RESUMEN
Azurin has a beta-barrel fold comprising eight beta-strands and one alpha helix. A disulfide bond between residues 3 and 26 connects the N-termini of beta strands beta1 and beta3. Three mutant proteins lacking the disulfide bond were constructed, C3A/C26A, C3A/C26I and a putative salt bridge (SB) in the C3A/S25R/C26A/K27R mutant. All three mutants exhibit spectroscopic properties similar to the wild-type protein. Furthermore, the crystal structure of the C3A/C26A mutant was determined at 2.0 A resolution and, in comparison to the wild-type protein, the only differences are found in the immediate proximity of the mutation. The mutants lose the 628 nm charge-transfer band at a temperature 10-22 degrees C lower than the wild-type protein. The folding of the zinc loaded C3A/C26A mutant was studied by guanidine hydrochloride (GdnHCl) induced denaturation monitored both by fluorescence and CD spectroscopy. The midpoint in the folding equilibrium, at 1.3 M GdnHCl, was observed using both CD and fluorescence spectroscopy. The free energy of folding determined from CD is -24.9 kJ.mol-1, a destabilization of approximately 20 kJ.mol-1 compared to the wild-type Zn2+-protein carrying an intact disulfide bond, indicating that the disulfide bond is important for giving azurin its stable structure. The C3A/C26I mutant is more stable and the SB mutant is less stable than C3A/C26A, both in terms of folding energy and thermal denaturation. The folding intermediate of the wild-type Zn2+-azurin is not observed for the disulfide-deficient C3A/C26A mutant. The rate of unfolding for the C3A/C26A mutant is similar to that of the wild-type protein, suggesting that the site of the mutation is not involved in an early unfolding reaction.
Asunto(s)
Azurina/química , Azurina/genética , Disulfuros , Dicroismo Circular , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Guanidina/farmacología , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oxidación-Reducción , Pliegue de Proteína , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Espectrometría de Fluorescencia , Temperatura , Rayos Ultravioleta , Zinc/farmacologíaRESUMEN
A monoclonal antibody, B1C1, binding to an epitope of antigenic site II of the herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, is a potent inhibitor of two important biological functions of gC-1: its binding to cell surface heparan sulfate and its binding to the receptor for complement factor C3b. Here, we have analyzed a B1C1-resistant HSV-1 variant (HSV-12762/B1C1B4.2), obtained after passage of wild type HSV-1 (HSV-12762) in the presence of high concentrations of B1C1. The transport of newly synthesized mutant gC-1 to the cell surface was comparable to that of wild type glycoprotein, but no binding of surface-associated mutant gC-1 to B1C1 was detected. However, mutant and wild type gC-1 bound equally well to other site II Mabs. Attachment of wild type but not mutant virus was inhibited by B1C1. Sequencing of the mutant gC-1 gene revealed only one nucleotide change, resulting in replacement of Thr150 by an Ile, in turn destroying an N-glycosylation site at Asn148. Loss of one complex type N-linked glycan was confirmed by endoglycosidase digestion and subsequent SDS-polyacrylamide gel electrophoresis. Circular dichroism analysis of purified gC-1 from cells infected with mutant or wild type virus did not reveal any difference in secondary structure between mutant and wild type gC-1. It was not possible to obtain a B1C1-resistant phenotype by nucleotide-directed mutagenesis of gC-1 where Asn148 was changed to a glutamine. These data demonstrated that the threonine of the glycosylation site and not the N-linked glycan in itself was essential for B1C1 binding
Asunto(s)
Epítopos/química , Proteínas del Envoltorio Viral/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Asparagina , Secuencia de Bases , Sitios de Unión de Anticuerpos , Conformación de Carbohidratos , Electroforesis en Gel de Poliacrilamida , Glicosilación , Heparitina Sulfato/metabolismo , Técnicas de Inmunoadsorción , Datos de Secuencia Molecular , Mutación Puntual , Polisacáridos/química , Estructura Secundaria de Proteína , Receptores de Complemento 3b/metabolismo , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Electrochemical measurements show that there are high-potential states of two copper proteins, Pseudomonas aeruginosa azurin and Thermus thermophilus CuA domain; these perturbed states are formed in guanidine hydrochloride (GuHCl) solution in which the proteins are still blue (azurin) and purple (CuA). In each case, the high-potential state forms reversibly. Absorption (azurin, CuA), visible circular dichroism (azurin, CuA), resonance-Raman (CuA), and EPR (CuA) spectra indicate that the structure of the oxidized copper site of each high-potential form is very similar to that of the native protein. It is proposed that GuHCl perturbs one or more H-bonds in the blue or purple copper active site, thereby allowing Cu(I) to adopt a more favorable coordination structure than that in the rigid cavity of the native protein.
Asunto(s)
Azurina/química , Cobre/química , Complejo IV de Transporte de Electrones/química , Pseudomonas aeruginosa/química , Thermus thermophilus/química , Proteínas Bacterianas/química , Dicroismo Circular , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Guanidina/farmacología , Enlace de Hidrógeno , Metaloproteínas/química , Estructura Molecular , Oxidación-Reducción , Desnaturalización Proteica , Pliegue de Proteína , Espectrofotometría , Espectrometría RamanRESUMEN
Major progress was made in 1997 in the understanding of the biological transport of copper. Blue copper and CuA sites have very low electron transfer reorganization energies. The mechanisms of copper-containing oxygenases and oxidases have been clarified by recent crystal structure determinations. Protein folding has been shown to tune the reduction potentials of blue copper proteins by hydrophobic encapsulation of the active sites and strict control of the axial ligation.
Asunto(s)
Cobre/fisiología , Metaloproteínas/química , Azurina/química , Proteínas Bacterianas/química , Sitios de Unión/fisiología , Transporte Biológico/fisiología , Cobre/química , Proteínas Fúngicas/química , Oxidación-Reducción , Oxidorreductasas/química , Oxigenasas/química , Pliegue de ProteínaRESUMEN
The unfolding by guanidine hydrochloride (GuHCl) and the refolding on dilution of zinc and apoazurin have been monitored by far-UV circular dichroism (CD). With the native protein, the unfolding was followed by CD and optical absorption in the visible spectral region. With the zinc protein, the reversible unfolding has also been followed by tryptophan fluorescence and NMR. The zinc and Cu2+ metal ions remain associated with the protein in the unfolded state. When the unfolding of the native protein is followed by CD, the initial, reversible transition due to unfolding is followed by a slow change associated with the reduction of Cu2+ by the thiol group of the ligand Cys112. The unfolding of apoazurin displays two CD transitions, which evidence suggests represent different folding domains, the least stable one including the metal-binding site and the other one the rest of the beta-sheet structure. Both occur at a lower GuHCl concentration than the unfolding of the native protein. The CD titrations also demonstrate that zinc azurin has a lower stability than the copper protein. Unfolding of zinc azurin followed by tryptophan fluorescence occurs at a much lower GuHCl concentration than the CD changes, and NMR spectra show that there is no loss of secondary and tertiary structure at this concentration, whereas the CD-detected loss of secondary structure correlates with the NMR changes. Thus, the fluorescence change is ascribed to a small local perturbation of the structure around the single tryptophan residue. The differences in stability of the three forms of azurin are discussed in terms of the rack mechanism. A bound metal ion stabilizes the native fold, and this stabilization is larger for Cu(II) than for Zn(II), reflecting the higher affinity of the protein for Cu(II).
Asunto(s)
Apoproteínas/química , Azurina/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Zinc/farmacología , Apoproteínas/metabolismo , Azurina/efectos de los fármacos , Azurina/metabolismo , Dicroismo Circular , Cobre/farmacología , Guanidina/farmacología , Cinética , Resonancia Magnética Nuclear Biomolecular , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , TriptófanoRESUMEN
Experimental data for the unfolding of cytochrome c and azurin by guanidinium chloride (GuHCl) are used to construct free-energy diagrams for the folding of the oxidized and reduced proteins. With cytochrome c, the driving force for folding the reduced protein is larger than that for the oxidized form. Both the oxidized and the reduced folded forms of yeast cytochrome c are less stable than the corresponding states of the horse protein. Due to the covalent attachment of the heme and its fixed tetragonal coordination geometry, cytochrome c folding can be described by a two-state model. A thermodynamic cycle leads to an expression for the difference in self-exchange reorganization energies for the folded and unfolded proteins. The reorganization energy for electron exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution. The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein suggests that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of CuI in the unfolded protein is linear or trigonal, whereas CuII prefers to be tetragonal. The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1, 10-phenanthroline)22+/+ reference system.