RESUMO
For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.
Assuntos
Técnicas Biossensoriais/instrumentação , Eletrônica/instrumentação , Ensaios Enzimáticos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , DNA , Desenho de Equipamento/instrumentação , Cinética , Dispositivos Lab-On-A-Chip , Miniaturização/instrumentação , Nanotecnologia/instrumentação , SemicondutoresRESUMO
Hybrid structural methods have been used in recent years to understand protein-protein or protein-ligand interactions where high resolution crystallography or NMR data on the protein of interest has been limited. For G protein-coupled receptors (GPCRs), high resolution structures of native structural forms other than rhodopsin have not yet been achieved; gaps in our knowledge have been filled by creative crystallography studies that have developed stable forms of receptors by multiple means. The neurotransmitter serotonin (5-hydroxytryptamine) is a key GPCR-based signaling molecule affecting many physiological manifestations in humans ranging from mood and anxiety to bowel function. However, a high resolution structure of any of the serotonin receptors has not yet been solved. Here, we used structural mass spectrometry along with theoretical computations, modeling, and other biochemical methods to develop a structured model for human serotonin receptor subtype 4(b) in the presence and absence of its antagonist GR125487. Our data confirmed the overall structure predicted by the model and revealed a highly conserved motif in the ligand-binding pocket of serotonin receptors as an important participant in ligand binding. In addition, identification of waters in the transmembrane region provided clues as to likely paths mediating intramolecular signaling. Overall, this study reveals the potential of hybrid structural methods, including mass spectrometry, to probe physiological and functional GPCR-ligand interactions with purified native protein.
Assuntos
Receptores 5-HT4 de Serotonina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Humanos , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Pegadas de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Células Sf9 , SpodopteraRESUMO
MsbA is an essential ATP-binding cassette transporter in Gram-negative bacteria that transports lipid A and lipopolysaccharide from the cytoplasmic leaflet to the periplasmic leaflet of the inner membrane. Here we report the X-ray structure of MsbA from Salmonella typhimurium at 2.8-Å resolution in an inward-facing conformation after cocrystallization with lipid A and using a stabilizing facial amphiphile. The structure displays a large amplitude opening in the transmembrane portal, which is likely required for lipid A to pass from its site of synthesis into the protein-enclosed transport pathway. Putative lipid A density is observed further inside the transmembrane cavity, consistent with a trap and flip model. Additional electron density attributed to lipid A is observed near an outer surface cleft at the periplasmic ends of the transmembrane helices. These findings provide new structural insights into the lipid A transport pathway through comparative analysis with existing MsbA structures.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Membrana Celular/química , Lipídeo A/química , Proteínas de Transferência de Fosfolipídeos/química , Salmonella typhimurium/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Lipídeo A/metabolismo , Modelos Moleculares , Periplasma/química , Periplasma/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/metabolismo , Especificidade por Substrato , TermodinâmicaRESUMO
Nicotinamide nucleotide transhydrogenase (TH) is an enzyme complex in animal mitochondria and bacteria that utilizes the electrochemical proton gradient across membranes to drive the production of NADPH. The enzyme plays an important role in maintaining the redox balance of cells with implications in aging and a number of human diseases. TH exists as a homodimer with each protomer containing a proton-translocating transmembrane domain and two soluble nucleotide binding domains that mediate hydride transfer between NAD(H) and NADP(H). The three-domain architecture of TH is conserved across species but polypeptide composition differs substantially. The complex domain coupling mechanism of TH is not fully understood despite extensive biochemical and structural characterizations. Herein the progress is reviewed, focusing mainly on structural findings from 3D crystallization of isolated soluble domains and more recently of the transmembrane domain and the holo-enzyme from Thermus thermophilus. A structural perspective and impeding challenges in further elucidating the mechanism of TH are discussed.
RESUMO
The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton-motive force to drive hydride transfer from NADH to NADP+ in bacteria and eukaryotes. Here we solved a 2.2-Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular dynamics simulations, we show that transient water flows across a narrow pore and a hydrophobic "dry" region in the middle of the membrane channel, with key residues His42α2 (chain A) being protonated and Thr214ß (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214ß to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , NADP Trans-Hidrogenases/química , Prótons , Membrana Celular/química , Membrana Celular/metabolismo , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Simulação de Dinâmica Molecular , Mutação , NAD/química , NAD/metabolismo , NADP/química , NADP/metabolismo , NADP Trans-Hidrogenases/genética , NADP Trans-Hidrogenases/metabolismo , Thermus thermophilus/enzimologiaRESUMO
Guanylyl cyclases (GC) are proteins that are essential for the production of the second messenger cyclic guanosine monophosphate (cGMP). Mammalian GC have attracted considerable interest due to their roles in important physiological processes such as vasodilation, vision, and bone growth. In addition, their link to disease and concomitant pharmaceutical potential have made these cyclases a long standing target for probing their intriguing mechanism of activation with the aim of drug development. The vasodilatory drugs nitroglycerin and nesiritide act through (different) GC pathways and have both been shown to provide beneficial relief for congestive heart failure patients. New structural insights are recently emerging on the activation mechanism and regulation of these receptors. The aim of this review is to discuss the interesting differences and similarities between members of the soluble and membrane bound GC in detail and put these in context with the structural knowledge that is available to date. These efforts contribute to an enhanced understanding of the GC and will likely lead to an increased success in structure-based therapeutic intervention.
Assuntos
Guanilato Ciclase/química , Receptores do Fator Natriurético Atrial/química , Receptores de Peptídeos/química , Animais , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Humanos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Receptores do Fator Natriurético Atrial/metabolismo , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase , Receptores de Peptídeos/metabolismoRESUMO
Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor-receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the ß2-adrenergic receptor (ß2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For ß2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a ß2-AR/T4-lysozyme chimera bound to the antagonist carazolol.
Assuntos
Receptores Adrenérgicos beta 2/química , Proteínas Recombinantes de Fusão/química , Rodopsina/química , Segmento Externo da Célula Bastonete/química , Sulfato de Amônio/química , Animais , Bovinos , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Cromatografia em Gel , Cristalização , Cristalografia por Raios X , Glucosídeos/química , Muramidase/química , Muramidase/genética , Propanolaminas/química , Multimerização Proteica , Estrutura Secundária de Proteína , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Rodopsina/isolamento & purificação , Células Sf9 , Spodoptera , Proteínas Virais/química , Proteínas Virais/genética , Acetato de Zinco/químicaRESUMO
The objective of this study was to determine the molecular factors that lead to beta-lactamase inhibitor resistance for the M69V variant in SHV-1 beta-lactamase. With mechanism-based inhibitors, the beta-lactamase forms an acyl-enzyme intermediate that consists of a trans-enamine derivative in the active site. This study focuses on these intermediates by introducing the E166A mutation that greatly retards deacylation. Thus, by comparing the properties of the E166A and M69V/E166A forms, we can explore the consequences of the resistance mutation at the level of the enamine acyl-enzyme forms. The reactions between the beta-lactamase and the inhibitors tazobactam, sulbactam, and clavulanic acid are followed in single crystals of the enzymes by using a Raman microscope. The resulting Raman difference spectroscopic data provide detailed information about conformational events involving the enamine species as well as an estimate of their populations. The Raman difference spectra for each of the inhibitors in the E166A and M69V/E166A variants are very similar. In particular, detailed analysis of the main enamine Raman vibration near 1595 cm(-1) reveals that the structure and flexibility of the enamine fragments are essentially identical for each of the three inhibitors in E166A and in the M69V/E166A double mutant. This finding is in accord with the X-ray-derived structures, presented herein at 1.6-1.75 A resolution, of the trans-enamine intermediates formed by the three inhibitors in M69V/E166A. However, a comparison of Raman results for M69V/E166A and E166A shows that the M69V mutation results in a 40%, 25%, and negligible reductions in the enamine population when the beta-lactamase crystals are soaked in 5 mM tazobactam, clavulanic acid, and sulbactam solutions, respectively. The levels of enamine from tazobactam and clavulanic acid can be increased by increasing the concentrations of inhibitor in the mother liquor. Thus, the sensitivity of population levels to the inhibitor concentration in the mother liquor focuses attention on the properties of the encounter complex preceding acylation. It is proposed that for small ligands, such as tazobactam, sulbactam, and clavulanic acid, the positioning of the lactam ring in the active site in the correct orientation for acylation is only one of a number of poorly defined conformations. For tazobactam and clavulanic acid, the correctly oriented encounter complex is even less likely in the M69V variant, leading to a reduction in the level of inhibition of the enzyme via formation of the acyl-enzyme intermediate and the onset of resistance. Analysis of the X-ray structures of the three intermediates in M69V/E166A demonstrates that, compared to the structures for the E166A form, the oxyanion hole becomes smaller, providing one explanation for why acylation may be less efficient following the M69V substitution.
Assuntos
Substituição de Aminoácidos , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/enzimologia , Klebsiella pneumoniae/enzimologia , Inibidores de beta-Lactamases , Sítios de Ligação/genética , Catálise , Cristalografia por Raios X/métodos , Farmacorresistência Bacteriana/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Klebsiella pneumoniae/química , Klebsiella pneumoniae/genética , Mutação de Sentido Incorreto , Ligação Proteica , Estrutura Terciária de Proteína , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
beta-Lactamases are one of the major causes of antibiotic resistance in Gram negative bacteria. The continuing evolution of beta-lactamases that are capable of hydrolyzing our most potent beta-lactams presents a vexing clinical problem, in particular since a number of them are resistant to inhibitors. The efficient inhibition of these enzymes is therefore of great clinical importance. Building upon our previous structural studies that examined tazobactam trapped as a trans-enamine intermediate in a deacylation deficient SHV variant, we designed a novel penam sulfone derivative that forms a more stable trans-enamine intermediate. We report here the 1.28 A resolution crystal structure of wt SHV-1 in complex with a rationally designed penam sulfone, SA2-13. The compound is covalently bound to the active site of wt SHV-1 similar to tazobactam yet forms an additional salt-bridge with K234 and hydrogen bonds with S130 and T235 to stabilize the trans-enamine intermediate. Kinetic measurements show that SA2-13, once reacted with SHV-1 beta-lactamase, is about 10-fold slower at being released from the enzyme compared to tazobactam. Stabilizing the trans-enamine intermediate represents a novel strategy for the rational design of mechanism-based class A beta-lactamase inhibitors.
Assuntos
Inibidores Enzimáticos/química , Ácido Penicilânico/análogos & derivados , Sulfonas/química , Inibidores de beta-Lactamases , Aminas/química , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Estabilidade de Medicamentos , Inibidores Enzimáticos/farmacologia , Cinética , Modelos Moleculares , Ácido Penicilânico/química , Ácido Penicilânico/farmacologia , Conformação Proteica , Sulfonas/síntese química , Sulfonas/farmacologia , Tazobactam , beta-Lactamases/químicaRESUMO
Antibiotic resistance mediated by constantly evolving beta-lactamases is a serious threat to human health. The mechanism of inhibition of these enzymes by therapeutic beta-lactamase inhibitors is probed using a novel approach involving Raman microscopy and x-ray crystallography. We have presented here the high resolution crystal structures of the beta-lactamase inhibitors sulbactam and clavulanic acid bound to the deacylation-deficient E166A variant of SHV-1 beta-lactamase. Our previous Raman measurements have identified the trans-enamine species for both inhibitors and were used to guide the soaking time and concentration to achieve full occupancy of the active sites. The two inhibitor-bound x-ray structures revealed a linear trans-enamine intermediate covalently attached to the active site Ser-70 residue. This intermediate was thought to play a key role in the transient inhibition of class A beta-lactamases. Both the Raman and x-ray data indicated that the clavulanic acid intermediate is decarboxylated. When compared with our previously determined tazobactam-bound inhibitor structure, our new inhibitor-bound structures revealed an increased disorder in the tail region of the inhibitors as well as in the enamine skeleton. The x-ray crystallographic observations correlated with the broadening of the O-C=C-N (enamine) symmetric stretch Raman band near 1595 cm(-1). Band broadening in the sulbactam and clavulanic acid inter-mediates reflected a heterogeneous conformational population that results from variations of torsional angles in the O-(C=O)-C=C=NH-C skeleton. These observations led us to conclude that the conformational stability of the trans-enamine form is critical for their transient inhibitory efficacy.
Assuntos
Ácido Clavulânico/farmacologia , Sulbactam/farmacologia , beta-Lactamases/química , Antibacterianos/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Farmacorresistência Bacteriana , Inibidores Enzimáticos/farmacologia , Klebsiella pneumoniae/metabolismo , Modelos Químicos , Modelos Moleculares , Mutação , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/farmacologia , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Serina/química , Análise Espectral Raman , Tazobactam , Fatores de Tempo , Raios X , Resistência beta-LactâmicaRESUMO
Advanced glycation end products (AGEs) from the Maillard reaction contribute to protein aging and the pathogenesis of age- and diabetes-associated complications. The alpha-dicarbonyl compound methylglyoxal (MG) is an important intermediate in AGE synthesis. Recent studies suggest that pyridoxamine inhibits formation of advanced glycation and lipoxidation products. We wanted to determine if pyridoxamine could inhibit MG-mediated Maillard reactions and thereby prevent AGE formation. When lens proteins were incubated with MG at 37 degrees C, pH 7.4, we found that pyridoxamine inhibits formation of methylglyoxal-derived AGEs concentration dependently. Pyridoxamine reduces MG levels in red blood cells and plasma and blocks formation of methylglyoxal-lysine dimer in plasma proteins from diabetic rats and it prevents pentosidine (an AGE derived from sugars) from forming in plasma proteins. Pyridoxamine also decreases formation of protein carbonyls and thiobarbituric-acid-reactive substances in plasma proteins from diabetic rats. Pyridoxamine treatment did not restore erythrocyte glutathione (which was reduced by almost half) in diabetic animals, but it enhanced erythrocyte glyoxalase I activity. We isolated a major product of the reaction between MG and pyridoxamine and identified it as methylglyoxal-pyridoxamine dimer. Our studies show that pyridoxamine reduces oxidative stress and AGE formation. We suspect that a direct interaction of pyridoxamine with MG partly accounts for AGE inhibition.
Assuntos
Arginina/análogos & derivados , Diabetes Mellitus Experimental/metabolismo , Lisina/análogos & derivados , Piridoxamina/farmacologia , Aldeído Pirúvico/metabolismo , Animais , Arginina/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Cristalinas/metabolismo , Dimerização , Eritrócitos/metabolismo , Glutationa/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Lactoilglutationa Liase/metabolismo , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Reação de Maillard , Modelos Químicos , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Substâncias Reativas com Ácido TiobarbitúricoRESUMO
The molecular chaperone function of alpha-crystallin in the lens prevents the aggregation and insolubilization of lens proteins that occur during the process of aging. We found that chemical modification of alpha-crystallin by a physiological alpha-dicarbonyl compound, methylglyoxal (MG), enhances its chaperone function. Protein-modifying sugars and ascorbate have no such effect and actually reduce chaperone function. Chaperone assay after immunoprecipitation or with immunoaffinity-purified argpyrimidine-alpha-crystallin indicates that 50-60% of the increased chaperone function is due to argpyrimidine-modified protein. Incubation of alpha-crystallin with DL-glyceraldehyde and arginine-modifying agents also enhances chaperone function, and we believe that the increased chaperone activity depends on the extent of arginine modification. Far- and near-UV circular dichroism spectra indicate modest changes in secondary and tertiary structure of MG-modified alpha-crystallin. LC MS/MS analysis of MG-modified alpha-crystallin following chymotryptic digestion revealed that R21, R49, and R103 in alphaA-crystallin were converted to argpyrimidine. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid binding, an indicator of hydrophobicity of proteins, increased in alpha-crystallin modified by low concentrations of MG (2-100 microM). MG similarly enhances chaperone function of another small heat shock protein, Hsp27. Our results show that posttranslational modification by a metabolic product can enhance the chaperone function of alpha-crystallin and Hsp27 and suggest that such modification may be a protective mechanism against environmental and metabolic stresses. Augmentation of the chaperone function of alpha-crystallin might have evolved to protect the lens from deleterious protein modifications associated with aging.
Assuntos
Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Ornitina/análogos & derivados , Aldeído Pirúvico/química , Aldeído Pirúvico/farmacologia , alfa-Cristalinas/química , alfa-Cristalinas/metabolismo , Álcool Desidrogenase/análise , Álcool Desidrogenase/metabolismo , Naftalenossulfonato de Anilina/análise , Naftalenossulfonato de Anilina/química , Naftalenossulfonato de Anilina/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Ácido Ascórbico/química , Ácido Ascórbico/farmacologia , Bovinos , Citrato (si)-Sintase/análise , Citrato (si)-Sintase/metabolismo , Humanos , Insulina/análise , Insulina/metabolismo , Cristalino/química , Lisina/química , Pessoa de Meia-Idade , Monossacarídeos/farmacologia , Ornitina/análise , Ornitina/química , Ornitina/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pirimidinas/análise , Pirimidinas/química , Pirimidinas/farmacologiaRESUMO
Many pathogenic bacteria develop antibiotic resistance by utilizing beta-lactamases to degrade penicillin-like antibiotics. A commonly prescribed mechanism-based inhibitor of beta-lactamases is tazobactam, which can function either irreversibly or in a transient manner. We have demonstrated previously that the reaction between tazobactam and a deacylation deficient variant of SHV-1 beta-lactamase, E166A, could be followed in single crystals using Raman microscopy [Helfand, M. S., et al. (2003) Biochemistry 42, 13386-13392]. The Raman data show that maximal populations of an enamine-like intermediate occur 20-30 min after "soaking in" has commenced. By flash-freezing crystals in this time frame, we were able to trap the enamine species. The resulting 1.63 A resolution crystal structure revealed tazobactam covalently bound in the trans-enamine intermediate state with close to 100% occupancy in the active site. The Raman data also indicated that tazobactam forms a larger population of enamine than sulbactam or clavulanic acid does and that tazobactam's intermediate is also the most long-lived. The crystal structure provides a rationale for this finding since only tazobactam is able to form favorable intra- and intermolecular interactions in the active site that stabilize this trans-enamine intermediate. These interactions involve both the sulfone and triazolyl groups that distinguish tazobactam from clavulanic acid and sulbactam, respectively. The observed stabilization of the transient intermediate of tazobactam is thought to contribute to tazobactam's superior in vitro and in vivo clinical efficacy. Understanding the structural details of differing inhibitor effectiveness can aid the design of improved mechanism-based beta-lactamase inhibitors.