RESUMO
Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK's lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.
Assuntos
Diacilglicerol Quinase/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Biocatálise , Domínio Catalítico , Membrana Celular/enzimologia , Diacilglicerol Quinase/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The seven-transmembrane-spanning G protein-coupled receptor (GPCR) superfamily plays many important roles in basic biology, human health, and human disease. Here, well-resolved solution NMR spectra are presented for a human GPCR, the vasopressin V2 receptor in detergent micelles. The quality of the NMR spectra indicates that backbone resonance assignments for a majority of resonances are feasible. The key to obtaining high quality spectra appears to be the coupling of methods for expressing the receptor into membranes rather than into inclusion bodies, with use of a biochemically mild lysolipid detergent for membrane extraction, protein purification, and NMR sample preparation.
Assuntos
Receptores Acoplados a Proteínas G/química , Receptores de Vasopressinas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Receptores de Vasopressinas/biossíntese , Receptores de Vasopressinas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , SoluçõesRESUMO
The first steps toward undertaking an NMR structural study of a new protein is very often to purify the protein and then to acquire an HSQC or TROSY NMR spectrum, the quality of which is used to assess the feasibility of an NMR-based structural determination. Relatively few integral membrane proteins (IMPs) have been subjected even to this very preliminary stage of NMR analysis. Here, NMR feasibility testing methods are outlined that are tailored for hexahistidine-tagged IMPs that have been expressed in Escherichia coli. Generally applicable protocols are presented for expression testing, purification, and NMR sample preparation. A 2D TROSY pulse sequence that has been optimized for use with IMPs is also presented.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/química , Detergentes , Deutério , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Proteínas de Escherichia coli/químicaRESUMO
The interactions of Bronsted acid sites of H-Y (FAU) with perdeuterated trimethylphosphine oxide (TMPO-d9) are studied with a set of high-resolution solid-state NMR experiments. Double- and triple-resonance MAS NMR techniques (such as CP, TRAPDOR, and REDOR) verify that the lines in the 31P MAS NMR spectrum are indeed from TMPO interacting with Bronsted acid sites. Replacement of acidic hydrogens in the sodalite cages with sodium cations results in the disappearance of one of the peaks, leading to final assignments of the resonances.