RESUMO
Bovine IF(1) is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F(1)F(0) ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF(1) (44-84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.
Assuntos
Proteínas/química , ATPases Translocadoras de Prótons/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Dimerização , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismo , Soluções , Termodinâmica , Proteína Inibidora de ATPaseRESUMO
Mutants of chymotrypsin inhibitor protein 2 have previously been studied in which 4 or 10 glutamine residues were inserted into the inhibitory loop of the protein between residues 59 and 60, as potential models for the behaviour of glutamine tracts in proteins associated with polyglutamine-expansion neurodegenarative diseases. These mutants form very stable monomers, dimers and trimers. Although the cause of oligomerisation was found to be domain-swapping, it was thought that the glutamine insertions might nevertheless show evidence of weak interglutamine interactions in solution that could mimic those occurring in disease-associated proteins. In the present NMR study, we used steady-state (15)N[(1)H] NOE measurements and chemical shift comparisons to characterise the motional properties of the inserted glutamines in these CI2 mutants. We found the glutamines to be highly mobile, with no evidence of interactions amongst them in either monomers or dimers.
Assuntos
Quimotripsina/antagonistas & inibidores , Glutamina/química , Peptídeos/química , Peptídeos/genética , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genética , Sequência de Aminoácidos , Estabilidade de Medicamentos , Glutamina/genética , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas de Plantas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sequências Repetitivas de Aminoácidos , SoluçõesRESUMO
NMR characterization of natural abundance (15)N in phosphorus-nitrogen compounds can be performed through (31)P using inverse detection methods. When the (31)P-(15)N scalar coupling is small, its observation is greatly disturbed by the residual signal coming from the 99.6% abundant (14)N isotopomer that usually is not completely suppressed by the phase cycle of the sequence. The combined use of pulsed field gradients to suppress this residual signal and the enhanced sensitivity (31)P, (15)N[(1)H]-esHSQC experiment affords artifact-free spectra with good signal-to-noise ratio, which allows the accurate measurement of (15)N NMR parameters such as chemical shifts and coupling constants with the benefits of phosphorus detection.
RESUMO
Macrolides are a group of antibiotics structurally characterized by a macrocyclic lactone to which one or several deoxy-sugar moieties are attached. The sugar moieties are transferred to the different aglycones by glycosyltransferases (GTF). The OleI GTF of an oleandomycin producer, Streptomyces antibioticus, catalyzes the inactivation of this macrolide by glycosylation. The product of this reaction was isolated and its structure elucidated. The donor substrate of the reaction was UDP-alpha-D-glucose, but the reaction product showed a beta-glycosidic linkage. The inversion of the anomeric configuration of the transferred sugar and other data about the kinetics of the reaction and primary structure analysis of several GTFs are compatible with a reaction mechanism involving a single nucleophilic substitution at the sugar anomeric carbon in the catalytic center of the enzyme.
Assuntos
Antibacterianos/antagonistas & inibidores , Antibacterianos/química , Glucosiltransferases/metabolismo , Oleandomicina/antagonistas & inibidores , Oleandomicina/química , Sequência de Aminoácidos , Antibacterianos/metabolismo , Configuração de Carboidratos , Glucose/química , Glucosiltransferases/genética , Glicosilação , Espectroscopia de Ressonância Magnética , Modelos Químicos , Oleandomicina/metabolismo , Homologia de Sequência de Aminoácidos , Streptomyces antibioticus/enzimologia , Streptomyces antibioticus/genéticaRESUMO
The oleD gene has been identified in the oleandomycin producer Streptomyces antibioticus and it codes a macrolide glycosyltransferase that is able to transfer a glucose moiety from UDP-glucose (UDP-Glc) to many macrolides. The glycosyltransferase coded by the oleD gene has been purified 371-fold from a Streptomyces lividans clone expressing this protein. The reaction product was isolated, and its structure determined by NMR spectroscopy. The kinetic mechanism of the reaction was analyzed using the macrolide antibiotic lankamycin (LK) as substrate. The reaction operates via a compulsory order mechanism. This has been shown by steady-state kinetic studies and by isotopic exchange reactions at equilibrium. LK binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated lankamycin (GS-LK). A pH study of the reaction was performed to determine values for the molecular pK values, suggesting possible amino acid residues involved in the catalytic process.