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Resonancia Magnética , Sondas Moleculares , TermodinámicaRESUMEN
Two-dimensional 1H NMR spectroscopy has been used to investigate the binding site, binding interactions, and the conformation of a 1:1 complex of aponeocarzinostatin (apo-NCS) with ethidium bromide in solution. The protein component in the complex has been sequence-specifically assigned using information derived from coherence transfer and two-dimensional homonuclear 1H NOESY experiments. The conformation of the protein in the complex has been found to be similar to the free form of the apoprotein, and intermolecular NOEs between the residues of the protein to protons on the ethidium bromide suggest that the ethidium bromide is bound to the protein in the same cleft in which the neocarzinostatin chromophore binds. Protons on ethidium show NOE interactions to the following protein residues: Trp-39, Leu-45, Cys-47, and Gln-94 which interact with the phenanthridine ring system of ethidium, Gly-102 and Asn-103 which interact with the alkyl chain of ethidium, and Phe-52 which interacts with the phenyl ring of ethidium. The orientation of ethidium in the cleft of apo-NCS is compared and contrasted to orientation of the chromophore as determined by high-resolution NMR and X-ray diffraction studies.
Asunto(s)
Apoproteínas/química , Cinostatina/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Etidio/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
Solid-state 2H NMR spectroscopy has been used to investigate the base dynamics of a RNA oligonucleotide with a defined sequence, [r(CGCGAAUUCGCG)]2, which contains the RNA analogue of the EcoRI binding site. The C8 protons of all purines in the self-complementary dodecamer were exchanged for deuterons. The quadrupole-echo lineshapes and spin-lattice relaxation times as a function of hydration for the sample in the form of the Na salt have previously been reported. In that study the 2H NMR lineshapes and T1 values of [r(CG*CG*A*A*UUCG*CG*)]2 were compared with those of the analogously labeled DNA sequence, [(CG*CG*A*A*TTCG*CG*)]2 (Wang et al., J. Am. Chem. Soc. 114, 6583, 1992). It was concluded that the amplitudes of purine motion for DNA and RNA are similar at all hydration levels; however, the rate difference observed at low-hydration levels may or may not persist at high hydration. Here the internal motions of the purine bases in the RNA oligomer have been thoroughly investigated. Three models were used to simulate the motion: (1) two-site jump, (2) diffusion in a cone, and (3) restricted diffusion on the surface of a cone. The purine motion is best simulated by the restricted-diffusion on a cone model with an amplitude of +/- 9.5 degrees and a rate between 8.0 x 10(6) rad/s at 90% RH and 8.4 x 10(8) rad/s at 0% RH. This small amplitude and fast rate of purine motion for RNA are similar to previous results obtained for DNA purines.
Asunto(s)
Espectroscopía de Resonancia Magnética , Purinas/química , ARN/química , Secuencia de Bases , Conformación de Ácido NucleicoRESUMEN
Lefevre et al. originally observed conformational transitions at the TpA step in the TTAA Pribnow box sequence of the trp promoter [Lefevre, J.-F., Lane, A. N., & Jardetzky, O. (1985) FEBS Lett. 190, 37-40]. In 500-MHz 1H NMR studies on the TnA(n)-containing DNA oligonucleotides [d(CGAGGTTTAAACCTCG)]2, [d(GCTCCTTTAAAGGAGC)]2, and [d(GCCGTTAACGGC)]2, we observe that, in addition to the H2 proton (which resides in the minor groove of DNA), the H8 proton of the first adenine (which resides in the major groove) is also broadened due to motion at the TpA junction. In analogous 16-mers where the T3A3 segment has been replaced by an A3T3 sequence, and therefore contains CA, GA, and AT steps (but no TA steps), all adenine proton resonances are narrow, indicating that the broadening occurs only at TpA steps. Assuming chemical exchange in the form of conformational dynamics, e.g., oscillation of the purine base about the glycosidic torsion angle, the experimental 500-MHz 1H T1 rho and 2D-NOESY data were used to constrain the correlation time of the internal motion to a range between the T1 and T1 rho minima. Calculated line shapes using a two-site exchange model indicate that the motion has an amplitude of 20-50 degrees with an associated tau c of ca. 1.6 x 10(-4) to 1.0 x 10(-5) s rad-1, respectively. The mobility appears to be a consequence of the structure at the TpA junction which is characterized by (1) a wide minor groove between two regions of narrow minor groove, (2) an unusual average orientation of the adenine heterocycle probably resulting from a poor base-stacking interaction of the adenine with the preceding thymine, and (3) a sharp discontinuity in the sugar conformation at the TpA step.
Asunto(s)
ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Secuencia de Bases , Sitios de Unión , Fosfatos de Dinucleósidos/química , Espectroscopía de Resonancia Magnética , Modelos Químicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/químicaRESUMEN
Backbone dynamics in the [5',5"-2H2]2'-deoxythymidine labeled duplex dodecamer [d-(CGCGAAT*T*CGC)]2 have been investigated by solid-state 2H NMR. Quadrupolar echo line shapes, spin-lattice relaxation, and quadrupolar echo decay times were obtained over hydration levels ranging from W = 0.0 to 25.2 (moles of H2O/mole of nucleotide). Variation of the line shape with changing hydration level was analyzed by using models employed in previous investigations of dodecamer base and sugar dynamics. Both fast local motions and a slower helix motion were present within the oligonucleotide. The fast motion was modeled as a four-site libration whose amplitude increased with hydration level. The root mean square amplitude of this librational model was 2-6 degrees larger than the amplitude observed in either the furanose ring or base labeled material for the entire range of hydration levels investigated. The observed line shape was inconsistent with a rapid three-site trans-gauche isomerization. A slow motion about the helix axis was observed at low water levels and increased in rate and amplitude with hydration. This motional model is in agreement with previous oligonucleotide studies.
Asunto(s)
Oligodesoxirribonucleótidos/química , Secuencia de Bases , Deuterio , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Movimiento (Física) , Conformación de Ácido Nucleico , Agua/químicaRESUMEN
Sequential assignments and secondary structural analysis have been accomplished for the 113-residue apoprotein of the antitumor drug neocarzinostatin (NCS) from Streptomyces carzinostaticus. A total of 98% of the main-chain and 77% of the side-chain resonances have been sequence specifically assigned by use of information from coherence transfer experiments and by sequential and interstrand NOEs. Because of the complexity of the NCS spectrum, several sequential assignment strategies were employed to complete the analysis. Apo-NCS consists of three antiparallel beta-sheeted domains by NMR analysis. There is an extensive four-strand antiparallel beta-sheet, and two two-stranded domains. One of the two-strand domains is contiguous, S72-N87, with chain reversal occurring through the region L77-R82. The other two-stranded domain has the section G16-A24 antiparallel with respect to the region S62-R70. This secondary structure is consistent with the crystal structure of holo-NCS at 2.8-A resolution.