RESUMO
Interactions that stabilize the native state of a protein have been studied by measuring the affinity between subdomain fragments with and without site-specific residue substitutions. A calbindin D(9k) variant with a single CNBr cleavage site at position 43 between its two EF-hand subdomains was used as a starting point for the study. Into this variant were introduced 11 site-specific substitutions involving hydrophobic core residues at the interface between the two EF-hands. The mutants were cleaved with CNBr to produce wild-type and mutated single-EF-hand fragments: EF1 (residues 1--43) and EF2 (residues 44--75). The interaction between the two EF-hands was studied using surface plasmon resonance (SPR) technology, which follows the rates of association and dissociation of the complex. Wild-type EF1 was immobilized on a dextran matrix, and the wild-type and mutated versions of EF2 were injected at several different concentrations. In another set of experiments, wild-type EF2 was immobilized and wild-type or mutant EF1 was injected. Dissociation rate constants ranged between 1.1 x 10(-5) and 1.0 x 10(-2) s(-1) and the association rate constants between 2 x 10(5) and 4.0 x 10(6) M(-1) s(-1). The affinity between EF1 and EF2 was as high as 3.6 x 10(11) M(-1) when none of them was mutated. For the 11 hydrophobic core mutants, a strong correlation (r = 0.999) was found between the affinity of EF1 for EF2 and the stability toward denaturation of the corresponding intact protein. The observed correlation implies that the factors governing the stability of the intact protein also contribute to the affinity of the bimolecular EF1-EF2 complex. In addition, the data presented here show that interactions among hydrophobic core residues are major contributors both to the affinity between the two EF-hand subdomains and to the stability of the intact domain.
Assuntos
Fragmentos de Peptídeos/química , Proteína G de Ligação ao Cálcio S100/química , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Calbindinas , Bovinos , Brometo de Cianogênio , Motivos EF Hand/genética , Humanos , Hidrólise , Cinética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína , Ratos , Proteína G de Ligação ao Cálcio S100/isolamento & purificação , Proteína G de Ligação ao Cálcio S100/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Ressonância de Plasmônio de Superfície , TermodinâmicaRESUMO
Strontium-82, produced by spallation reaction with medium-energy proton beams, was used to evaluate Bio-Rex 70 and Chelex-100 ion-exchange resins for use in a compact Rb-82 generator. Adsorption of Sr-82 to the resin column, Rb-82 elution yields, Sr breakthrough, and 82Rb-Sr separation factors were determined for newly prepared columns and for longterm elution conditions. Separation factors of 10(7) to 10(8) were obtained with 2% NaCl elutions from Bio-Rex 70 resin columns while the separation factors was about 5 X 10(4) with the Chelex-100 resin column.
