RESUMEN
The ferrous iron transporter FeoB is an important factor in the iron metabolism of many bacteria. Although several structural studies have been performed on its cytosolic GTPase domain (NFeoB), the full-length structure of FeoB remains elusive. Based on a crystal packing analysis that was performed on crystals of NFeoB, a trimeric structure of the FeoB channel was proposed, where the transport pore runs along the trimer axis. Because this trimer has not been observed in some subsequently solved structures of NFeoB homologs, it remains unclear whether or not the trimer is indeed functionally relevant. Here, pulsed electron-electron double resonance spectroscopy, negative stain electron microscopy, and native mass spectrometry are used to analyze the oligomeric state of different soluble and full-length FeoB constructs. The results show that the full-length protein is predominantly monomeric, whereas dimers and trimers are formed to a small percentage. Furthermore, the solution structure of the switch I region is analyzed by pulsed electron-electron double resonance spectroscopy and a new, to our knowledge, crystal structure of NFeoB from Escherichia coli BL21 is presented.
Asunto(s)
Proteínas de Transporte de Catión/química , Proteínas de Escherichia coli/química , Dicroismo Circular , Cristalografía por Rayos X , Escherichia coli , Espectrometría de Masas , Microscopía Electroquímica de Rastreo , Dominios Proteicos , Multimerización de Proteína , SolucionesRESUMEN
The ferrous iron transporter FeoB is an important factor in the iron metabolism of various bacteria. As a membrane bound GTPase it also represents an interesting evolutionary link between prokaryotic and eukaryotic membrane signalling pathways. To date, structural information for FeoB is limited to the cytosolic GTPase domain and structural features such as the oligomeric state of the transporter in the membrane, and thereby the nature of the transport pore are a matter of constant debate. Recently, EPR distance measurements have become an important tool to investigate such questions in frozen solution. As a prerequisite for these experiments, we designed protocols to express and purify both the cytosolic domain of FeoB (NFeoB) and full-length FeoB from Escherichia coli BL21 in purity, quantity and quality needed for EPR studies. Since FeoB from E. coli contains 12 native cysteines, we incorporated the unnatural amino acid para-acetylphenylalanine (pAcF) into the protein. We spin labelled the mutant protein using the HO4120 spin label and performed preliminary EPR experiments using cw-X-band EPR spectroscopy. Our results provide new insights concerning the oligomeric state of full-length FeoB.
Asunto(s)
Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/aislamiento & purificación , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Transporte de Catión/análisis , Proteínas de Transporte de Catión/metabolismo , Clonación Molecular , Espectroscopía de Resonancia por Spin del Electrón , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Marcadores de SpinRESUMEN
Metal ions play an important role in the catalysis and folding of proteins and oligonucleotides. Their localization within the three-dimensional fold of such biomolecules is therefore an important goal in understanding structure-function relationships. A trilateration approach for the localization of metal ions by means of long-range distance measurements based on electron paramagnetic resonance (EPR) is introduced. The approach is tested on the Cu(2+) center of azurin, and factors affecting the precision of the method are discussed.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Magnetismo , Metales/química , Proteínas/químicaRESUMEN
BACKGROUND: EPR-based distance measurements between spin labels in proteins have become a valuable tool in structural biology. The direct translation of the experimental distances into structural information is however often impaired by the intrinsic flexibility of the spin labelled side chains. Different algorithms exist that predict the approximate conformation of the spin label either by using pre-computed rotamer libraries of the labelled side chain (rotamer approach) or by simply determining its accessible volume (accessible volume approach). Surprisingly, comparisons with many experimental distances have shown that both approaches deliver the same distance prediction accuracy of about 3 Å. RESULTS: Here, instead of comparing predicted and experimental distances, we test the ability of both approaches to predict the actual conformations of spin labels found in a new high-resolution crystal structure of spin labelled azurin (T21R1). Inside the crystal, the label is found in two very different environments which serve as a challenging test for the in silico approaches. CONCLUSIONS: Our results illustrate why simple and more sophisticated programs lead to the same prediciton error. Thus, a more precise treatment of the complete environment of the label and also its interactions with the environment will be needed to increase the accuracy of in silico spin labelling algorithms.
Asunto(s)
Algoritmos , Azurina/química , Proteínas Bacterianas/química , Pseudomonas aeruginosa/metabolismo , Biología Computacional , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Marcadores de SpinRESUMEN
The role of heme as a cofactor in enzymatic reactions has been studied for a long time and in great detail. Recently it was discovered that heme can also serve as a signalling molecule in cells but so far only few examples of this regulation have been studied. In order to discover new potentially heme-regulated proteins, we screened protein sequence databases for bacterial proteins that contain sequence features like a Cysteine-Proline (CP) motif, which is known for its heme-binding propensity. Based on this search we synthesized a series of these potential heme regulatory motifs (HRMs). We used cw EPR spectroscopy to investigate whether these sequences do indeed bind to heme and if the spin state of heme is changed upon interaction with the peptides. The corresponding proteins of two potential HRMs, FeoB and GlpF, were expressed and purified and their interaction with heme was studied by cw EPR and UV-Visible (UV-Vis) spectroscopy.
Asunto(s)
Cisteína/metabolismo , Hemo/metabolismo , Péptidos/metabolismo , Proteínas/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Acuaporinas/química , Acuaporinas/genética , Acuaporinas/metabolismo , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Cisteína/química , Cisteína/genética , Espectroscopía de Resonancia por Spin del Electrón/métodos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hemo/química , Hemo/genética , Hemoproteínas/química , Hemoproteínas/genética , Hemoproteínas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Homología de Secuencia de Aminoácido , Espectrofotometría/métodosRESUMEN
A new series of 1-alkyl-8-(piperazine-1-sulfonyl)phenylxanthines was designed, synthesized, and characterized in radioligand binding and functional assays at A(2B) adenosine receptors. A(2B) antagonists with subnanomolar affinity and high selectivity were discovered. The most potent compounds were 1-ethyl-8-(4-(4-(4-trifluoromethylbenzyl)piperazine-1-sulfonyl)phenyl)xanthine (24, PSB-09120, K(i) (human A(2B)) = 0.157 nM) and 8-(4-(4-(4-chlorobenzyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine (17, PSB-0788, K(i) (human A(2B)) = 0.393 nM). Moreover, 8-(4-(4-(4-chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine (35, PSB-603) was developed as an A(2B)-specific antagonist exhibiting a K(i) value of 0.553 nM at the human A(2B) receptor and virtually no affinity for the human and rat A(1) and A(2A) and the human A(3) receptors up to a concentration of 10 microM. A tritiated form of the compound was prepared as a new radioligand and characterized in kinetic, saturation, and competition studies. It was shown to be a useful pharmacological tool for the selective labeling of human as well as rodent A(2B) receptors (K(D) human A(2B) 0.403 nM, mouse A(2B) 0.351 nM).