RESUMO
[alpha-(15)N]Lysine-labeled rhodopsin, prepared by expression of a synthetic gene in HEK293 cells, was investigated both by conventional and transverse relaxation optimized spectroscopy-type heteronuclear single quantum correlation spectroscopy. Whereas rhodopsin contains 11 lysines, 8 in cytoplasmic loops and 1 each in the C-terminal peptide sequence and the intradiscal and transmembrane domains, only a single sharp peak was observed in dodecyl maltoside micelles. This result did not change when dodecyl maltoside was replaced by octyl glucoside or octyl glucoside-phospholipid-mixed micelles. Additional signals of much lower and variable intensity appeared at temperatures above 20 degrees C and under denaturing conditions. Application of the transverse relaxation optimized spectroscopy sequence resulted in sharpening of resonances but also losses of signal intensity. The single peak observed has been assigned to the C-terminal Lys-339 from the following lines of evidence. First, the signal is observed in HNCO spectra of rhodopsin, containing the labeled [(13)C]Ser-338/[(15)N]Lys-339 dipeptide. Second, addition of a monoclonal anti-rhodopsin antibody that binds to the C-terminal 8 aa of rhodopsin caused disappearance of the peak. Third, truncated rhodopsin lacking the C-terminal sequence Asp-330-Ala-348 showed no signal, whereas the enzymatically produced peptide fragment containing the above sequence showed the single peak. The results indicate motion in the backbone amide groups of rhodopsin at time scales depending on their location in the sequence. At the C terminus, conformational averaging occurs at the nanosecond time scale but varies from microsecond to millisecond in other parts of the primary sequence. The motions reflecting conformational exchange may be general for membrane proteins containing transmembrane helical bundles.
Assuntos
Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Rodopsina/química , Rodopsina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Bovinos , Linhagem Celular , Detergentes , Dissulfetos/metabolismo , Humanos , Luz , Lisina/química , Micelas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Rodopsina/genética , Rodopsina/imunologia , Espalhamento de Radiação , Soluções , Espectrometria de Fluorescência , TemperaturaRESUMO
19F nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution (19)F NMR spectra of fluorine-labeled rhodopsin in detergent micelles were described, demonstrating the applicability of this technique to studies of tertiary structure in the cytoplasmic domain. To quantitate tertiary contacts we have applied a transient one-dimensional difference NOE solution (19)F NMR experiment to this system, permitting assessment of proximities between fluorine labels specifically incorporated into different regions of the cytoplasmic face. Three dicysteine substitution mutants (Cys-140-Cys-316, Cys-65-Cys-316, and Cys-139-Cys-251) were labeled by attachment of the trifluoroethylthio group through a disulfide linkage. Each mutant rhodopsin was prepared (8-10 mg) in dodecylmaltoside and analyzed at 20 degrees C by solution (19)F NMR. Distinct chemical shifts were observed for all of the rhodopsin (19)F labels in the dark. An up-field shift of the Cys-316 resonance in the Cys-65-Cys-316 mutant suggests a close proximity between the two residues. When analyzed for (19)F-(19)F NOEs, a moderate negative enhancement was observed for the Cys-65-Cys-316 pair and a strong negative enhancement was observed for the Cys-139-Cys-251 pair, indicating proximity between these sites. No NOE enhancement was observed for the Cys-140-Cys-316 pair. These NOE effects demonstrate a solution (19)F NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.
Assuntos
Citoplasma/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Bovinos , Cisteína/genética , Cisteína/metabolismo , Escuridão , Detergentes , Dissulfetos/química , Dissulfetos/metabolismo , Flúor/metabolismo , Glucosídeos , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutação/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rodopsina/genética , Soluções , Espectrofotometria UltravioletaRESUMO
Obtaining well ordered crystals of membrane proteins is the single most serious stumbling block in the pursuit of their high-resolution structures. The applicability of lipidic cubic phase-mediated crystallization is demonstrated on a diverse set of bacterial membrane proteins: two photosynthetic reaction centres, a light-harvesting complex and two retinal proteins, halorhodopsin and bacteriorhodopsin. Despite marked differences in molecular dimensions, subunit composition and membrane origin, one single lipid, monoolein, is sufficient to form a crystallization matrix for all the aforementioned systems. Therefore, the lipidic cubic phase approach is proposed as a general method for crystallizing membrane proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana/química , Bacteriorodopsinas/química , Cristalização , Cristalografia por Raios X , Halobacterium salinarum , Halorrodopsinas , Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides , RodopseudomonasRESUMO
We report high resolution solution (19)F NMR spectra of fluorine-labeled rhodopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trifluoroethylthio (TET), CF(3)-CH(2)-S, group through a disulfide linkage. TET-labeled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 316 in rhodopsin were thus prepared. Purified mutant rhodopsins (6-10 mg), in dodecylmaltoside, were analyzed at 20 degrees C by solution (19)F NMR spectroscopy. The spectra recorded in the dark showed the following chemical shifts relative to trifluoroacetate: Cys-67, 9.8 ppm; Cys-140, 10.6 ppm; Cys-245, 9.9 ppm; Cys-248, 9.5 ppm; Cys-311, 9.9 ppm; and Cys-316, 10.0 ppm. Thus, all mutants showed chemical shifts downfield that of free TET (6.5 ppm). On illumination to form metarhodopsin II, upfield changes in chemical shift were observed for (19)F labels at positions 67 (-0.2 ppm) and 140 (-0.4 ppm) and downfield changes for positions 248 (+0.1 ppm) and 316 (+0.1 ppm) whereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those originally observed in the dark. The results demonstrate the applicability of solution (19)F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation.
Assuntos
Rodopsina/química , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular Transformada , Cisteína/química , Cisteína/genética , Escuridão , Detergentes/química , Dimiristoilfosfatidilcolina/química , Radioisótopos de Flúor , Glucosídeos/química , Luz , Memantina/análogos & derivados , Dados de Sequência Molecular , Mutagênese , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Rodopsina/genética , SoluçõesRESUMO
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
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 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.
Assuntos
Carboidratos/química , Glicoproteínas/química , Gelo , Lectinas/química , Animais , Proteínas Anticongelantes , Sequência de Bases , Sítios de Ligação , Peixes , Congelamento , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Pichia/genética , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut(s) clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut+ clones. The mixed-feeding/Mut(s) strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l.
Assuntos
Proteínas Anticongelantes Tipo II , Proteínas de Transporte/biossíntese , Pichia/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Aminoácidos/metabolismo , Animais , Meios de Cultura , Fermentação , Peixes/genética , Glicerol/metabolismo , Concentração de Íons de Hidrogênio , Metanol/metabolismo , Oxigênio/metabolismo , PlasmídeosRESUMO
The most abundant isoform (HPLC-6) of type I antifreeze protein (AFP1) in winter flounder is a 37-amino-acid-long, alanine-rich, alpha-helical peptide, containing four Thr spaced 11 amino acids apart. It is generally assumed that HPLC-6 binds ice through a hydrogen-bonding match between the Thr and neighboring Asx residues to oxygens atoms on the {2021} plane of the ice lattice. The result is a lowering of the nonequilibrium freezing point below the melting point (thermal hysteresis). HPLC-6, and two variants in which the central two Thr were replaced with either Ser or Val, were synthesized. The Ser variant was virtually inactive, while only a minor loss of activity was observed in the Val variant. CD, ultracentrifugation, and NMR studies indicated no significant structural changes or aggregation of the variants compared to HPLC-6. These results call into question the role of hydrogen bonds and suggest a much more significant role for entropic effects and van der Waals interactions in binding AFP to ice.