RESUMO
Extensive use of quantum chemical calculations has been made to rationally design a molecule whose spin state can be switched reversibly using light of two different wavelengths at room temperature in solution. Spin change is induced by changing the coordination number of a nickel complex. The coordination number in turn is switched using a photochromic ligand that binds in one configuration and dissociates in the other. We demonstrate that successful design relies on a precise geometry fit and delicate electronic tuning. Our designer complex exhibits an extremely high long-term switching stability (more than 20 000 cycles) and a high switching efficiency. The high-spin state is extraordinarily stable with a half-life of 400 days at room temperature. Switching between the dia- and paramagnetic state is achieved with visible light (500 and 430 nm). The compound can also be used as a molecular logic gate with light and pH as input and the magnetic state as non-destructive read-out.
RESUMO
Besides the cannabinoid mimetic JWH-073, a novel 4 methylnaphthoyl homologue of JWH-073 was detected in a herbal mixture. The structure of the compound was elucidated after thin layer chromatographic enrichment from the herbal mixture by nuclear magnetic resonance (NMR) and gas chromatographic mass spectrometric (GC-MS) analysis. The paper outlines data after GC-MS, liquid chromatography mass spectrometry (LC-MS) and NMR spectroscopy, and describes the structure elucidation.
RESUMO
Magnetic bistability, as manifested in the magnetization of ferromagnetic materials or spin crossover in transition metal complexes, has essentially been restricted to either bulk materials or to very low temperatures. We now present a molecular spin switch that is bistable at room temperature in homogeneous solution. Irradiation of a carefully designed nickel complex with blue-green light (500 nanometers) induces coordination of a tethered pyridine ligand and concomitant electronic rearrangement from a diamagnetic to a paramagnetic state in up to 75% of the ensemble. The process is fully reversible on irradiation with violet-blue light (435 nanometers). No fatigue or degradation is observed after several thousand cycles at room temperature under air. Preliminary data show promise for applications in magnetic resonance imaging.
RESUMO
The EGF receptor is the prototype for four highly related receptors constituting the ErbB family. The EGF receptor is normally targeted to the basolateral membrane in polarized epithelial cells, where it relays information from underlying tissues. Two basolateral sorting signals have been mapped to the cytoplasmic juxtamembrane region of the receptor, a dominant signal comprised of a polyproline core (667-PXXP) and a preceding basic residue (Arg662), and a consensus leucine-based signal (658-LL) responsible for residual sorting when the 667-PXXP signal is absent or defective. The goal of this study was to define the structure of these signals, and gain some insights into how these structures might be regulated by cellular microenvironment. Structural information was acquired for two peptides corresponding to EGF receptor residues Arg645 and Ala674 in aqueous solution or in the presence of membrane-mimicking dodecylphosphocholine micelles, using a variety of NMR and CD spectroscopic methods. Chemical shift data indicate that the 667-PXXP signal does not bind to the micelles and is in random coil state in both aqueous solution and a micellar environment, raising the possibility that 667-PXXP switches to an ordered structure during interaction with the basolateral sorting machinery. In contrast, the adjacent region including 658-LL does bind to micelles mediated by a highly positively charged region located between Arg645 and Arg656. The micelle-bound region also includes Thr654, a known substrate for PKC. This suggests a distinct mode of regulation for this signal involving membrane association and/or phosphorylation.
Assuntos
Polaridade Celular , Receptores ErbB/química , Micelas , Conformação Proteica , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Água/químicaRESUMO
Membrane protein misfolding is related to the etiology of many diseases, but is poorly understood, particularly from a structural standpoint. This study focuses upon misfolding of a mutant form of diacylglycerol kinase (s-DAGK), a 40 kDa homotrimeric protein having nine transmembrane segments. Preparations of s-DAGK sometimes contain a kinetically trapped misfolded population, as evidenced by lower-than-expected enzyme activity (with no accompanying change in substrate K(m)) and by the appearance of a second band in electrophoresis gels. Misfolding of s-DAGK may take place during cellular overexpression, but can also be reproduced using the purified enzyme. TROSY NMR spectra of s-DAGK as a 100 kDa complex with detergent micelles exhibit a single additional set of resonances from the misfolded form, indicating a single misfolded conformational state. The relative intensities of these extra resonances correlate with the percent reduction in enzyme activity below the maximum observed for fully folded s-DAGK. Misfolded s-DAGK exhibits a modest difference in its far-UV CD spectrum compared to the folded enzyme, consistent with a small degree of variance in secondary structural content between the two forms. However, differences in NMR chemical shift dispersion and temperature-dependent line widths exhibited by folded and misfolded s-DAGK support the notion that they represent very different structural states. Cross-linking experiments indicate that both the correctly folded enzyme and the kinetically trapped misfolded form are homotrimers. This work appears to represent the first documentation of conformationally specific misfolding of an integral membrane protein.
Assuntos
Diacilglicerol Quinase/química , Proteínas de Membrana/química , Dobramento de Proteína , Dicroísmo Circular , Reagentes de Ligações Cruzadas/química , Diacilglicerol Quinase/genética , Suscetibilidade a Doenças , Escherichia coli/enzimologia , Escherichia coli/genética , Glutaral/química , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/química , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Antifreeze proteins (AFPs) are a group of structurally very diverse proteins with the unique capability of binding to the surface of seed ice crystals and inhibiting ice crystal growth. The AFPs bind with high affinity to specific planes of the ice crystal. Previously, this affinity of AFPs has been ascribed to the formation of multiple hydrogen bonds across the protein-ice interface, but more recently van der Waals interactions have been suggested to be the dominant energetic factors for the adsorption. To determine whether van der Waals interactions are also responsible for the binding specificities of AFPs, the protein-ice interaction of the helical AFP Type I from winter flounder (HPLC6) was studied using a Monte Carlo rigid body docking approach. HPLC6 binds in the [1102] direction of the [2021] plane, with the Thr-Ala-Asn surface comprising the protein's binding face. The binding of HPLC6 to this ice plane is highly preferred, but the protein is also found to bind favorably to the [1010] prism plane using a different protein surface comprised of Thr and Ala residues. The results show that van der Waals interactions, despite accounting for most of the intermolecular energy (>80%), are not sufficient to completely explain the AFP binding specificity.
Assuntos
Proteínas Anticongelantes/química , Gelo , Algoritmos , Cromatografia Líquida de Alta Pressão , Modelos Biológicos , Modelos Moleculares , Método de Monte Carlo , Propriedades de Superfície , Água/químicaRESUMO
Prion propagation in transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrP(C), into a pathogenic conformer, PrP(Sc). Hereditary forms of the disease are linked to specific mutations in the gene coding for the prion protein. To gain insight into the molecular basis of these disorders, the solution structure of the familial Creutzfeldt-Jakob disease-related E200K variant of human prion protein was determined by multi-dimensional nuclear magnetic resonance spectroscopy. Remarkably, apart from minor differences in flexible regions, the backbone tertiary structure of the E200K variant is nearly identical to that reported for the wild-type human prion protein. The only major consequence of the mutation is the perturbation of surface electrostatic potential. The present structural data strongly suggest that protein surface defects leading to abnormalities in the interaction of prion protein with auxiliary proteins/chaperones or cellular membranes should be considered key determinants of a spontaneous PrP(C) --> PrP(Sc) conversion in the E200K form of hereditary prion disease.
Assuntos
Síndrome de Creutzfeldt-Jakob/genética , Príons/química , Sequência de Bases , Humanos , Dados de Sequência Molecular , Mutação , Dobramento de Proteína , Estrutura Secundária de Proteína , SoluçõesRESUMO
Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and oxalacetate. Within the multi-subunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier and also binds the other two subunits to assist in the overall assembly of the enzyme. The 1.3S subunit is a 123 amino acid polypeptide (12.6 kDa) to which biotin is covalently attached at Lys 89. The three-dimensional solution structure of the full-length holo-1.3S subunit of TC has been solved by multidimensional heteronuclear NMR spectroscopy. The C-terminal half of the protein (51-123) is folded into a compact all-beta-domain comprising of two four-stranded antiparallel beta-sheets connected by short loops and turns. The fold exhibits a high 2-fold internal symmetry and is similar to that of the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase, but lacks an extension that has been termed "protruding thumb" in BCCP. The first 50 residues, which have been shown to be involved in intersubunit interactions in the intact enzyme, appear to be disordered in the isolated 1.3S subunit. The molecular surface of the folded domain has two distinct surfaces: one side is highly charged, while the other comprises mainly hydrophobic, highly conserved residues.
Assuntos
Carboxil e Carbamoil Transferases/química , Fragmentos de Peptídeos/química , Propionibacterium/enzimologia , Acetil-CoA Carboxilase/química , Proteínas de Transporte/química , Cristalografia por Raios X , Di-Hidrolipoamida Desidrogenase/química , Ácido Graxo Sintase Tipo II , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/isolamento & purificação , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Soluções , Eletricidade Estática , Propriedades de SuperfícieRESUMO
BACKGROUND: THP12 is an abundant and extraordinarily hydrophilic hemolymph protein from the mealworm Tenebrio molitor and belongs to a group of small insect proteins with four highly conserved cysteine residues. Despite their sequence homology to odorant-binding proteins and pheromone-binding proteins, the function of these proteins is unclear. RESULTS: The first three-dimensional structure of THP12 has been determined by multidimensional NMR spectroscopy. The protein has a nonbundle helical structure consisting of six alpha helices. The arrangement of the alpha helices has a 'baseball glove' shape. In addition to the hydrophobic core, electrostatic interactions make contributions to the overall stability of the protein. NMR binding studies demonstrated the binding of small hydrophobic ligands to the single hydrophobic groove in THP12. Comparing the structure of THP12 with the predicted secondary structure of homologs reveals a common fold for this new class of insect proteins. A search with the program DALI revealed extensive similarity between the three-dimensional structure of THP12 and the N-terminal domain (residues 1-95) of recoverin, a member of the family of calcium-binding EF-hand proteins. CONCLUSIONS: Although the biological function of this new class of proteins is as yet undetermined, a general role as alpha-helical carrier proteins for small hydrophobic ligands, such as fatty acids or pheromones, is proposed on the basis of NMR-shift perturbation spectroscopy.
Assuntos
Proteínas de Transporte/química , Proteínas de Insetos/química , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Dicroísmo Circular , Proteínas de Insetos/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Hmu O, a heme degradation enzyme in Corynebacterium diphtheriae, forms a stoichiometric complex with iron protoporphyrin IX and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Using a multitude of spectroscopic techniques, we have determined the axial ligand coordination of the heme-Hmu O complex. The ferric complex shows a pH-dependent reversible transition between a water-bound hexacoordinate high spin neutral pH form and an alkaline form, having high spin and low spin states, with a pK(a) of 9. (1)H NMR, EPR, and resonance Raman of the heme-Hmu O complex establish that a neutral imidazole of a histidine residue is the proximal ligand of the complex, similar to mammalian heme oxygenase. EPR of the deoxy cobalt porphyrin IX-Hmu O complex confirms this proximal histidine coordination. Oxy cobalt-Hmu O EPR reveals a hydrogen-bonding interaction between the O(2) and an exchangeable proton in the Hmu O distal pocket and two distinct orientations for the bound O(2). Mammalian heme oxygenase has only one O(2) orientation. This difference and the mixed spin states at alkaline pH indicate structural differences in the distal environment between Hmu O and its mammalian counterpart.
Assuntos
Proteínas de Bactérias/química , Corynebacterium diphtheriae/enzimologia , Heme Oxigenase (Desciclizante) , Sítios de Ligação , Cobalto/química , Espectroscopia de Ressonância de Spin Eletrônica , Hemina/química , Histidina/química , Concentração de Íons de Hidrogênio , Ferro/química , Espectroscopia de Ressonância Magnética , Complexos Multienzimáticos , Protoporfirinas/química , Espectrofotometria , Análise Espectral RamanRESUMO
Neuronal voltage-dependent calcium channels are integral components of cellular excitation and neurosecretion. In addition to mediating the entry of calcium across the plasma membrane, both N-type and P/Q-type voltage-dependent calcium channels have been shown to form stable complexes with synaptic vesicle and presynaptic membrane proteins, indicating a structural role for the voltage-dependent calcium channels in secretion. Recently, detailed structural analyses of N-type calcium channels have identified residues amino acids 718-963 as the site in the rat alpha1B subunit that mediates binding to syntaxin, synaptosome-associated protein of 25,000 mol. wt and synaptotagmin [Sheng et al. (1996) Nature 379, 451-454]. The purpose of this study was to employ site-directed antibodies to target domains within and outside of the interaction site on the rat alpha1B to probe potential binding sites for syntaxin/SNAP-25/synaptotagmin. Our results demonstrate that both antibodies employed in this study have access to their epitopes on the alpha1B as evidenced by equivalent immunoprecipitation of native [125I]omega-conotoxin GVIA-labeled alpha1B protein from CHAPS-solubilized preparations. The N-type voltage-dependent calcium channel immunoprecipitated by Ab CW14, the antibody directed to a domain outside of the synprint site, is associated with syntaxin and SNAP-25 with the recovery of these proteins, increasing in parallel to the recovery of alpha1B. However, when we used the antibody raised to an epitope within the synprint site (Ab CW8) to immunoprecipitate N-type calcium channels, the alpha1B was depleted of more than 65% of syntaxin and 80% of SNAP-25 when compared to the recovery of these proteins using Ab CW14. This is the first report of a defined epitope on the alpha1B subunit II-III loop (amino acids 863-875) whose perturbation by a site-directed antibody influences the dissociation of SNAP-25 and syntaxin.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Especificidade de Anticorpos , Sítios de Ligação , Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/isolamento & purificação , Membrana Celular/metabolismo , Humanos , Substâncias Macromoleculares , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/isolamento & purificação , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Estrutura Secundária de Proteína , Proteínas Qa-SNARE , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína 25 Associada a Sinaptossoma , SinaptotagminasRESUMO
Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alpha-helix. Leu and Asn flank the central two of four regularly spaced ice-binding Thr in the i-1 and i + 3 positions, respectively. All three residues project from the same side of the helix to form the protein's putative ice-adsorption site and are considered in some models to act together as an "ice-binding motif". Replacement of Asn by residues with shorter side chains resulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. However, substitution of Asn by its slightly larger homologue (Gln) abolished thermal hysteresis activity. The Gln-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu was replaced by Ala, two of the three were more active than their Leu-containing counterparts, but all three variants began to precipitate as the peptide concentration increased. None of the seven variants tested showed dramatic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventing peptide aggregation at the antifreeze protein concentrations (10 mg/mL) normally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as traditionally thought. Nevertheless, the dramatic loss of activity in the Asn-to-Gln replacement demonstrates the steric restriction on residues in or near the ice-binding site of the peptide.
Assuntos
Glicoproteínas/metabolismo , Gelo , Sequência de Aminoácidos , Proteínas Anticongelantes , Asparagina/metabolismo , Sítios de Ligação , Glicoproteínas/química , Leucina/metabolismo , Dados de Sequência MolecularRESUMO
A suite of programs called CAMRA (Computer Aided Magnetic Resonance Assignment) has been developed for computer assisted residue-specific assignments of proteins. CAMRA consists of three units: ORB, CAPTURE and PROCESS. ORB predicts NMR chemical shifts for unassigned proteins using a chemical shift database of previously assigned homologous proteins supplemented by a statistically derived chemical shift database in which the shifts are categorized according to their residue, atom and secondary structure type. CAPTURE generates a list of valid peaks from NMR spectra by filtering out noise peaks and other artifacts and then separating the derived peak list into distinct spin systems. PROCESS combines the chemical shift predictions from ORB with the spin systems identified by CAPTURE to obtain residue specific assignments. PROCESS ranks the top choices for an assignment along with scores and confidence values. In contrast to other auto-assignment programs, CAMRA does not use any connectivity information but instead is based solely on matching predicted shifts with observed spin systems. As such, CAMRA represents a new and unique approach for the assignment of protein NMR spectra. CAMRA will be particularly useful in conjunction with other assignment methods and under special circumstances, such as the assignment of flexible regions in proteins where sufficient NOE information is generally not available. CAMRA was tested on two medium-sized proteins belonging to the chemokine family. It was found to be effective in predicting the assignment providing a database of previously assigned proteins with at least 30% sequence identity is available. CAMRA is versatile and can be used to include and evaluate heteronuclear and three-dimensional experiments.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Software , Sequência de Aminoácidos , Substituição de Aminoácidos , Quimiocina CXCL12 , Quimiocinas CXC/química , Interleucina-8/análogos & derivados , Interleucina-8/química , Dados de Sequência Molecular , Mutação PuntualRESUMO
Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier. The 1.3S is a 123-amino acid polypeptide (12.6 kDa), to which biotin is covalently attached at Lys 89. We have expressed 1.3S in Escherichia coli with uniform 15N labeling. The backbone structure and dynamics of the protein have been characterized in aqueous solution by three-dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. The secondary structure elements in the protein were identified based on NOE information, secondary chemical shifts, homonuclear 3J(HNHalpha) coupling constants, and amide proton exchange data. The protein contains a predominantly disordered N-terminal half, while the C-terminal half is folded into a compact domain comprising eight beta-strands connected by short loops and turns. The topology of the C-terminal domain is consistent with the fold found in both carboxyl carrier and lipoyl domains, to which this domain has approximately 26-30% sequence similarity.
Assuntos
Carboxil e Carbamoil Transferases/química , Propionibacterium/enzimologia , Sequência de Aminoácidos , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Isótopos de Nitrogênio/análise , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/químicaRESUMO
Translational diffusion coefficients and catalytic activities were measured for the integral membrane protein diacylglycerol kinase (DAGK) in a variety of types of detergent micelles. Despite the structural diversity of the detergents examined, the translational diffusion coefficients observed for DAGK spanned a fairly limited range of values: 2.7 to 4.7 (x10(-7) cm2/s). No general correlation was observed between the diffusion coefficients for the detergent-DAGK aggregates and the sizes of the corresponding protein-free micelles. These results indicate that the effective molecular weights of the DAGK-detergent aggregates were determined more by the structural properties of the protein than by the properties of the detergents. The catalytic activity of DAGK in detergents having medium-length alkyl chains such as dodecylphosphocholine or decylmaltoside was usually observed to be substantially higher than in short-chain detergents such as octylphosphocholine or octylglucoside. Taken together, the diffusion and activity results indicate that medium-chain detergents are generally preferred for use in NMR studies of complex membrane proteins because they are no worse than short-chained detergents in terms of increasing the effective molecular weight of the protein of interest while they are considerably better at maintaining native-like protein conformation. Among the 10 detergents examined, only sodium dodecylsulfate was observed to be unable to support DAGK activity under any conditions examined, suggest that this well-known protein denaturant should be used with care in studies of complex membrane proteins.
Assuntos
Detergentes , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Diacilglicerol Quinase/química , Diacilglicerol Quinase/metabolismo , Difusão , Ativação Enzimática , Estabilidade Enzimática , Proteínas de Membrana/metabolismo , MicelasRESUMO
The adenovirus E3-13.7 protein interferes with endosomal protein sorting to down-regulate the epidermal growth factor receptor and related tyrosine kinase receptors. The cytoplasmic C terminus of this protein contains three protein sorting motifs which are related to the function of E3-13.7. In this study, the structure of a 23-residue polypeptide corresponding to this domain was examined using solution NMR and CD spectroscopic methods. The peptide was observed to exist in a mostly random structural state in aqueous solution but underwent high affinity association with dodecylphosphocholine micelles, where it adopted an ordered structure. The affinity of this peptide for the micellar surface and the structure of the bound peptide were independent of pH variation, surface charge, or attachment of a myristoyl anchor to the N-terminal. Studies with phospholipid vesicles suggested that the micellar structural results can be extrapolated to a true lipid bilayer. On the micellar surface all three sorting motifs are closely associated with the water/apolar interface: 72-YLRH and 87-LL lie within interfacial amphipathic helices, while 76-HPQY is non-helical and dimples just above the surface. These results contribute to the development of an understanding of the basis for specificity in recognition of sorting motifs by components of the cellular protein trafficking machinery.
Assuntos
Proteínas E3 de Adenovirus/metabolismo , Regulação para Baixo , Endocitose , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Proteínas E3 de Adenovirus/química , Sequência de Aminoácidos , Membrana Celular/metabolismo , Dicroísmo Circular , Citosol/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Micelas , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The alpha-helical antifreeze protein (AFP) from winter flounder inhibits ice growth by binding to a specific set of pyramidal surface planes that are not otherwise macroscopically expressed. The 37-residue AFP contains three 11-amino acid repeats that make a stereo-specific fit to the ice lattice along the <01-12> direction of the (20-21) and equivalent binding planes. When the AFP was shortened to delete two of the three 11-amino acid ice-binding repeats, the resulting 15-residue peptide and its variants were less helical and showed no antifreeze activity. However, when the helicity of the peptide was reinforced by an internal lactam bridge between Glu-7 and Lys-11, the minimized AFP was able to stably express the pyramidal plane (20-21) on the surface of growing ice crystals. This dynamic shaping of the ice surface by a single ice-binding repeat provides evidence that AFP adsorption to the ice lattice is not an "all-or-nothing" interaction. Instead, a partial interaction can help develop the binding site on ice to which the remainder of the AFP (or other AFP molecules) can orient and bind.
Assuntos
Glicoproteínas/química , Gelo , Oligopeptídeos/química , Proteínas Anticongelantes , Dicroísmo Circular , Cristalografia , Temperatura Alta , Desnaturação Proteica , Estrutura Secundária de ProteínaRESUMO
A recombinant form of the sea raven type II antifreeze protein (SRAFP) has been produced using the Pichia pastoris expression system. The antifreeze activity of recombinant SRAFP is indistinguishable from that of the wild-type protein. The global fold of SRAFP has been determined by two-dimensional 1H homonuclear and three-dimensional 1H-¿15N¿ heteronuclear NMR spectroscopy using 785 NOE distance restraints and 47 angular restraints. The molecule folds into one globular domain that consists of two helices and nine beta-strands in two beta-sheets. The structure confirms the proposed existence of five disulfide bonds. The global fold of SRAFP is homologous to C-type lectins and pancreatic stone proteins, even though the sequence identity is only approximately 20%.
Assuntos
Proteínas Anticongelantes Tipo II , Proteínas de Transporte/química , Lectinas/química , Sequência de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Congelamento , Espectroscopia de Ressonância Magnética , Modelos Químicos , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Antifreeze proteins (AFPs) are a structurally diverse class of proteins that bind to ice and inhibit its growth in a noncolligative manner. This adsorption-inhibition mechanism operating at the ice surface results in a lowering of the (nonequilibrium) freezing point below the melting point. A lowering of approximately 1 degree C, which is sufficient to prevent fish from freezing in ice-laden seawater, requires millimolar AFP levels in the blood. The solubility of AFPs at these millimolar concentrations and the small size of the AFPs (typically 3-15 kDa) make them ideal subjects for NMR analysis. Although fish AFPs are naturally abundant, seasonal expression, restricted access to polar fishes, and difficulties in separating numerous similar isoforms have made protein expression the method of choice for producing AFPs for structural studies. Expression of recombinant AFPs has also facilitated NMR analysis by permitting isotopic labeling with 15N and 13C and has permitted mutations to be made to help with the interpretation of NMR data. NMR analysis has recently solved two AFP structures and provided valuable information about the disposition of ice-binding side chains in a third. The potential exists to solve other AFP structures, including the newly described insect AFPs, and to use solid-state NMR techniques to address fundamental questions about the nature of the interaction between AFPs and ice.
Assuntos
Glicoproteínas/química , Espectroscopia de Ressonância Magnética , Conformação Proteica , Adsorção , Animais , Proteínas Anticongelantes , Cristalização , Peixes/metabolismo , Congelamento , Glicoproteínas/classificação , Gelo , Modelos Moleculares , Maleabilidade , Dobramento de Proteína , Isoformas de Proteínas/química , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , TermodinâmicaRESUMO
Antifreeze proteins lower the freezing point of their solution by binding to ice and inhibiting its growth. One of several structurally different antifreeze proteins in fishes (type II) is homologous to the carbohydrate-recognition domain of Ca2+-dependent lectins and adopts the same three-dimensional fold. Type II antifreeze proteins from herring and smelt require Ca2+ for binding to ice, whereas this same antifreeze protein in sea raven binds to ice in the absence of Ca2+ and has only two of the five Ca2+-liganding amino acids that are present in the lectin. To locate the ice-binding site, site-directed mutants of the 15 kDa, globular, disulfide-bonded sea raven antifreeze protein were produced by secretion from Pichia pastoris. Pairs of amino acid replacements, insertions, and a peptide loop swap were made in the region equivalent to the sugar-binding site of the lectin that encompasses loops 3 and 4 and beta-sheets 7 and 8. Even the most extensive mutation caused only a 25% decrease in antifreeze activity and demonstrated that the residues corresponding to the Ca2+-binding site are only peripherally involved in ice binding. When adjacent surface residues were mutated, the replacement of one residue, Ser120 by His, caused a 35% decrease in activity by itself and an 80% loss in conjunction with the peptide loop swap mutation. This pivotal sea raven antifreeze protein amino acid does not coincide with the herring ice-binding epicenter, but is located within the region corresponding to the proposed CaCO3-binding surface of a third homologue, the pancreatic stone protein. Intron and exon structure of the sea raven AFP gene also suggests that it might be more closely related to the stone protein gene than to the lectin gene. These results support the notion that this family of proteins has evolved more than one binding surface from the same protein scaffold.