RESUMO
Voltage-gated sodium channels are targets for many analgesic and antiepileptic drugs whose therapeutic mechanisms and binding sites have been well characterized. We describe the identification of a previously unidentified receptor site within the NavMs voltage-gated sodium channel. Tamoxifen, an estrogen receptor modulator, and its primary and secondary metabolic products bind at the intracellular exit of the channel, which is a site that is distinct from other previously characterized sodium channel drug sites. These compounds inhibit NavMs and human sodium channels with similar potencies and prevent sodium conductance by delaying channel recovery from the inactivated state. This study therefore not only describes the structure and pharmacology of a site that could be leveraged for the development of new drugs for the treatment of sodium channelopathies but may also have important implications for off-target health effects of this widely used therapeutic drug.
Assuntos
Modelos Moleculares , Tamoxifeno/química , Canais de Sódio Disparados por Voltagem/química , Células HEK293 , HumanosRESUMO
Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole-mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane-active peptides.
Assuntos
Éxons/genética , Membranas/química , Schistosoma mansoni/genética , Sequência de Aminoácidos , Animais , Varredura Diferencial de Calorimetria/métodos , Dicroísmo Circular/métodos , Peptídeos/química , Conformação Proteica em alfa-Hélice , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/genética , Esquistossomose mansoni/metabolismoRESUMO
The phage shock protein (Psp) response maintains integrity of the inner membrane (IM) in response to extracytoplasmic stress conditions and is widely distributed amongst enterobacteria. Its central component PspA, a member of the IM30 peripheral membrane protein family, acts as a major effector of the system through its direct association with the IM. Under non-stress conditions PspA also negatively regulates its own expression via direct interaction with the AAA+ ATPase PspF. PspA has a counterpart in cyanobacteria called Vipp1, which is implicated in protection of the thylakoid membranes. PspA's and Vipp1's conserved N-terminal regions contain a putative amphipathic helix a (AHa) required for membrane binding. An adjacent amphipathic helix b (AHb) in PspA is required for imposing negative control upon PspF. Here, purified peptides derived from the putative AH regions of PspA and Vipp1 were used to directly probe their effector and regulatory functions. We observed direct membrane-binding of AHa derived peptides and an accompanying change in secondary structure from unstructured to alpha-helical establishing them as bona fide membrane-sensing AH's. The peptide-binding specificities and their effects on membrane stability depend on membrane anionic lipid content and stored curvature elastic stress, in agreement with full length PspA and Vipp1 protein functionalities. AHb of PspA inhibited the ATPase activity of PspF demonstrating its direct regulatory role. These findings provide new insight into the membrane binding and function of PspA and Vipp1 and establish that synthetic peptides can be used to probe the structure-function of the IM30 protein family.
Assuntos
Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Proteínas de Membrana/química , Peptídeos/química , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Transativadores/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/síntese química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estresse Fisiológico , Synechocystis/química , Synechocystis/metabolismo , Transativadores/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismoRESUMO
Cystic fibrosis transmembrane conductance regulator (CFTR) is a multidomain membrane protein that functions as a phosphorylation-regulated anion channel. The interface between its two cytosolic nucleotide binding domains and coupling helices conferred by intracellular loops extending from the channel pore domains has been referred to as a transmission interface and is thought to be critical for the regulated channel activity of CFTR. Phosphorylation of the regulatory domain of CFTR by protein kinase A (PKA) is required for its channel activity. However, it was unclear if phosphorylation modifies the transmission interface. Here, we studied purified full-length CFTR protein using spectroscopic techniques to determine the consequences of PKA-mediated phosphorylation. Synchrotron radiation circular dichroism spectroscopy confirmed that purified full-length wild-type CFTR is folded and structurally responsive to phosphorylation. Intrinsic tryptophan fluorescence studies of CFTR showed that phosphorylation reduced iodide-mediated quenching, consistent with an effect of phosphorylation in burying tryptophans at the transmission interface. Importantly, the rate of phosphorylation-dependent channel activation was compromised by the introduction of disease-causing mutations in either of the two coupling helices predicted to interact with nucleotide binding domain 1 at the interface. Together, these results suggest that phosphorylation modifies the interface between the catalytic and pore domains of CFTR and that this modification facilitates CFTR channel activation.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células HEK293 , Humanos , Fosforilação/genética , Domínios ProteicosRESUMO
Crystallographic and electrophysiological studies have recently provided insight into the structure, function, and drug binding of prokaryotic sodium channels. These channels exhibit significant sequence identities, especially in their transmembrane regions, with human voltage-gated sodium channels. However, rather than being single polypeptides with four homologous domains, they are tetramers of single domain polypeptides, with a C-terminal domain (CTD) composed of an inter-subunit four helix coiled coil. The structures of the CTDs differ between orthologues. In NavBh and NavMs, the C-termini form a disordered region adjacent to the final transmembrane helix, followed by a coiled-coil region, as demonstrated by synchrotron radiation circular dichroism (SRCD) and double electron-electron resonance electron paramagnetic resonance spectroscopic measurements. In contrast, in the crystal structure of the NavAe orthologue, the entire C-terminus is comprised of a helical region followed by a coiled coil. In this study, we have examined the CTD of the NsvBa from Bacillus alcalophilus, which unlike other orthologues is predicted by different methods to have different types of structures: either a disordered region adjacent to the transmembrane region, followed by a helical coiled coil, or a fully helical CTD. To discriminate between the two possible structures, we have used SRCD spectroscopy to experimentally determine the secondary structure of the C-terminus of this orthologue and used the results as the basis for modeling the open and closed conformations of the channel.
Assuntos
Bacillus , Proteínas de Bactérias/química , Homologia de Sequência de Aminoácidos , Canais de Sódio/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biologia Computacional , Modelos Moleculares , Domínios Proteicos , Canais de Sódio/metabolismoRESUMO
Voltage-gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na(+) ≈ Li(+) â« K(+), Ca(2+), Mg(2+)) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.
Assuntos
Canais de Sódio/química , Canais de Sódio/metabolismo , Sódio/metabolismo , Alphaproteobacteria/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cátions/metabolismo , Cristalografia por Raios X , Ácido Glutâmico/genética , Células HEK293 , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Mutação , Técnicas de Patch-Clamp , Permeabilidade , Conformação Proteica , Canais de Sódio/genética , Eletricidade EstáticaRESUMO
Circular dichroism (CD) spectroscopy is a valuable method for defining canonical secondary structure contents of proteins based on empirically-defined spectroscopic signatures derived from proteins with known three-dimensional structures. Many proteins identified as being "Intrinsically Disordered Proteins" have a significant amount of their structure that is neither sheet, helix, nor turn; this type of structure is often classified by CD as "other", "random coil", "unordered", or "disordered". However the "other" category can also include polyproline II (PPII)-type structures, whose spectral properties have not been well-distinguished from those of unordered structures. In this study, synchrotron radiation circular dichroism spectroscopy was used to investigate the spectral properties of collagen and polyproline, which both contain PPII-type structures. Their native spectra were compared as representatives of PPII structures. In addition, their spectra before and after treatment with various conditions to produce unfolded or denatured structures were also compared, with the aim of defining the differences between CD spectra of PPII and disordered structures. We conclude that the spectral features of collagen are more appropriate than those of polyproline for use as the representative spectrum for PPII structures present in typical amino acid-containing proteins, and that the single most characteristic spectroscopic feature distinguishing a PPII structure from a disordered structure is the presence of a positive peak around 220nm in the former but not in the latter. These spectra are now available for inclusion in new reference data sets used for CD analyses of the secondary structures of soluble proteins.
Assuntos
Peptídeos/análise , Peptídeos/química , Dicroísmo Circular , Desnaturação Proteica , Estrutura Secundária de Proteína , TemperaturaRESUMO
Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.
Assuntos
Membrana Celular/química , Ouabaína/química , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/ultraestrutura , Cardiotônicos , Ativação Enzimática , Estabilidade Enzimática , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , TemperaturaRESUMO
The membrane-bound cation-transporting P-type Na,K-ATPase isolated from pig kidney membranes is much more resistant towards thermal inactivation than the almost identical membrane-bound Na,K-ATPase isolated from shark rectal gland membranes. The loss of enzymatic activity is correlated well with changes in protein structure as determined using synchrotron radiation circular dichroism (SRCD) spectroscopy. The enzymatic activity is lost at a 12°C higher temperature for pig enzyme than for shark enzyme, and the major changes in protein secondary structure also occur at T(m)'s that are ~10-15°C higher for the pig than for the shark enzyme. The temperature optimum for the rate of hydrolysis of ATP is about 42°C for shark and about 57°C for pig, both of which are close to the temperatures for onset of thermal unfolding. These results suggest that the active site region may be amongst the earliest parts of the structure to unfold. Detergent-solubilized Na,K-ATPases from the two sources show the similar differences in thermal stability as the membrane-bound species, but inactivation occurs at a lower temperature for both, and may reflect the stabilizing effect of a bilayer versus a micellar environment.
Assuntos
Proteínas de Membrana/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Dicroísmo Circular , Estabilidade Enzimática , Bicamadas Lipídicas , Proteínas de Membrana/metabolismo , Tubarões , SuínosRESUMO
Osteopontin (OPN) is an acidic hydrophilic glycophosphoprotein that was first identified as a major sialoprotein in bones. It functions as a cell attachment protein displaying a RGD cell adhesion sequence and as a cytokine that signals through integrin and CD44 cell adhesion molecules. OPN is also implicated in human tumor progression and cell invasion. OPN has intrinsic transforming activity, and elevated OPN levels promote metastasis. OPN gene expression is also strongly activated in avian fibroblasts simultaneously transformed by the v-myc and v-mil(raf) oncogenes. Here we have investigated the solution structure of a 220-amino acid recombinant OPN protein by an integrated structural biology approach employing bioinformatic sequence analysis, multidimensional nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism spectroscopy, and small-angle X-ray scattering. These studies suggest that OPN is an intrinsically unstructured protein in solution. Although OPN does not fold into a single defined structure, its conformational flexibility significantly deviates from random coil-like behavior. OPN comprises distinct local secondary structure elements with reduced conformational flexibility and substantially populates a compact subspace displaying distinct tertiary contacts. These compacted regions of OPN encompass the binding sites for α(V)ß(III) integrin and heparin. The conformational flexibility combined with the modular architecture of OPN may represent an important structural prerequisite for its functional diversity.
Assuntos
Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Metástase Neoplásica/patologia , Proteínas de Neoplasias/fisiologia , Osteopontina/química , Osteopontina/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Dicroísmo Circular , Humanos , Ligantes , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Mapeamento de Interação de Proteínas , Desdobramento de Proteína , CodornizRESUMO
Abstract The hepatitis C virus (HCV) encodes the p7 protein that oligomerizes to form an ion channel. The 63 amino acid long p7 monomer is an integral membrane protein predominantly found in the endoplasmic reticulum (ER). Although it is currently unknown whether p7 is incorporated into secreted virions, its presence is crucial for the release of infectious virus. The molecular and biophysical mechanism employed by the p7 ion channel is largely unknown, but in vivo it is likely to be embedded in membranes undergoing changes in lipid composition. In this study we analyze the influence of the lipid environment on p7 ion channel structure and function using electrophysiology and synchrotron radiation circular dichroism (SRCD) spectroscopy. We incorporated chemically synthesized p7 polypeptides into artificial planar membranes of various lipid compositions. A lipid bilayer composition comprising phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (4:1 PC:PE) led to burst-like patterns in the channel recordings with channel openings lasting up to 0.5 s. The reverse ratio of PC:PE (1:4) gave rise to individual channels continuously opening for up to 8 s. SRCD spectroscopy of p7 embedded into liposomes of corresponding lipid compositions suggests there is a structural effect of the lipid composition on the p7 protein.
Assuntos
Hepacivirus/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Lipídeos/química , Proteínas Virais/química , Proteínas Virais/metabolismo , Dicroísmo Circular , Ativação do Canal Iônico , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Relação Estrutura-Atividade , SíncrotronsRESUMO
Molecular-dynamics simulations were carried out to ascertain which of the potential multimeric forms of the transmembrane peptaibol channel, antiamoebin, is consistent with its measured conductance. Estimates of the conductance obtained through counting ions that cross the channel and by solving the Nernst-Planck equation yield consistent results, indicating that the motion of ions inside the channel can be satisfactorily described as diffusive. The calculated conductance of octameric channels is markedly higher than the conductance measured in single channel recordings, whereas the tetramer appears to be nonconducting. The conductance of the hexamer was estimated to be 115 ± 34 pS and 74 ± 20 pS, at 150 mV and 75 mV, respectively, in satisfactory agreement with the value of 90 pS measured at 75 mV. On this basis, we propose that the antiamoebin channel consists of six monomers. Its pore is large enough to accommodate K⺠and Clâ» with their first solvation shells intact. The free energy barrier encountered by K⺠is only 2.2 kcal/mol whereas Clâ» encounters a substantially higher barrier of nearly 5 kcal/mol. This difference makes the channel selective for cations. Ion crossing events are shown to be uncorrelated and follow Poisson statistics.
Assuntos
Condutividade Elétrica , Canais Iônicos/química , Canais Iônicos/metabolismo , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/metabolismo , Fenômenos Biomecânicos , Difusão , Ativação do Canal Iônico , Transporte de Íons , Lipídeos/química , Peptaibols , Maleabilidade , Porosidade , Estabilidade Proteica , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Termodinâmica , Água/químicaRESUMO
The structure of proteins at interfaces is a key factor determining the stability as well as organoleptic properties of food emulsions. While it is widely believed that proteins undergo conformational changes at interfaces, the measurement of these structural changes remains a significant challenge. In this study, the conformational changes of beta-lactoglobulin (beta-Lg) upon adsorption to the interface of hexadecane oil-in-water emulsions were investigated using synchrotron radiation circular dichroism (SRCD) spectroscopy. Far-UV SRCD spectra showed that adsorption of beta-Lg to the O/W interface caused a significant increase in non-native alpha-helix structure, accompanied by a concomitant loss of beta-sheet structure. Near-UV SRCD spectra revealed that a considerable disruption of beta-Lg tertiary structure occurred upon adsorption. Moreover, heat-induced changes to the non-native beta-Lg conformation at the oil/water interface were very small compared to the dramatic loss of beta-Lg secondary structure that occurred during heating in solution, suggesting that the interface has a stabilizing effect on the structure of non-native beta-Lg. Overall, our findings provide insight into the conformational behavior of proteins at oil/water interfaces and demonstrate the applicability of SRCD spectroscopy for measuring the conformation of adsorbed proteins in optically turbid emulsions.
Assuntos
Dicroísmo Circular/métodos , Emulsões , Lactoglobulinas/química , Síncrotrons , Adsorção , Tamanho da Partícula , Estrutura Secundária de Proteína , Espectrofotometria UltravioletaRESUMO
Extramembranous domains play important roles in the structure and function of membrane proteins, contributing to protein stability, forming association domains, and binding ancillary subunits and ligands. However, these domains are generally flexible, making them difficult or unsuitable targets for obtaining high-resolution X-ray and NMR structural information. In this study we show that the highly sensitive method of synchrotron radiation circular dichroism (SRCD) spectroscopy can be used as a powerful tool to investigate the structure of the extramembranous C-terminal domain (CTD) of the prokaryotic voltage-gated sodium channel (Na(V)) from Bacillus halodurans, NaChBac. Sequence analyses predict its CTD will consist of an unordered region followed by an alpha-helix, which has a propensity to form a multimeric coiled-coil motif, and which could form an association domain in the homotetrameric NaChBac channel. By creating a number of shortened constructs we have shown experimentally that the CTD does indeed contain a stretch of approximately 20 alpha-helical residues preceded by a nonhelical region adjacent to the final transmembrane segment and that the efficiency of assembly of channels in the membrane progressively decreases as the CTD residues are removed. Analyses of the CTDs of 32 putative prokaryotic Na(V) sequences suggest that a CTD helical bundle is a structural feature conserved throughout the bacterial sodium channel family.
Assuntos
Bacillus/química , Dicroísmo Circular/métodos , Canais de Sódio/química , Síncrotrons , Canal de Sódio Disparado por Voltagem NAV1.5 , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Canais de Sódio/metabolismoRESUMO
CD (circular dichroism) spectroscopy is a well-established technique in structural biology. SRCD (synchrotron radiation circular dichroism) spectroscopy extends the utility and applications of conventional CD spectroscopy (using laboratory-based instruments) because the high flux of a synchrotron enables collection of data at lower wavelengths (resulting in higher information content), detection of spectra with higher signal-to-noise levels and measurements in the presence of absorbing components (buffers, salts, lipids and detergents). SRCD spectroscopy can provide important static and dynamic structural information on proteins in solution, including secondary structures of intact proteins and their domains, protein stability, the differences between wild-type and mutant proteins, the identification of natively disordered regions in proteins, and the dynamic processes of protein folding and membrane insertion and the kinetics of enzyme reactions. It has also been used to effectively study protein interactions, including protein-protein complex formation involving either induced-fit or rigid-body mechanisms, and protein-lipid complexes. A new web-based bioinformatics resource, the Protein Circular Dichroism Data Bank (PCDDB), has been created which enables archiving, access and analyses of CD and SRCD spectra and supporting metadata, now making this information publicly available. To summarize, the developing method of SRCD spectroscopy has the potential for playing an important role in new types of studies of protein conformations and their complexes.
Assuntos
Dicroísmo Circular/métodos , Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Síncrotrons , Animais , Dicroísmo Circular/instrumentação , Bases de Dados de Proteínas , Humanos , Metabolismo dos Lipídeos/fisiologia , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica/fisiologia , Dobramento de Proteína , Mapeamento de Interação de Proteínas/instrumentaçãoRESUMO
Equinatoxin II (EqtII) is a protein toxin that lyses both red blood cells and artificial membranes. Lysis is dependent on the lipid composition, with small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC) and sphingomyelin (SM) (1:1 molar) being lysed more readily than those of phosphatidylcholine alone. Removing the N-terminus of EqtII prevents pore formation, but does not prevent membrane binding. A peptide corresponding to residues 1-32 of EqtII was found using NMR to adopt a helical structure in micelles. To further understand the structural changes that accompany membrane insertion, synchrotron radiation circular dichroism spectra of the N-terminal peptide in a range of model membranes have been analysed. The peptide structure was examined in water, dodecylphosphocholine (DPC) and DPC:SM (5:1) micelles, and SUVs composed of dioleoylphosphatidylcholine (DOPC) or DMPC, together with SM and cholesterol (Chol). The peptide adopted different conformations in different lipids. Although the presence of SM did not affect the conformation in micelles, inclusion of SM in the bilayer-forming lipid increased the helicity of the peptide. This effect was abolished when Chol was added in DOPC but not in DMPC, which may relate to liquid ordered versus disordered phase properties of the lipid. SM may act as a promoter of membrane organisation necessary for membrane lysis by EqtII.
Assuntos
Venenos de Cnidários/química , Lipídeos/química , Síncrotrons , Sequência de Aminoácidos , Colesterol/química , Dicroísmo Circular , Bicamadas Lipídicas/química , Micelas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosfatidilcolinas/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Conformação Proteica , Esfingomielinas/química , Água/químicaRESUMO
Circular dichroism (CD) spectroscopy is a well-established technique for the study of proteins. Synchrotron radiation circular dichroism (SRCD) spectroscopy extends the utility of conventional CD spectroscopy (i.e. using laboratory-based instruments) because the high light flux from a synchrotron enables collection of data to lower wavelengths, detection of spectra with higher signal-to-noise levels and measurements in the presence of strongly absorbing non-chiral components such as salts, buffers, lipids and detergents. This review describes developments in instrumentation, methodologies and bioinformatics that have enabled new applications of the SRCD technique for the study of proteins. It includes examples of the use of SRCD spectroscopy for providing static and dynamic structural information on molecules, including determinations of secondary structures of intact proteins and domains, assessment of protein stability, detection of conformational changes associated with ligand and drug binding, monitoring of environmental effects, examination of the processes of protein folding and membrane insertion, comparisons of mutant and modified proteins, identification of intermolecular interactions and complex formation, determination of the dispositions of proteins in membranes, identification of natively disordered proteins and their binding partners and examination of the carbohydrate components of glycoproteins. It also discusses how SRCD can be used in conjunction with macromolecular crystallography and other biophysical techniques to provide a more complete picture of protein structures and functions, including how proteins interact with other macromolecules and ligands. This review also includes a discussion of potential new applications in structural and functional genomics using SRCD spectroscopy and future instrumentation and bioinformatics developments that will enable such studies. Finally, the appendix describes a number of computational/bioinformatics resources for secondary structure analyses that take advantage of the improved data quality available from SRCD. In summary, this review discusses how SRCD can be used for a wide range of structural and functional studies of proteins.
Assuntos
Dicroísmo Circular/métodos , Proteínas/química , SíncrotronsRESUMO
New high-flux synchrotron radiation circular dichroism (SRCD) beamlines are providing important information for structural biology, but can potentially cause denaturation of the protein samples under investigation. This effect has been studied at the new CD1 dedicated SRCD beamline at ISA in Denmark, where radiation-induced thermal damage effects were observed, depending not only on the radiation flux but also on the focal spot size of the light. Comparisons with similar studies at other SRCD facilities worldwide has lead to the estimation of a flux density threshold under which SRCD beamlines should be operated when samples are to be exposed to low-wavelength vacuum ultraviolet radiation for extended periods of time.
Assuntos
Dicroísmo Circular , Desnaturação Proteica/efeitos da radiação , Síncrotrons , Humanos , Albumina Sérica/química , Albumina Sérica/efeitos da radiaçãoRESUMO
Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the 'bound' state and the 'free' state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.
Assuntos
Dicroísmo Circular/métodos , Venenos de Cnidários/química , Lipídeos/química , Conformação Proteica , Animais , Bicamadas Lipídicas/química , Micelas , Síncrotrons , Lipossomas Unilamelares/química , Vasoconstritores/químicaRESUMO
Age-related cataract is a result of crystallins, the predominant lens proteins, forming light-scattering aggregates. In the low protein turnover environment of the eye lens, the crystallins are susceptible to modifications that can reduce stability, increasing the probability of unfolding and aggregation events occurring. It is hypothesized that the alpha-crystallin molecular chaperone system recognizes and binds these proteins before they can form the light-scattering centres that result in cataract, thus maintaining the long-term transparency of the lens. In the present study, we investigated the unfolding and aggregation of (wild-type) human and calf betaB2-crystallins and the formation of a complex between alpha-crystallin and betaB2-crystallins under destabilizing conditions. Human and calf betaB2-crystallin unfold through a structurally similar pathway, but the increased stability of the C-terminal domain of human betaB2-crystallin relative to calf betaB2-crystallin results in the increased population of a partially folded intermediate during unfolding. This intermediate is aggregation-prone and prevents constructive refolding of human betaB2-crystallin, while calf betaB2-crystallin can refold with high efficiency. alpha-Crystallin can effectively chaperone both human and calf betaB2-crystallins from thermal aggregation, although chaperone-bound betaB2-crystallins are unable to refold once returned to native conditions. Ordered secondary structure is seen to increase in alpha-crystallin with elevated temperatures up to 60 degrees C; structure is rapidly lost at temperatures of 70 degrees C and above. Our experimental results combined with previously reported observations of alpha-crystallin quaternary structure have led us to propose a structural model of how activated alpha-crystallin chaperones unfolded betaB2-crystallin.