RESUMEN
To determine its potential for interacting with other components of the casein micelle, the N-terminal section of bovine alphas1-casein-B, residues 1-23, was investigated with nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies, and molecular modeling. NMR data were not consistent with conventional alpha-helical or beta-sheet structures, but changes in N-H proton chemical shifts suggested thermostable structures. Both CD and FTIR predicted a range of secondary structures for the peptide (30-40% turns, 25-30% extended) that were highly stable from 5 degrees C to 25 degrees C. Other conformational elements, such as loops and polyproline II helix, were indicated by FTIR only. Molecular dynamics simulation of the peptide predicted 32% turns and 27% extended, in agreement with FTIR and CD predictions and consistent with NMR data. This information is interpreted in accord with recent spectroscopic evidence regarding the nature of unordered conformations, leading to a possible role of alphas1-casein (1-23) in facilitating casein-casein interactions.
Asunto(s)
Caseínas/química , Fragmentos de Péptidos/química , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
We employ NMR structure determination, thermodynamics, and enzymatics to uncover the structural, thermodynamic and enzymatic properties of alpha/beta-ODNs containing 3'-3' and 5'-5' linkages. RNase H studies show that alpha/beta-gapmers that are designed to target erbB-2 efficiently elicit RNase H activity. NMR structures of DNA.DNA and DNA.RNA duplexes reveal that single alpha-anomeric residues fit well into either duplex, but alter the dynamic properties of the backbone and deoxyriboses as well as the topology of the minor groove in the DNA.RNA hybrid.
Asunto(s)
Oligonucleótidos Antisentido/química , Animales , ADN/química , Activación Enzimática , Humanos , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Oligonucleótidos Antisentido/farmacología , ARN/química , Ribonucleasa H/metabolismo , TermodinámicaRESUMEN
Nucleic acid duplexes featuring a single alpha-anomeric thymidine inserted into each DNA strand via 3'-3' and 5'-5' phosphodiester linkages exhibit local conformational dynamics that are not adequately depicted by conventional restrained molecular dynamics (rMD) methods. We have used molecular dynamics with time-averaged NMR restraints (MDtar) to explore its applicability to describing the conformational dynamics of two alpha-containing duplexes--d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2 and d(ATGG-3'-3'-alphaT-5'-5'-GCTC) x r(gagcaccau). In contrast to rMD, enforcing NOE-based distance restraints over a period of time in MDtar rather than instantaneously results in better agreement with the experimental NOE and J-data. This conclusion is based on the dramatic decreases in average distance and coupling constant violations (delta d(av), J(rms), and delta J(av)) and improvements in sixth-root R-factors (R(X)). In both duplexes, the deoxyribose ring puckering behavior predicted independently by pseudorotation analysis is portrayed remarkably well using this approach compared to rMD. This indicates that the local dynamic behavior is encoded within the NOE data, although this is not obvious from the local R(X) values. In both systems, the backbone torsion angles comprising the 3'-3' linkage as well as the (high S-) sugars of the alpha-nucleotide and preceding residue (alpha - 1) are relatively static, while the conformations of the 5'-5' linkage and the sugar in the neighboring beta-nucleotide (alpha + 1) show enhanced flexibility. To reduce the large ensembles generated by MDtar to more manageable clusters we utilized the PDQPRO program. The resulting PDQPRO clusters (in both cases, 13 structures and associated probabilities extracted from a pool of 300 structures) adequately represent the structural and dynamic characteristics predicted by the experimental data.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Oligonucleótidos/química , Disparidad de Par Base , Emparejamiento Base , Desoxirribosa/química , Isomerismo , Modelos Moleculares , Termodinámica , Factores de TiempoRESUMEN
We report the thermodynamic and structural properties of an alpha-containing DNA.RNA nonamer hybrid duplex, d(ATGG-3'-3'-alphaT-5'-5'-GCTC).r(gagcaccau). The RNA strand corresponds to the core of the initiation sequence for the transcript of the erbB-2 oncogene. The tandem anomeric and polarity changes in the DNA strand result in a slight decrease in thermostability (DeltaT(m) = -2.8 degrees C) compared to the unmodified control hybrid. The three-dimensional solution structure determination of the alpha-containing DNA.RNA hybrid, conducted via restrained molecular dynamics using interproton distance (nuclear Overhauser enhancement) and furanose ring torsion angle (J-based) restraints, converged to a final ensemble of structures from unique starting models. In agreement with hyperchromicity and circular dichroism data, the final average structure derived from this ensemble is consistent with an overall A-like motif featuring Watson-Crick base pairing and base stacking across the entire sequence, albeit with localized B-like traits within the DNA strand. Comparative pseudorotation analyses of the J-coupling data for this hybrid and its unmodified control reveal a surprising increase in S-puckering for two nucleotides immediately upstream of the 3'-3' linkage, and the associated narrowing of the minor groove in this portion of the hybrid. Other structural perturbations are localized to and diagnostic of the central alpha-nucleotide and juxtaposed polarity reversals. The structural information presented here has direct relevance to the design of future antisense oligonucleotides composed of these modifications.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Oligodesoxirribonucleótidos/química , ARN/química , Timidina/análogos & derivados , Timidina/química , Dicroismo Circular , Simulación por Computador , Cristalografía por Rayos X , Desoxirribosa/química , Glicósidos/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Soluciones , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
We present the high-resolution solution structures of a self-complementary DNA decamer duplex featuring a single alpha-anomeric nucleotide per strand encompassed by a set of 3'-3' and 5'-5' phosphodiester linkages, d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2, alphaT, and its unmodified control, d(GCGAATTCGC)2, obtained by restrained molecular dynamics. Interproton distance and deoxyribose ring torsion angle restraints were deduced from homonuclear NOESY and DQF-COSY data, respectively. For both the control and alphaT duplexes, excellent global convergence was observed from two different (A- and B-) starting models. The final average structures of the two duplexes are highly homologous, and overall possess the traits characteristic of right-handed B-DNA duplexes. However, localized differences between the two structures stem from the enhanced conformational exchange in the deoxyribose ring of the cytidine following the 5'-5' linkage, the C3'- exo pseudorotation phase angle of the alpha-nucleotide, and unusual backbone torsions in the 3'-3' and 5'-5' phosphodiester linkages. The structural data reported here are relevant to the design of antisense therapeutics comprised of these modifications.
Asunto(s)
Sondas de ADN/química , Oligonucleótidos/química , ADN Complementario , Espectroscopía de Resonancia Magnética , Conformación de Ácido NucleicoRESUMEN
We present a summary of the quadrupolar metal ion NMR studies of metalloproteins conducted in our laboratory in recent years. The approaches we employ can be subdivided into two categories: (i) the use of low-frequency metal nuclei to probe metal ion binding sites in small proteins, exemplified by 43Ca NMR studies of alpha-lactalbumins and calcium-binding lysozymes, and (ii) the novel detection of the central transition of half-integer quadrupolar nuclei of moderate frequency bound to large metalloproteins, typified by 27Al, 45Sc, 69,71Ga, and 51V NMR studies of the transferrins. We highlight the chemical information regarding the nature of metal ion binding sites that can be obtained from this technique and emphasize the salient parameters that an investigator must consider to successfully apply quadrupolar NMR to the study of biological macromolecules.
Asunto(s)
Iones , Espectroscopía de Resonancia Magnética , Metaloproteínas/química , Animales , Sitios de Unión , Humanos , Lactalbúmina/química , Metales , Muramidasa/químicaRESUMEN
We present a summary of our research to date on a family of self-complementary DNA decamers containing single alpha-anomeric nucleotides flanked by 3'-3' and 5'-5' phosphodiester linkages from the perspective of the salient NMR techniques employed to shed light on the structural and dynamic properties of these sequences. Research into this class of synthetic alpha-/beta-oligonucleotides containing mixed strand disposition may have medical relevance given their recently documented efficacy as antisense therapeutics.
Asunto(s)
ADN/química , Espectroscopía de Resonancia Magnética , Conformación de Ácido Nucleico , Nucleótidos/química , Algoritmos , Emparejamiento Base , Fenómenos Químicos , Química Física , Dicroismo Circular , Modelos Moleculares , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/química , Oligonucleótidos Antisentido/química , Isótopos de Fósforo , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
We present a thermodynamic, enzymatic, and spectroscopic study of three self-complementary DNA decamer duplexes, d[GCGAATT-3'-3'-(alphaC)-5'-5'-GC]2 (alphaC), d[GCG-3'-3'-(alphaA)-5'-5'-ATTCGC]2 (alphaA), and d[GC-3'-3'-(alphaG)-5'-5'-AATTCGC]2 (alphaG), which are identical in sequence but contain one alpha-anomeric nucleotide per strand in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds; the results are placed in the context of our recent studies on the other members of this series, namely alphaT, d[GCGAAT-3'-3'-(alphaT)-5'-5'-CGC]2, and the unmodified control [Aramini, J. M., et al. (1996) Biochemistry 35, 9355-9365]. On the basis of UV hyperchromicity and melting profiles as well as 1H and 31P nuclear magnetic resonance (NMR) spectroscopic data, we conclude that all five constructs form stable duplexes, with very comparable structural features that are consistent with an overall right-handed, antiparallel B-DNA motif and Watson-Crick base pairing throughout. However, each of the alpha-containing sequences exhibits unique thermodynamic and structural differences ascribed to the nature (and position) of the alpha-nucleotide. First, the thermostability of these duplexes decreases from the control to alphaC in the following series: control > alphaT approximately alphaA approximately alphaG > alphaC. Second, in each of the four alpha-duplexes, 1H and 31P chemical shift differences compared to those of the control duplex are largely confined to the region encompassing the alpha-nucleotide and unnatural phosphodiester linkages, as well as neighboring nucleotides. Surprisingly, for alphaC, these modifications result in a significant alteration to the backbone conformation at the phosphodiester group directly across from the 3'-3' linkage. Finally, spin-spin (J) coupling data, specifically Sigma1', indicate that the vast majority of the furanose rings in these duplexes display a high propensity for adopting the S pucker. However, in alphaC, alphaA, and alphaT (but not alphaG), the sugar ring conformation in the nucleotide immediately following the 5'-5' linkage is described by an approximately equal distribution between the N and S conformers.
Asunto(s)
ADN/química , Desoxirribonucleótidos/química , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Oligonucleótidos Antisentido/química , Termodinámica , Composición de Base , Conformación de Carbohidratos , Nucleótidos de Desoxiadenina/química , Nucleótidos de Desoxicitosina/química , Nucleótidos de Desoxiguanina/química , Desoxirribonucleasa EcoRI , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Isótopos de Fósforo , Espectrofotometría UltravioletaRESUMEN
The interaction between CD4 and major histocompatibility complex class II proteins provides a critical co-receptor function for the activation of CD4(+) T cells implicated in the pathogenesis of a number of autoimmune diseases and transplantation responses. A small synthetic cyclic heptapeptide was designed and shown by high resolution NMR spectroscopy to closely mimic the CD4 domain 1 CC' surface loop. This peptide effectively blocked stable CD4-major histocompatibility complex class II interaction, possessed significant immunosuppressive activity in vitro and in vivo, and strongly resisted proteolytic degradation. These results demonstrate the therapeutic potential of this peptide as a novel immunosuppressive agent and suggest a general strategy of drug design by using small conformationally constrained peptide mimics of protein surface epitopes to inhibit protein interactions and biological functions.
Asunto(s)
Antígenos CD4/química , Antígenos CD4/efectos de los fármacos , Epítopos/química , Linfocitos/inmunología , Oligopéptidos/química , Oligopéptidos/farmacología , Péptidos Cíclicos/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Animales , Sitios de Unión , Adhesión Celular/efectos de los fármacos , Gráficos por Computador , Diseño de Fármacos , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Genes MHC Clase II , Rechazo de Injerto/inmunología , Supervivencia de Injerto/efectos de los fármacos , Enfermedad Injerto contra Huésped , Humanos , Prueba de Cultivo Mixto de Linfocitos , Linfocitos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Modelos Moleculares , Oligopéptidos/síntesis química , Péptidos Cíclicos/síntesis química , Trasplante de Piel/inmunologíaRESUMEN
We report a comparative spectroscopic study of a novel self-complementary duplex decamer, d(GCGAAT-3'-3'-(alpha T)-5'-5'-CGC)2, in which an alpha-anomeric nucleotide has been inserted into the sequence in a parallel orientation via 3'-3' and 5'-5' phosphodiester bonds, and its unmodified B-DNA analog, d(GCGAATTCGC)2. Plots of the hyperchromicity and circular dichroism of these oligonucleotides are virtually identical, indicating that the overall base stacking and handedness are preserved in the alpha duplex. Thermodynamic parameters extracted from UV melting experiments show that the alpha duplex is only slightly less stable than the control. A near complete set of 1H and 31P nuclear magnetic resonance (NMR) assignments were obtained for both duplexes using classical one- and two-dimensional approaches. Several lines of evidence, in particular, imino 1H, 31P, nuclear Overhauser enhancement, and deoxyribose ring proton spin-spin coupling data, convincingly demonstrate that the overall structural integrity of the alpha and control duplexes are quite comparable, with any perturbations in the former localized to the regions of the construct encompassing the alpha-nucleotide and the unique backbone linkages. Specifically, the alpha duplex exhibits normal Watson-Crick type base pairing, it remains antiparallel except at the inverted nucleotide, all bases are in the anti orientation, and the sugar ring puckering is predominantly "S"-type. However, the J-coupling information for the alpha-nucleotide and the neighboring (3') cytidine are notably different, and reflect a decrease in the amplitude of the sugar pucker in alpha T7, and a significant shift in the conformational equilibrium of the furanose ring in C8 toward the "N"-type pucker. The feasibility of synthesizing oligodeoxynucleotides containing a combination of alpha sugars and short parallel stranded segments, their propensity for forming stable duplexes, and the structural insights into such complexes reported here are of potential importance in the area of antisense therapy.
Asunto(s)
ADN/química , Oligodesoxirribonucleótidos/química , Oligonucleótidos Antisentido/química , Composición de Base , Secuencia de Bases , Dicroismo Circular , Simulación por Computador , ADN Complementario/química , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Oligonucleótidos Antisentido/síntesis química , Oligonucleótidos Antisentido/metabolismo , Organofosfatos/química , Programas Informáticos , Espectrofotometría , TermodinámicaRESUMEN
1H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to alpha-lactalbumins from bovine, goat, and human sources. We have identified two 1H-NMR markers diagnostic of metal ion binding to the high-affinity Ca2+-binding site of bovine alpha-lactalbumin, namely the signals corresponding to the delta-CH3 groups of Met-90, and a leucine, tentatively assigned to Leu-96. A number of metal ions other than Ca2+ bind to this site in either slow (La3+, Lu3+, Y3+, Sr2+, Sc3+) or fast (Cd2+, Ba2+, Pb2+) exchange. From competition experiments using this approach, we have determined an affinity series for metal ion binding at this site, in which lanthanides and Y3+ bind the strongest (Y3+>La3+, Lu3+>Ca2+>Sr2+>Cd2+, Pb2+, Ba2+>Sc3+). Several metal ions do not alter the 1H spectrum of bovine alpha-lactalbumin, retaining the protein in an 'apo-like' state. Evidence is given to support the notion that the paramagnetic divalent metal ions Co2+ and Cu2+ bind to a second distinct site, termed the 'zinc site', and that His-68 is involved in metal ion coordination. Finally, stopped-flow techniques using the indicator Xylenol orange were employed to obtain lanthanide off-rates for bovine, human, and goat alpha-lactalbumins (bovine and goat alpha-LA: k(off)(s-1) approximately 0.2 to 0.01 from La3+ to Lu3+; human alpha-LA: k(off)(s-1) approximately 0.02 to 0.001 from La3+ to Lu3+). In each case, we found that k(off) values decreased by an order of magnitude across the series, meaning that the dissociation constants for these metal ions are relatively constant. Data for the bovine and goat proteins are virtually identical, while the off-rates for human alpha-lactalbumin are appreciably slower. In addition, these rates are much slower than the Ca2+ off-rate for the bovine protein (k(off)(s-1) approximately 2 to 5), determined using the fluorescent indicator, BAPTA.
Asunto(s)
Lactalbúmina/química , Espectroscopía de Resonancia Magnética , Metales/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Biomarcadores , Calcio/metabolismo , Bovinos , Cobalto/metabolismo , Cobre/metabolismo , Ácido Edético/farmacología , Cabras , Humanos , Concentración de Iones de Hidrógeno , Cinética , Lactalbúmina/metabolismo , Metales de Tierras Raras/metabolismo , Metionina/química , Metionina/metabolismo , Unión ProteicaRESUMEN
A number of reports in recent years have demonstrated the feasibility of detecting quadrupolar metal ions bound tightly to rather large proteins via the quadrupolar central transition (QCT) NMR approach. In this article, an in-depth investigation of several interesting properties of transferrin-bound 27Al NMR signals, namely, their dependence on temperature, viscosity, and molecular size is presented. It is shown that (1) decreasing temperature and (2) increasing viscosity by adding reagents such as glycerol and ethylene glycol perturb only the linewidths of transferrin-bound 27Al signals, and, in fact, produce a decrease in signal linewidth. These effects are in accord with quadrupolar relaxation theory, which predicts that the linewidth of the central transition of a half-integer quadrupolar nucleus should decrease with increasing correlation time of the protein under nonextreme narrowing conditions. Furthermore, it is demonstrated that these trends, which are completely opposite to those generally observed in NMR spectroscopy, can be exploited to monitor ovotransferrin half-molecule reassociation reactions. In combination with the peculiar properties of transferrin-bound quadrupolar nuclei reported in the literature to date, the phenomena described here provide the basis for understanding the conditions and experimental parameters which may facilitate the application of the QCT NMR technique to the study of other quadrupolar nuclei and proteins.
Asunto(s)
Espectroscopía de Resonancia Magnética , Transferrina/química , Aluminio/química , Animales , Conalbúmina/química , Glicol de Etileno , Glicoles de Etileno/química , Glicerol/química , Humanos , Lactoferrina/química , Conformación Molecular , Unión Proteica , Solventes/química , Temperatura , ViscosidadRESUMEN
The intercellular messenger nitric oxide is produced through the action of nitric oxide synthases, a class of enzymes that is regulated by calcium-calmodulin (CaM). In this work, the interaction of CaM with a 23-amino-acid residue synthetic peptide, encompassing the CaM-binding domain of constitutive rat cerebellar nitric oxide synthase (cNOS), was investigated by various NMR methods. Cadmium-113 NMR studies showed that binding of the cNOS peptide increased the affinity of CaM for metal ions and induced interdomain cooperativity in metal ion binding as earlier observed for complexes of CaM with myosin light chain kinase (MLCK) peptides. By using specific isotopically labeled [13C]methyl-Met and selenomethionine-substituted CaM in two-dimensional proton-detected 13C and 77Se NMR studies, we obtained evidence for the involvement of the Met residues of CaM in the binding of the cNOS peptide. These residues form two hydrophobic surface areas on CaM, and they are also involved in the binding of other target proteins. A nitroxide spin-labeled version of the cNOS peptide caused broadening only for NMR resonances in the N-terminal half of CaM, showing that the peptide binds with a C to N orientation to the N- and C-terminal domains of CaM. pH titration experiments of CaM dimethylated with [13C]formaldehyde show that Lys-75 (and Lys-148) experience a large increase in pKa upon peptide binding; this indicates an unraveling of part of the helical linker region of CaM upon cNOS peptide binding. Taken together, our data show that the cNOS and MLCK peptides bind in a closely analogous fashion to CaM.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Calmodulina/metabolismo , Cerebelo/enzimología , Aminoácido Oxidorreductasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calmodulina/química , Espectroscopía de Resonancia Magnética , Metionina/análisis , Datos de Secuencia Molecular , Óxido Nítrico Sintasa , Péptidos/química , Péptidos/metabolismo , Conformación Proteica , Ratas , Especificidad por SustratoRESUMEN
The high-affinity calcium-binding sites of bovine and human alpha-lactalbumin as well as equine lysozyme were analyzed by 113Cd NMR spectroscopy. In the case of equine lysozyme, the addition of isotopically enriched 113Cd2+ results in a signal at delta = -75.9 ppm corresponding to the metal ion bound to the lone Ca(2+)-binding site of the protein. A peak at virtually the identical resonance position (delta = -77.1 ppm) was observed in the analogous experiment with bovine alpha-lactalbumin. In addition, a signal upfield of these (delta = -94.7 ppm) was observed for 113Cd(2+)-substituted human alpha-lactalbumin. The chemical shifts of these proteins are in the vicinity of those reported for other Ca(2+)-binding proteins. The field dependence of the 113Cd signals for all three proteins and bovine calmodulin were compared. At each field, the 113Cd signal linewidths for the alpha-lactalbumins and the lysozyme are somewhat broader than those observed for the EF-hand protein. In addition, the 113Cd linewidths for the lactalbumins and the lysozyme, especially bovine alpha-lactalbumin, increase dramatically with the square of the magnetic field strength, indicative of the presence of nuclear relaxation via chemical shift anisotropy and chemical exchange. The protein-bound 113Cd signals for the alpha-lactalbumins are also markedly affected by changes in the amount of K+ present, since Cd2+ and K+ can compete for occupation of the high-affinity Ca(2+)-site. Their linewidths also to some extent depend on the concentration of the protein itself.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Lactalbúmina/química , Muramidasa/química , Animales , Cadmio , Bovinos , Caballos , Humanos , Isótopos , Transducción de Señal , VolumetríaRESUMEN
We have examined the binding of Tl3+ to human serotransferrin and chicken ovotransferrin in the presence of carbonate and oxalate by 205Tl and 13C NMR spectroscopy. With carbonate as the synergistic anion, one observes two 205Tl NMR signals due to the bound metal ion in the two high-affinity iron-binding sites of each protein. When the same adducts are prepared with 13C-labeled carbonate, one finds two closely spaced doublets in the carbonyl region of the 13C NMR spectrum of serotransferrin; these correspond to the labeled anion directly bound to the metal ion in both sites of the protein. The analogous resonances in ovotransferrin are completely degenerate, and only one doublet can be detected. The magnitudes of the spin-spin coupling between the bound metal ion and carbonate range from 2J(205Tl-13C) approximately 270 to 290 Hz. We have used the proteolytic half-molecules of ovotransferrin and the recombinant N-terminal half-molecule of serotransferrin to assign the 205Tl and 13C NMR signals due to the bound metal ion and anion in both proteins. From titration studies, we found that Tl3+ is bound with a greater affinity at the C-terminal site of serotransferrin, whereas no site preference can be noted for ovotransferrin. When oxalate is used as the anion instead of carbonate, the 205Tl NMR signals arising from the bound metal ion in the sites of ovotransferrin are shifted downfield and become almost degenerate. A very complex pattern of resonances is observed for bound 13C2O4(2-) in the 13C NMR spectra of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Conalbúmina/química , Conalbúmina/metabolismo , Espectroscopía de Resonancia Magnética , Talio/metabolismo , Transferrina/química , Transferrina/metabolismo , Animales , Isótopos de Carbono , Carbonatos/farmacología , Pollos , Humanos , Oxalatos/farmacologíaRESUMEN
The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by 43Ca NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective 43Ca NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta approximately -3 to -7 ppm) and quadrupole coupling constants (chi approximately 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pKa values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine alpha-lactalbumin approximately human alpha-lactalbumin greater than pigeon lysozyme greater than equine lysozyme (KD approximately 5 x 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (KD = 3 x 10(-3) M) was obtained for bovine alpha-lactalbumin, but not for the other proteins studied.(ABSTRACT TRUNCATED AT 250 WORDS)