RESUMEN
Automating the determination of novel macromolecular structures via X-ray crystallographic methods involves building a model into an electron-density map. Unfortunately, the conventional crystallographic asymmetric unit volumes are usually not well matched to the biological molecular units. In most cases, the facets of the asymmetric unit cut the molecules into a number of disconnected fragments, rendering interpretation by the crystallographer significantly more difficult. The FINDMOL algorithm is designed to quickly parse the arrangement of trace points (pseudo-atoms) derived from a skeletonized electron-density map without requiring higher level prior information such as sequence information or number of molecules in the asymmetric unit. The algorithm was tested with a variety of density-modified maps computed with medium- to low-resolution data. Typically, the resulting volume resembles the biological unit. In the remaining cases the number of disconnected fragments is very small. In all examples, secondary-structural elements such as alpha-helices or beta-sheets are easily identifiable in the defragmented arrangement. FINDMOL can greatly assist a crystallographer during manual model building or in cases where automatic model building can only build partial models owing to limitations of the data such as low resolution and/or poor phases.
Asunto(s)
Algoritmos , Cristalografía por Rayos X/métodos , Sustancias Macromoleculares/química , alfa-Globulinas/química , Análisis por Conglomerados , Electrones , Modelos MolecularesRESUMEN
The small blue copper protein rusticyanin from Thiobacillus ferrooxidans contains a type 1 Cu centre with a single axial ligand, Met148, which together with the His-Cys-His trigonal planar ligands produces a distorted trigonal pyramidal coordination geometry to copper. Type 1 Cu sites are found in cupredoxins and several multicopper proteins, including oxidases and nitrite reductases. The role of the axial ligand has been extensively debated in terms of its function in the fine tuning of the redox potential and spectroscopic properties of type 1 Cu sites. Numerous mutations of the Met ligand in azurins have been studied, but interpretation of the results has been complicated by the presence of the additional carbonyl oxygen ligand from Gly45, a neighbouring residue to the coordinating His46. The importance of the axial ligand has been further emphasized by the finding that the type 1 centre in Rhus vernicifera stellacyanin, with the lowest redox potential in a type 1 Cu site of 184 mV, has Gln as the axial ligand, whilst fungal laccase and ceruloplasmin, which have redox potentials of 550-800 mV, have a Leu in this position. Here, the crystal structure of the M148Q mutant of rusticyanin at 1.5 A resolution is presented. This is a significantly higher resolution than that of the structures of native rusticyanin. In addition, the M148Q structure is that of the oxidized protein while the native structures to date are of the reduced protein. The mutant protein crystallizes with two molecules per asymmetric unit, in contrast to the one present in the native crystal form. This mutant's redox potential (550 mV at pH 3.2) is lowered compared with that of the native protein ( approximately 670 mV at pH 3.2) by about 120 mV. The type 1 Cu site of M148Q closely mimics the structural characteristics of the equivalent site in non-glycosylated cucumber stellacyanin (redox potential approximately 260 mV) and, owing to the absence in rusticyanin of the fifth, carbonyl ligand present in azurin, may provide a better model for the R. vernicifera stellacyanin (redox potential approximately 184 mV) type 1 Cu site, which also lacks the fifth ligand. Furthermore, the presence of two molecules in the asymmetric unit cell indicates a potential binding region of the redox partners.
Asunto(s)
Azurina/análogos & derivados , Azurina/química , Metaloproteínas/química , Proteínas de Plantas/química , Thiobacillus/química , Sustitución de Aminoácidos , Azurina/genética , Sitios de Unión , Cobre/química , Cristalización , Cristalografía por Rayos X , Ácido Glutámico/genética , Metionina/genética , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Pliegue de ProteínaRESUMEN
Type 1 Cu centers in cupredoxins, nitrite reductases, and multi-copper oxidases utilize the same trigonal core ligation to His-Cys-His, with a weak axial ligand generally provided by a Met sulfur. In azurin, an additional axial ligand, a carbonyl oxygen from a Gly, is present. The importance of these axial ligands and in particular the Met has been debated extensively in terms of their role in fine-tuning the redox potential, spectroscopic properties, and rack-induced or entatic state properties of the copper sites. Extensive site-directed mutagenesis of the Met ligand has been carried out in azurin, but the presence of an additional carbonyl oxygen axial ligand has made it difficult to interpret the effects of these substitutions. Here, the axial methionine ligand (Met148) in rusticyanin is replaced with Leu, Gln, Lys, and Glu to examine the effect on the redox potential, acid stability, and copper site geometry. The midpoint redox potential varies from 363 (Met148Lys) to 798 mV (Met148Leu). The acid stability of the oxidized proteins is reduced except for the Met148Gln mutant. The Gln mutant remains blue at all pH values between 2.8 and 8, and has a redox potential of 563 mV at pH 3.2. The optical and rhombic EPR properties of this mutant closely resemble those of stellacyanin, which has the lowest redox potential among single-type 1 copper proteins (185 mV). The Met148Lys mutant exhibits type 2 Cu EPR and optical spectra in this pH range. The Met148Glu mutant exhibits a type 2 Cu EPR spectrum above pH 3 and a mixture of type 1 and type 2 Cu spectra at lower pH. The Met148Leu mutant exhibits the highest redox potential ( approximately 800 mV at pH 3.2) which is similar to the values in fungal laccase and in the type 1 Cu site of ceruloplasmin where this axial ligand is also a Leu.
Asunto(s)
Azurina/análogos & derivados , Proteínas Bacterianas/metabolismo , Cobre/química , Metionina/metabolismo , Azurina/química , Azurina/genética , Azurina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Cartilla de ADN , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Metionina/química , Metionina/genética , Mutagénesis Sitio-Dirigida , Oxidación-ReducciónRESUMEN
The expression of rusticyanin in Escherichia coli and a number of mutants for Ser86 is reported. Mutations of Ser86 to Asn, Asp, Gln, and Leu were undertaken as this is an Asn residue in other structurally characterized cupredoxins, and it has been suggested that this may be partly responsible for the high redox potential (680 mV) and extreme acid stability of rusticyanin. N-Terminal sequence analysis, together with other biochemical and spectrochemical characterization, shows that the recombinant wild-type protein is indistinguishable from native rusticyanin. All four mutants retain the rhombic nature of the EPR spectra and a significant absorption maximum at approximately 450 nm, thus confirming that the overall geometry of the Cu ligands is essentially maintained. The oxidized form of all four mutants is less acid stable than the wild-type protein, although the detailed mechanism of lability varies. Ser86Leu readily loses copper as the pH is reduced from 4.0, but the protein does not denature. A significant proportion (approximately 30%) of Ser86Gln is denatured at lower pH values, whereas Ser86Asn and Ser86Asp are stable as the reduced (CuI) protein. The redox potential also varies by approximately 110 mV (590-702 mV) upon these single point mutations, thus providing direct experimental support to the idea that this residue is at least in part responsible for the acid stability and the highest redox potential of rusticyanin in the cupredoxin family.