RESUMEN
It is generally considered that electrostatic interactions on the protein surface, such as ion pairs, contribute little to protein stability, although they may play important roles in conformational specificity. We found that the tenth fibronectin type III domain of human fibronectin (FNfn10) is more stable at acidic pH than neutral pH, with an apparent midpoint of transition near pH 4. Determination of pK(a)'s for all the side chain carboxyl groups of Asp and Glu residues revealed that Asp 23 and Glu 9 have an upshifted pK(a). These residues and Asp 7 form a negatively charged patch on the surface of FNfn10, with Asp 7 centrally located between Asp 23 and Glu 9, suggesting repulsive electrostatic interactions among these residues at neutral pH. Mutant proteins, D7N and D7K, in which Asp 7 was replaced with Asn and Lys, respectively, exhibited a modest but significant increase in stability at neutral pH, compared to the wild type, and they no longer showed pH dependence of stability. The pK(a)'s of Asp 23 and Glu 9 in these mutant proteins shifted closer to their respective unperturbed values, indicating that the unfavorable electrostatic interactions have been reduced in the mutant proteins. Interestingly, the wild-type and mutant proteins were all stabilized to a similar degree by the addition of 1 M sodium chloride at both neutral and acidic pH, suggesting that the repulsive interactions between the carboxyl groups cannot be effectively shielded by 1 M sodium chloride. These results indicate that repulsive interactions between like charges on the protein surface can destabilize a protein, and protein stability can be significantly improved by relieving these interactions.
Asunto(s)
Fibronectinas/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Ácido Aspártico , Clonación Molecular , Escherichia coli , Ácido Glutámico , Calor , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Electricidad Estática , Propiedades de Superficie , TermodinámicaRESUMEN
Omega (omega)-loop A, residues 18-32 in wild-type yeast iso-1-cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase. Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low-temperature spectroscopy and cannot grow on nonfermentable media. Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures. In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native-like cytochrome c conformation, but are less stable than the wild-type protein. Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone. A single-site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.
Asunto(s)
Grupo Citocromo c/química , Citocromos c , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Dicroismo Circular , Estabilidad de Enzimas , Calor , Datos de Secuencia Molecular , Conformación Proteica , Saccharomyces cerevisiae/crecimiento & desarrollo , Espectrofotometría UltravioletaRESUMEN
Fractures of the most distal part of the olecranon process differ from more proximal fractures because a plane of instability exists between the humerus and the radial head. Thus, the fixation of these fractures must resist the deforming forces of the forearm flexor muscles. The investigators fixed 10 paired cadaver ulnae with either the tension-band wire technique or a one-third tubular plate, and tested the specimens to simulate the effect of the biceps and brachialis muscles. The average maximum fixation stiffness for specimens fixed with the one-third tubular plate was found to be 163 N/mm compared with 53 N/mm for the tension-band wire group. This study indicates that for fractures of the distal part of the olecranon, fixation with screws and a one-third tubular plate affords better resistance to the forces applied by the brachialis and the biceps brachii than the tension-band wire technique.
Asunto(s)
Lesiones de Codo , Fijación Interna de Fracturas/métodos , Fracturas Óseas/cirugía , Fenómenos Biomecánicos , Placas Óseas , Tornillos Óseos , Hilos Ortopédicos , Fijación Interna de Fracturas/instrumentación , Fracturas Óseas/clasificación , Fracturas Óseas/diagnóstico por imagen , Humanos , RadiografíaRESUMEN
In experiment 1, splenectomized mice exposed to 1 X 10(9) colony-forming units (cfu) of type 3 Streptococcus pneumoniae experienced no mortality when given 40,000 or 4,000 units of procaine penicillin IM starting 2 days before exposure and continuing for 4 days. On day 5, Bicillin was given once in the same dosages. In experiment 2, splenectomized mice exposed to 2.2 X 10(9) cfu were significantly protected by 400 but not 40 units of penicillin when compared to controls (P less than 0.005). In experiment 3, splenectomized mice exposed to 2.7 X 10(9) cfu were significantly protected when compared to controls if 4,000 units of procaine penicillin were given either 2 days postexposure for 2 days followed by 1 day of Bicillin in the same dosage or 4 days postexposure with the same schedule and dosage (P less than 0.005 and 0.01, respectively) but not 6 days postexposure.
Asunto(s)
Penicilinas/uso terapéutico , Infecciones Neumocócicas/mortalidad , Esplenectomía , Aerosoles , Animales , Inyecciones Intramusculares , Ratones , Penicilina G Benzatina/administración & dosificación , Penicilina G Benzatina/uso terapéutico , Penicilina G Procaína/administración & dosificación , Penicilina G Procaína/uso terapéutico , Infecciones Neumocócicas/prevención & controlRESUMEN
Splenosis has been shown to occur after traumatic injury to the spleen. It is postulated that this is the mechanism for the low incidence of bacterial infection in this group of patients when compared to those who undergo splenectomy for other reasons. Therefore, we studied the effect of exposure to an aerosolized suspension of type III Streptococcus pneumoniae on splenectomized mice who had either all or half of their splenic tissue cut up and reimplanted into the abdominal cavity 8 wk prior to bacterial exposure. It was determined that the mortality experience of these two groups of mice was similar to each other and no different from the sham control group, although all three groups had a statistically significant lower mortality experience than the splenectomized control group. This study demonstrates that splenosis in mice can protect against aerosolized bacterial infection.