RESUMEN
Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.
Asunto(s)
Colesterol/química , Riñón/enzimología , ATPasa Intercambiadora de Sodio-Potasio/química , Animales , Membrana Celular/enzimología , Colesterol/metabolismo , Difenilhexatrieno/química , Activación Enzimática , Polarización de Fluorescencia , Membrana Dobles de Lípidos/química , Liposomas/química , Lípidos de la Membrana/química , Modelos Químicos , Fosfatidilcolinas/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de Fluorescencia , Porcinos , Agua/químicaRESUMEN
Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Animales , Unión Competitiva , Cromatografía Líquida de Alta Presión , Dinaminas , Colorantes Fluorescentes , GTP Fosfohidrolasas/química , Guanosina Difosfato/análogos & derivados , Hidrólisis , Cinética , Unión Proteica , Conformación Proteica , Ratas , Ultracentrifugación , ortoaminobenzoatosRESUMEN
In the present study, structural aspects of the two soluble transducers, HtrX and HtrXI, from the archaeon H. salinarum have been examined using UV circular dichroism and steady-state fluorescence spectroscopies. Circular dichroism (CD) data indicate that both HtrX and HtrXI exhibit salt-dependent protein folding. Under low-ionic-strength conditions (0.2 M NaCl or KCl) the CD spectra of HtrXI is similar to that of the Gdn-HCl- or urea-denatured forms and is indicative of random coil structure. In contrast, the CD spectrum of HtrX under low-ionic-strength conditions contains roughly 85% alpha-helical character, indicating a significant degree of folding. Addition of NaCl or KCl to solutions of HtrX or HtrXI results in CD features consistent with predominately alpha-helical character (>95%) for both proteins. In addition, the transition points (i.e., ionic strengths at which the protein converts from random coil to alpha-helical character) are quite distinct and dependent upon the type of salt present (i.e., either NaCl or KCl). Accessibility of tryptophan residues to the solvent was also examined for both HtrX and HtrXI in both folded and unfolded states using KI quenching. The Stem-Volmer constants obtained suggest that the tryptophans (Trp35 in HtrX and both Trp47 and Trp74 in HtrXI) are partially exposed to the solvent, indicating that they are located near the surface of the protein in all three cases. Furthermore, fluorescence quenching with the single Trp mutants Trp74AIa and Trp47AIa of HtrXI indicates different environments for these two residues.
Asunto(s)
Proteínas Arqueales/química , Halobacterium salinarum/química , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Proteínas de la Membrana/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Transducción de Señal , Cloruro de Sodio/metabolismo , Cloruro de Sodio/farmacología , Volumetría , Triptófano/químicaRESUMEN
The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized by a tetramer/monomer equilibrium with an equilibrium constant of 1.67 x 10(17) M(-3). Stopped-flow fluorescence experiments show that the association rate constant for 2'(3')-O-N-methylanthraniloyl (mant)GTP is 7.0 x 10(-5) M(-1) s(-1) and the dissociation rate constant is 2.1 s(-1), whereas the dissociation rate constant for mantdeoxyGDP is 93 s(-1). We also demonstrate the cooperativity of dynamin binding and GTPase activation on a microtubule lattice. Our results indicate that dynamin self-association is not a sufficient condition for the expression of maximal GTPase activity, which suggests that dynamin molecules must be in the proper conformation or orientation if they are to form an active oligomer.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Animales , Encéfalo/enzimología , Bovinos , Relación Dosis-Respuesta a Droga , Dinaminas , Cinética , Microtúbulos/metabolismo , Modelos Biológicos , Cloruro de Sodio/farmacología , Factores de Tiempo , Tubulina (Proteína)/metabolismo , UltracentrifugaciónRESUMEN
Site-directed mutagenesis was utilized to construct mutants, containing one or two tryptophan residues, of the bifunctional enzyme fructose 6-phosphate,2-kinase-fructose 2,6-bisphosphatase. Two of the single-tryptophan mutants (W15 and W64) had the tryptophan residue located in the kinase domain, which is in the N-terminal half, and two (W299 and W320) had the tryptophan residue located in the phosphatase domain, which is in the C-terminal half. The double-tryptophan mutants were W15/W64, W15/W299, W64/W299, and W299/W320. Dynamic polarization data indicated that these tryptophan residues had varying degrees of local mobility. Steady-state polarization data revealed energy transfer between the tryptophan residues in the double mutant W299/W320 but not in the W15/W64, W15/W299, or W64/W299 mutants, indicating the proximity of the W299 and W320 residues. The binding of fructose-6-phosphate resulted in a significant increase in the anisotropy of the W15 mutants, but did not affect the anisotropies of any of the other single-tryptophan mutants. Binding of fructose-2,6-bisphosphate also significantly increased the anisotropy of W15. In the case of fructose-6-phosphate binding, the increased anisotropy was shown to be due to a restriction of the tryptophan residue's local mobility in the presence of bound ligand, which suggests that the N-terminus is located near the kinase active site. These increases in anisotropies were used to estimate the dissociation constants of fructose-6-phosphate and fructose-2,6-bisphosphate, which were 29 +/- 3 and 2.1 +/- 0.3 microM, respectively. These observations are considered in light of the recently published crystal structure for this bifunctional enzyme.
Asunto(s)
Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Fosfotransferasas/química , Fosfotransferasas/genética , Conformación Proteica , Testículo/enzimología , Sustitución de Aminoácidos/genética , Animales , Polarización de Fluorescencia , Fructosadifosfatos/metabolismo , Fructosafosfatos/metabolismo , Ligandos , Masculino , Fenilalanina/genética , Fosfofructoquinasa-2 , Unión Proteica , Ratas , Triptófano/genéticaRESUMEN
MantATP [2'(3')-O-(-N-methylanthraniloyl)-adenosine 5'-triphosphate] was employed as a fluorescence probe of the nucleotide-binding sites of dynein from sea urchin sperm flagella. MantATP binds specifically with enhanced fluorescence (approximately 2.2-fold), homogeneous lifetime (8.4 ns), and high anisotropy (r approximately 0.38) to dynein and can be displaced by ATP and ADP added to the medium. The association constants of mantATP complexed with dynein were determined from anisotropy titration data. Using a multiple stepwise equilibrium model, the average values of the first two association constants are K1 = 2.7 x 10(5) M-1 and K2 = 1.8 x 10(4) M-1. This value of K1 is 7-8 times higher than that found previously for unsubstituted ATP, whereas K2 is little changed [Mocz and Gibbons (1996) Biochemistry 35, 9204-9211]. The lower-affinity binding sites, K3 and K4, observed previously could not be studied with mantATP within the available protein concentrations. The alpha and beta heavy chain subfractions have binding parameters similar to those of intact dynein. Formation of the stable ternary complex of mantATP with dynein and monomeric vanadate is accompanied by only a moderate increase in the binding affinities. Oligomeric vanadate reduces the binding affinities by approximately 50%. Addition of TritonX-100, methanol, or various salts changes the binding affinities by up to 50%, suggesting that the microenvironment of the nucleotide-binding sites involves significant contributions from both polar and apolar interactions. The distinct affinities of the individual binding sites are consistent with a physiological role in regulating nucleotide binding.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Dineínas/química , Colorantes Fluorescentes/química , ortoaminobenzoatos/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Marcadores de Afinidad/química , Marcadores de Afinidad/metabolismo , Animales , Sitios de Unión , Dineínas/metabolismo , Flagelos/enzimología , Polarización de Fluorescencia , Colorantes Fluorescentes/metabolismo , Masculino , Sales (Química) , Erizos de Mar , Solventes , Espermatozoides/enzimología , ortoaminobenzoatos/metabolismoRESUMEN
The role of dityrosine as a fluorescent crossbridge between adjacent calmodulin molecules within the high molecular mass polymers that are generated by Arthromyces peroxidase-catalyzed cross-linking [Malencik, D. A., and Anderson, S. R. (1996) Biochemistry 35, 4375] has been examined in frequency domain fluorescence anisotropy studies. Measurements on a polymer fraction possessing a range of molecular masses > 96 000 in NaDodSO4 polyacrylamide gel electrophoresis demonstrate predominating fast local rotations involving the dityrosyl moieties. Normal distribution analyses of the results show peak rotational correlation times of 0.6 ns (zero Ca2+) and 1.2 ns (+Ca2+), values that are smaller than the principal correlation times determined for the global rotation of the free calmodulin monomer in either the presence or absence of Ca2+. The intermolecularly cross-linked segments of the polymers retain a degree of the mobility that is characteristic of the tyrosine-containing sequences of native calmodulin. The half-widths of the normal distribution curves range from 13 ns (zero Ca2+) to approximately 90 ns (5 mM Ca2+), thus encompassing varying rates of segmental motion within the polymers. When Ca2+ is present, possible contributions from the global rotations of polymer molecules are detected near the operating limits of the method. Experiments with the intramolecularly cross-linked calmodulin monomer give global rotational correlation times of 7.9 ns (zero Ca2+) and 11.4 ns (+Ca2+), which compare to values of 7.2 ns and 9.9 ns found previously in time domain measurements [Small, E. W., and Anderson, S. R. (1988) Biochemistry 27, 419]. Rotations of apparent phi2 = 0.2 to 0.3 ns also are detected, accounting for 31% (-Ca2+) to 23% (+Ca2+) of the anisotropy.
Asunto(s)
Calmodulina/química , Reactivos de Enlaces Cruzados/química , Tirosina/análogos & derivados , Animales , Calmodulina/metabolismo , Bovinos , Reactivos de Enlaces Cruzados/metabolismo , Polarización de Fluorescencia , Hongos Mitospóricos , Peroxidasas/química , Peroxidasas/metabolismo , Polímeros/química , Polímeros/metabolismo , Estructura Terciaria de Proteína , Espectrofotometría , Tirosina/químicaRESUMEN
Human serum albumin (HSA) contains a single tryptophan residue at position 214. The emission properties of tryptophan 214 from recombinant albumins, namely, normal HSA, FDH-HSA and a methionine 218 HSA were examined. In all cases, the excited state lifetimes were best described by a two component model consisting mainly of a Lorentzian distribution. The centers of these distributions were 5.60 ns for HSA, 4.23 ns for FDH-HSA, and 6.08 ns for Met-218 HSA. The global rotational correlation times of the three HSAs were near 41 ns while the amplitude and rate of the local motion varied. These changes in the lifetimes and mobilities suggest perturbation in the local protein environment near tryptophan 214 as a consequence of the amino acid substitutions.
Asunto(s)
Mutagénesis Sitio-Dirigida , Albúmina Sérica/química , Albúmina Sérica/genética , Espectrometría de Fluorescencia , Polarización de Fluorescencia , Humanos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Albúmina Sérica/metabolismo , Triptófano/químicaRESUMEN
Elongation factor Tu from Escherichia coli is known to polymerize at slightly acidic pH and low ionic strength. The structure and dynamics of these aggregates have been examined using imaging and spectroscopic methodologies. Electron microscopy provides evidence for two-dimensional sheets and bundled filaments of EF-Tu, whereas fluorescence microscopy of EF-Tu covalently labeled with tetramethylrhodamine isothiocyanate showed highly branched polymers of EF-Tu several microns in diameter. These polymers were studied using quasi-elastic light scattering to determine the evolution of the translational diffusion coefficient during the polymerization process. The rotational dynamics of the aggregate were investigated using phosphorescence anisotropy of EF-Tu covalently labeled with erythrosin isothiocyanate. A high infinite-time anisotropy was observed, suggesting a lack of motion or entanglement of EF-Tu polymers. A sub-microsecond motion which was slowed in the presence of glycerol may be due to local flexibility of the polymers. The possible relevance of polymeric EF-Tu to its function in vivo is discussed.
Asunto(s)
Escherichia coli/química , Factor Tu de Elongación Peptídica/química , Polímeros/química , Anisotropía , Glicerol/farmacología , Concentración de Iones de Hidrógeno , Luz , Mediciones Luminiscentes , Microscopía Fluorescente , Factor Tu de Elongación Peptídica/aislamiento & purificación , Factor Tu de Elongación Peptídica/ultraestructura , Dispersión de RadiaciónRESUMEN
Elongation factor Tu (EF-Tu) from Escherichia coli is shown here to polymerize under conditions of low ionic strength and slightly acidic pH. Several factors, such as decreasing pH, decreasing ionic strength, increasing temperature, and increasing protein concentration (up to 2.5 microM) enhance the rate of polymerization. Both EF-Tu.GTP and EF-Tu.GDP polymerized equally well under the conditions studied. A lag time was observed between the lowering of the pH and the onset of measurable polymerization, which was not overcome by addition of preformed polymer "seeds." Finally, addition of unpolymerized EF-Tu.GDP to a solution of polymerized EF-Tu.GDP appears to lead to formation of new polymers instead of addition to preexisting ones, which may suggest a size limit for polymers of EF-Tu.GDP.