RESUMO
The stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges. Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding. The recombinant major cold shock protein of Escherichia coli CspA an all-beta protein unfolds reversible in a two-state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5-9.0 is linked to protonation of an amino group. This amino group appears to be the N-terminal alpha-amino group of the CspA molecule. It undergoes a 1.6 U shift in pKa values between native and unfolded states. Although this shift in pKa is expected to contribute approximately 5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only approximately 1 kJ/mol. This discrepancy is related to a strong enthalpy-entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the alpha-amino group.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Soluções Tampão , Varredura Diferencial de Calorimetria , Primers do DNA/genética , Estabilidade de Medicamentos , Entropia , Escherichia coli/química , Escherichia coli/genética , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Desnaturação Proteica , Dobramento de Proteína , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , TermodinâmicaRESUMO
The first 5 amino acids of the catalytic alpha 1 isoform from Na,K-ATPase are cleaved enzymatically during or after translation. To evaluate the structural requirements for that cleavage, we constructed amino-terminal mutants of alpha 1 in which an epitope tag from the c-myc oncogene product was added. Immunoblots of isolated membranes from transfected monkey kidney cells revealed binding of an antibody specific for the first 9 residues of the alpha 1 nascent protein. Because this antibody does not recognize the shorter sequence corresponding to the processed polypeptide, these results indicate that the epitope tag prevented normal processing, a conclusion confirmed by the observed binding of an anti-myc antibody. In contrast, membranes from cells expressing deletion mutants that lack residues 10-24 and 10-31 of the nascent chain failed to bind the amino-terminal-directed antibody, suggesting that the mutants were cleaved normally and that amino acids downstream of the first 9 are not required for proteolysis. Amino-terminal mutants produced in other laboratories have shown an anomalous stimulation of ATPase activity by K+ when measured in low ATP concentrations. The myc-tagged and downstream deletion mutants were sensitive to K+ in the range from 0.05 to 5 mM, similar to wild-type enzyme, despite the differences in posttranslational processing. A mutant missing the first 40 residues of the nascent chain, however, displayed an activation by K+. These results suggest that amino-terminal processing of the alpha 1 isoform was prevented by mutation, yet that processing had little influence on the kinetic parameter most likely to be influenced by such changes.
Assuntos
Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional/genética , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Catálise , Linhagem Celular , Epitopos/genética , Homeostase/genética , Humanos , Transporte de Íons/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Deleção de Sequência , ATPase Trocadora de Sódio-Potássio/metabolismo , TransfecçãoAssuntos
Processamento de Proteína Pós-Traducional , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Linhagem Celular , Chlorocebus aethiops , Isoenzimas/química , Isoenzimas/metabolismo , Substâncias Macromoleculares , Oligopeptídeos/química , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoAssuntos
Grânulos Cromafim/metabolismo , Grupo dos Citocromos b/metabolismo , Dopamina beta-Hidroxilase/metabolismo , Transporte de Elétrons , Proteínas/metabolismo , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/enzimologia , Glândulas Suprarrenais/metabolismo , Animais , Bovinos , Grânulos Cromafim/enzimologia , Cobre/análise , Concentração de Íons de Hidrogênio , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Proteínas/químicaRESUMO
A soft method of purification of cytochrome-561 from the membranes of chromaffin granules has been developed. It permits isolating a protein in its natural microsurroundings, i.e. a complex with lipids, provided that a buffer with high ionic force is used without a detergent. This method helps obtaining an electrophoretically homogeneous preparation as a high-molecular lipoprotein hexamer whose molecular weight is about 400 kDa. Basic physicochemical parameters of this preparation (subunit composition, content and composition of lipids, heme content, spectra of optical absorption of the oxidized and reduced forms) are determined. Possible presence of two forms of cytochrome b-561 in the chromaffin granules is discussed.
Assuntos
Grânulos Cromafim/enzimologia , Grupo dos Citocromos b/isolamento & purificação , Animais , Bovinos , Cromatografia em Gel , Grupo dos Citocromos b/química , Eletroforese em Gel de Poliacrilamida , Heme/análise , Lipídeos/análise , Peso MolecularRESUMO
The interaction of acidic copper-containing protein from the membranes of chromaffin granules has been investigated with cytochrome b-561 and dopamine-beta-monooxygenase from the same source. By the use of spectral and polarographic measurements it was demonstrated that the acidic copper-containing protein acts as an electron acceptor for cytochrome b-561 and as electron donor in the reactions, catalyzed by dopamine-beta-monooxygenase. According to the data obtained the possible function of the acidic copper-containing protein in vivo on the area of electron transfer chain between cytochrome b-561 and dopamine-beta-monooxygenase are discussed. The activation or inhibition of the electron transfer reactions by a variety of phospholipids, analogs of membrane lipids of chromaffin granules has been established. The experiments were performed in a model systems by the use of highly purified preparations of proteins and bilamellar liposomes and micelles, prepared from the corresponding phospholipids.