Structural and dynamic aspects of beta-glycosidase from mesophilic and thermophilic bacteria by multitryptophanyl emission decay studies

The tryptophanyl emission decay of beta-glycosidase from the extremophilic archaeon Sulfolobus solfataricus (Sbetagly) has been investigated by frequency domain fluorometry. The data were analyzed in terms of sum of discrete lifetimes as well as in terms of quasi- continuous lifetime distributions of different shape. At neutral pH the emission decay is characterized by two components: a long-lived component, centered at 7.4 ns, and a short one at 2.7 ns, irrespective of the decay scheme used for the interpretation of the experimental results. The effects of an irreversible inhibitor, that is, cyclophellitol, and that of a powerful denaturant such as guanidinium hydrochloride on the dynamics of Sbetagly has been investigated by observing the changes induced in the two components of the tryptophanyl emission decay. The addition of cyclophellitol to native Sbetagly reduces the contribution of the short-lived component but does not affect the long-lived one. Increasing concentrations of guanidinium hydrochloride differently affect the contributions of the two emission components. Higher concentrations were required to unfold the molecular regions containing the long-lived indolic fluorophores. These results indicate that the long-lived contribution arises from tryptophanyl residues deeply clustered in the interior of the protein matrix, whereas the short-lived one includes residues located in less rigid and more solvent accessible regions, some of which might be located in functionally important parts of protein. The knowledge of the crystallographic structure of Sbetagly allowed us to evaluate some average parameters for each tryptophanyl microenvironment in the Sbetagly such as hydrophobicity, structural flexibility, and ability of side chains to act as fluorescence quenchers. These results permitted to divide the tryptophanyl fluorescence of Sbetagly in the contribution of two emitting groups: one consisting of eight closely clustered tryptophans, that is, Trp 33, 36, 60, 84, 151 174, 425, and 433, responsible for the long-lived emission component and the other one, composed of nine tryptophans nearer to the subunit surface, that is, Trp 12, 156, 192, 287, 288, 316, 361, 376, 455, associable to the short-lived emission component. Finally, the examination of the tryptophanyl emission decay of the mesophilic beta-galactosidase from Escherichia coli (Cbetagal) and the Arrhenius analysis of its dependence on temperature indicated that the tryptophanyl environments of the mesophilic enzyme are rather homogeneous in consequence of a larger protein dynamics.     

Regulation of intracellular protein degradation in IMR-90 human diploid fibroblasts

Human diploid fibroblasts (IMR-90) regulate their overall rates of proteolysis in response to the composition of the culture medium and the ambient temperature. The magnitude and, in some cases, the direction of the response depend on the half-lives of the cellular proteins that are radioactively labeled and the time chosen for measurements of protein degradation. Fetal calf serum, insulin, fibroblast growth factor, epidermal growth factor, and amino acids selectively regulate catabolism of long-lived proteins without affecting degradation of short-lived proteins. Fetal calf serum reduces degradative rates of long-lived proteins and is maximally effective at a concentration of 20%, but the effect of serum on proteolysis is evident only for the first 24 hr. Insulin inhibits degradation of long-lived proteins in the presence or absence of glucose and amino acids in the medium, but is maximally effective only at high concentrations (10(-5) M). Amino acid deprivation increases degradative rates of long-lived proteins for the first 6 hr, but then decreases their catabolism for the subsequent 20 hr. Lowered temperature is the only condition tested that significantly alters degradative rates of short-lived proteins. Although cells incubated at 27 degrees C have reduced rates of degradation for both short-lived and long-lived proteins compared to cells at 37 degrees C, lowered temperature reduces catabolism of long-lived proteins to a greater extent.     

Fluorescence studies on the coat protein of Alfalfa Mosaic Virus

Abstract

The intrinsic luminescence of different forms of the alfalfa mosaic virus (AMV) strain 425 coat protein has been studied, both statically and time resolved. It was found that the emission of the protein (M_r24,250), which contains two tryptophans at positions 54 and 190 and four tyrosines, is completely dominated by tryptophan fluorescence. The high fluorescence quantum yield indicates that both tryptophans are emitting. Surprisingly, the fluorescence decay is found to be strictly exponential, with a lifetime of 5.1 nsec. Similar results were obtained for various other forms of the protein, i.e. the 30-S polymer, the mildly trypsinised forms of the protein lacking the N-terminal part and the protein assembled into viral particles. Virus particles and proteins of stains S and VRU gave similar results, as well as the VRU protein polymerised into tubular structures. The fluorescence decay is also monoexponential in the presence of various concentrations of the quenching molecules acrylamide and potassium iodide. Stern-Volmer plots were linear and yield for the coat protein dimer with acrylamide a quenching constant of 4.5 ^* 10⁸ M^?1sec^?1. This indicates that the tryptophans are moderately accessible for acrylamide. For the 30-S polymer a somewhat smaller value was found, whereas in the viral Top a particles the accessibility of the tryptophans is still further reduced. From the decay of the polarisation anisotropy of the fluorescence of the coat protein dimer the rotational correlation time was obtained as 35 nsec. Since this roughly equals the expected rotational correlation time of the dimer as a whole, it suggests that the tryptophans are contained rigidly in the dimer.

The results show that in the excited state of the protein the two tryptophans are strongly coupled and suggest that the trp-trp distance is smaller than 10 A. Because the coat protein occurs as a dimer, the coupling can be inter- or intramolecular. The implications for the viral structure are discussed.     

Long-lived tryptophan fluorescence in phosphoglycerate mutase

Phosphoglycerate mutase (PGM; EC 2.7.5.3) isolated from rat and rabbit muscle has been shown to possess an unusually long-lived fluorescence component when excited by ultraviolet light below 310 nm. On the basis of spectral and physical measurements, this 16.4 (+/- 0.2) ns fluorescence lifetime at room temperature is assigned to a tryptophan residue in an unusual environment. The emission profile of this long-lived tryptophan is red shifted from the other tryptophans of PGM by approximately 25 nm. PGM has been crystallized and sequenced from yeast where it has been shown to be a tetramer with 29K subunits. However, we have not been able to detect the existence of an unusually long-lived fluorescence component in the yeast isomer. The long fluorescence lifetime is lost upon denaturation of rabbit PGM and is partially restored upon introduction of the protein to a nondenaturing environment, suggesting the long lifetime is not the result of a covalent modification. The PGM molecule was studied by a number of techniques including time-resolved tryptophan fluorescence, quenching studies of tryptophan fluorescence, and enzyme activity studies. The long-lived fluorescence has been shown to be statistically quenched by Br-, I-, and Cu2+ in the submillimolar region while the acrylamide quenching shows the tryptophan is marginally accessible to solvent. Characterization of the long-lived fluorescence and its possible sources are discussed.     

Temperature dependence of solute transport and enzyme activities in hog renal brush border membrane vesicles

The temperature dependence of sodium-dependent and sodium-independent d-glucose and phosphate uptake by renal brush border membrane vesicles has been studied under tracer exchange conditions. For sodium-dependent d-glucose and phosphate uptake, discontinuities in the Arrhenius plot were observed. The apparent activation energy for both processes increased at least 4-fold with decreasing temperature. The most striking change in the slope of the Arrhenius plot occurred between 12 and 15°C. The sodium-independent uptake of d-glucose and phosphate showed a linear Arrhenius plot over the temperature range tested (35–5°C). The behavior of the transport processes was compared to the temperature dependence of typical brush border membrane enzymes. Alkaline phosphatase as intrinsic membrane protein showed a nonlinear Arrhenius plot with a transition temperature at 12.4°C. Aminopeptidase M, an extrinsic membrane protein exhibited a linear Arrhenius plot. These data indicate that the sodium-glucose and sodium-phosphate cotransport systems are intrinsic brush border membrane proteins, and that a change in membrane organization alters the activity of a variety of intrinsic membrane proteins simultaneously.     

Effect of Ca2+-Mg2+ exchange on the flexibility and/or conformation of the small domain in monomeric actin.

A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F(DA)), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature over the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26 degrees C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26 degrees C that was not detected in the case of Mg-G-actin. In the temperature range between 6 degrees C and 26 degrees C the slope of the temperature profile of f' is the same for Ca-G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin.     

Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence studies on the coat protein of alfalfa mosaic virus

The intrinsic luminescence of different forms of the alfalfa mosaic virus (AMV) strain 425 coat protein has been studied, both statically and time resolved. It was found that the emission of the protein (Mr 24,250), which contains two tryptophans at positions 54 and 190 and four tyrosines, is completely dominated by tryptophan fluorescence. The high fluorescence quantum yield indicates that both tryptophans are emitting. Surprisingly, the fluorescence decay is found to be strictly exponential, with a lifetime of 5.1 nsec. Similar results were obtained for various other forms of the protein, i.e. the 30-S polymer, the mildly trypsinized forms of the protein lacking the N-terminal part and the protein assembled into viral particles. Virus particles and proteins of stains S and VRU gave similar results, as well as the VRU protein polymerised into tubular structures. The fluorescence decay is also monoexponential in the presence of various concentrations of the quenching molecules acrylamide and potassium iodide. Stern-Volmer plots were linear and yield for the coat protein dimer with acrylamide a quenching constant of 4.5* 10(8) M-1 sec-1. This indicates that the tryptophans are moderately accessible for acrylamide. For the 30-S polymer a somewhat smaller value was found, whereas in the viral Top a particles the accessibility of the tryptophans is still further reduced. From the decay of the polarisation anisotropy of the fluorescence of the coat protein dimer the rotational correlation time was obtained as 35 nsec. Since this roughly equals the expected rotational correlation time of the dimer as a whole, it suggests that the tryptophans are contained rigidly in the dimer. The results show that in the excited state of the protein the two tryptophans are strongly coupled and suggest that the trp-trp distance is smaller than 10 A. Because the coat protein occurs as a dimer, the coupling can be inter- or intramolecular. The implications for the viral structure are discussed.     

Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20–28% of the initial fluorescence with (K_SV+V)0.55 M^–1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka=5 M^–1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weberet al., 1989).     

Temperature-dependencies of various catalytic activities of membrane-bound Na+/K+-ATPase from ox brain, ox kidney and shark rectal gland and of C12E8-solubilized shark Na+/K+-ATPase

The temperature dependence of ouabain-sensitive ATPase and phosphatase activities of membrane fragments containing the Na+/K+-ATPase were investigated in tissue from ox kidney, ox brain and from shark rectal glands. The shark enzyme was also tested in solubilized form. Arrhenius plots of the Na+/K+-ATPase activity seem to be linear up to about 20 degrees C, and non-linear above this temperature. The Arrhenius plots of mammalian enzyme (ox brain and kidney) were steeper, especially at temperatures below 20-30 degrees C, than that of shark enzyme. The Na+-ATPase activity showed a weaker temperature-dependence than the Na+/K+-ATPase activity. The phosphatase reactions measured, K+-stimulated, Na+/K+-stimulated and Na+/K+/ATP-stimulated, also showed a weaker temperature-dependence than the overall Na+/K+-ATPase activity. Among the phosphatase reactions, the largest change in slope of the Arrhenius plot was observed with the Na+/K+/ATP)-stimulated phosphatase reaction. The Arrhenius plots of the partial reactions were all non-linear. Solubilization of shark enzyme in C12E8 did not change the curvature of Arrhenius plots of the Na+/K+-ATPase activity or the K+-phosphatase activity. Since solubilization involves a disruption of the membrane and an 80% delipidation, the observed curvature of the Arrhenius plot can not be attributed to a property of the membrane as such.     

Hyperthermia stimulates energy-proteasome-dependent protein degradation in cultured myotubes

Previous studies suggest that elevated temperature stimulates protein degradation in skeletal muscle, but the intracellular mechanisms are not fully understood. We tested the role of different proteolytic pathways in temperature-dependent degradation of long- and short-lived proteins in cultured L6 myotubes. When cells were cultured at different temperatures from 37 to 43 degrees C, the degradation of both classes of proteins increased, with a maximal effect noted at 41 degrees C. The effect of high temperature was more pronounced on long-lived than on short-lived protein degradation. By using blockers of individual proteolytic pathways, we found evidence that the increased degradation of both long-lived and short-lived proteins at high temperature was independent of lysosomal and calcium-mediated mechanisms but reflected energy-proteasome-dependent degradation. mRNA levels for enzymes and other components of different proteolytic pathways were not influenced by high temperature. The results suggest that hyperthermia stimulates the degradation of muscle proteins and that this effect of temperature is regulated by similar mechanisms for short- and long-lived proteins. Elevated temperature may contribute to the catabolic response in skeletal muscle typically seen in sepsis and severe infection.     

Importance of membrane fluidity in the induction of alkaline phosphatase,a periplasmic enzyme,in Escherichiacoli

An unsaturated fatty acid auxotroph was supplemented with either elaidate or oleate. After derepression of alkaline phosphatase by phosphate limitation at 38°C, the cells were shifted to incubation at various temperatures. Arrhenius plots of the rate of enzyme induction gave a steeper negative slope in the temperature range from 30°C to 35°C with elaidate-supplemented cells than with oleate-supplemented cells. At 25°C the induction was arrested in the former cells, while it was continued at a considerable rate in the latter. The arrest was released upon shift-back to 38°C, and precursors convertible to the active enzyme were not accumulated during incubation at 25°C. There was no marked difference in slope of Arrhenius plots of the rate of bulk protein synthesis between both types of cells, and the slope was almost equal to that of the rate of enzyme induction in the oleate-supplemented cells. The rate of β-galactosidase induction in the elaidate cells showed a similar temperature dependence to that of bulk protein synthesis.     

Surface-modified mutants of cytochrome P450cam: enzymatic properties and electrochemistry.

We report the electrochemistry of genetic variants of the haem monooxygenase cytochrome P450cam. A surface cysteine-free mutant (abbreviated as SCF) was prepared in which the five surface cysteine residues Cys-58, Cys-85, Cys-136, Cys-148 and Cys-334 were changed to alanines. Four single surface cysteine mutants with an additional mutation, R72C, R112C, K344C or R364C, were also prepared. The haem spin-state equilibria, NADH turnover rates and camphor-hydroxylation properties, as well as the electrochemistry of these mutants are reported. The coupling of a redox-active label, N-ferrocenylmaleimide, to the single surface cysteine mutant SCF-K344C, and the electrochemistry of this modified mutant are also described.     

Temperature and Ca(2+)-dependence of the sarcoplasmic reticulum Ca(2+)-ATPase in haddock,salmon, rainbow trout and zebra cichlid

Temperature dependence of Ca(2+)-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degrees C. Whether the break point arises as a result of temperature dependent changes in the enzyme or its membrane lipid environment is still a matter of discussion. In this study we compared the temperature dependence and Ca(2+)-dependence of SR Ca(2+)-ATPase in haddock (Melanogrammus aeglefinus), salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and zebra cichlid (Cichlasoma nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degrees C, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the temperature area, 6-14 degrees C. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca(2+)-ATPase activity. The temperature range of the plateau was 14-21 and 18-25 degrees C in salmon and rainbow trout, respectively. Ca(2+)-dependence in the four different fish species investigated was very similar with half maximal activation (K(0.5)) between 0.2 and 0.6 micro M and half maximal inhibition (I(0.5)) between 60 and 250 micro M. Results indicated that interaction between SR Ca(2+)-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated.     

Temperature dependence of dielectric relaxations in alpha-elastin coacervate: evidence for a peptide librational mode

The dielectric permittivity of alpha-elastin coacervate is reported over the frequency range of 1 MHz to 1000 MHz and the temperature dependence from 6.8 degrees C to 70 degrees C is also reported. A temperature-dependent simple Debye-type relaxation is observed with a correlation time of 8 nsec (40 degrees C) which is similar to that of the polypentapeptide of elastin (i.e. 7 nsec at 40 degrees C) where the band has been assigned to a peptide librational mode. By analogy this allows for the first assignment of a peptide librational mode in a naturally occurring polypeptide or protein. The strong spectrally localized band indicates a regularity of structure. The low temperature dependence of the correlation time, giving a 1.7 kcal/mole enthalpy of activation, is consistent with torsional motions associated with a peptide librational mode.     

Spectroscopic observations of ferric enterobactin transport

We characterized the uptake of ferric enterobactin (FeEnt), the native Escherichia coli ferric siderophore, through its cognate outer membrane receptor protein, FepA, using a site-directed fluorescence methodology. The experiments first defined locations in FepA that were accessible to covalent modification with fluorescein maleimide (FM) in vivo; among 10 sites that we tested by substituting single Cys residues, FM labeled W101C, S271C, F329C, and S397C, and all these exist within surface-exposed loops of the outer membrane protein. FeEnt normally adsorbed to the fluoresceinated S271C and S397C mutant FepA proteins in vivo, which we observed as quenching of fluorescence intensity, but the ferric siderophore did not bind to the FM-modified derivatives of W101C or F329C. These in vivo fluorescence determinations showed, for the first time, consistency with radioisotopic measurements of the affinity of the FeEnt-FepA interaction; K(d) was 0.2 nm by both methods. Analysis of the FepA mutants with AlexaFluor(680), a fluorescein derivative with red-shifted absorption and emission spectra that do not overlap the absorbance spectrum of FeEnt, refuted the possibility that the fluorescence quenching resulted from resonance energy transfer. These and other data instead indicated that the quenching originated from changes in the environment of the fluor as a result of loop conformational changes during ligand binding and transport. We used the fluorescence system to monitor FeEnt uptake by live bacteria and determined its dependence on ligand concentration, temperature, pH, and carbon sources and its susceptibility to inhibition by the metabolic poisons. Unlike cyanocobalamin transport through the outer membrane, FeEnt uptake was sensitive to inhibitors of electron transport and phosphorylation, in addition to its sensitivity to proton motive force depletion.     

Functional interactions of lipids and proteins in rat intestinal microvillus membranes.

Interactions of lipids and proteins in isolated rat intestinal microvillus membranes were examined by studying the temperature dependence of enzyme activities and of D-glucose transport in relation to the membrane lipid thermotropic transition observed by fluorescence polarization (26 +/- 2 degrees C) and differential scanning calorimetry (23--39 degrees C). Two groups of activities were defined. Enzymes of the first group, comprising lactase, maltase, sucrase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, all yielded a single slope on the Arrhenius plot in the range 10--40 degrees C and did not appear to experience functionally the effects of the lipid thermotropic transition. Each activity of the second group, comprising calcium- and magnesium-dependent adenosine triphosphatases, p-nitrophenylphosphatase, and D-glucose transport, showed a change in the slope of the Arrhenius plot in the range 25--30 degrees C, corresponding to the lower region of the lipid transition. The terms "extrinsic" and "intrinsic" activities could be applied to these groups. Delipidation of the particulate p-nitrophenylphosphatase removed the discontinuity in the Arrhenius plot. Subsequent relipidation with a variety of lipids restored a break point, but the temperature corresponded to the original discontinuity (25--29 degrees C) rather than to the phase transition temperature of the exogenous lipid added.     

Does fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole incorporated into sarcoplasmic reticulum ATPase monitor putative E1-E2 conformational transition?

When a purified preparation of sarcoplasmic reticulum Ca2(+)-ATPase was labeled with 0.3 mM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in 1 mM AMPPNP and 1 mM CaCl2 at 25 degrees C and pH 7.0 for 60 min and then was treated with 10 mM dithiothreitol for 7 min, about 1 mol of NBD was incorporated per mol of the enzyme, and this inhibited the enzyme activity by 90 to 95%. The modified residue was identified as Cys-344 that is located near the phosphorylation site of the ATPase, Asp-351. The NBD-inhibition of enzyme activity could be reversed by treatment with membrane-acting agents such as C12E8, suggesting that Cys-344 is not directly involved in enzyme catalysis. A detailed study of partial reactions of ATP hydrolysis by the modified enzyme and associated changes in the fluorescence intensity of the incorporated NBD label revealed that a predominant effect of the NBD-modification was the inhibition of Ca2+ release from the ADP-sensitive phosphoenzyme intermediate and that two major fluorescent states of the enzyme alternated during ATP hydrolysis. The latter fluorescent data are consistent with the E1-E2 model of Ca2(+)-ATPase reaction.     

Coupled responses of the regions near cysteine-190 and the carboxy terminus of rabbit cardiac tropomyosin: fluorescence and circular dichroism studies

Rabbit cardiac tropomyosin was labeled at Cys-190 with either N-(1-pyrenyl)iodoacetamide (Py) or 6-acryloyl-2-(dimethylamino)naphthalene (AD, acrylodan). Half of the labeled sample then was treated with carboxypeptidase A to produce an identically labeled nonpolymerizable form of tropomyosin, NPTM. Investigation of temperature-dependent changes in pyrene excimer emission, acrylodan fluorescence polarization, and tyrosyl circular dichroism in different samples of tropomyosin and NPTM reveals that absence of the COOH-terminal portion of tropomyosin modifies the response of the Cys-190 region to heat. Removal of the COOH terminus releases certain conformational constraints from the coiled-coil back to and including the Cys-190 region without causing a severe drop in the net alpha-helical content of the protein. Observation of changes in pyrene excimer fluorescence and in fluorescence polarization of acrylodan with time after addition of carboxypeptidase A to samples of labeled tropomyosin directly demonstrates this relaxation process. Thermally induced reduction in tyrosyl circular dichroism, together with consideration of the distribution of tyrosyl residues on tropomyosin, also supports the proposal.     

Saturation-transfer electron spin resonance studies on the mobility of spin-labeled sodium and potassium ion activated adenosinetriphosphatase in membranes from Squalus acanthias

The sodium and potassium ion activated adenosinetriphosphatase [(Na+,K+)-ATPase] in membranous preparations from Squalus acanthias has been spin-labeled on sulfhydryl groups after prelabeling with N-ethylmaleimide. Saturation-transfer electron spin resonance spectroscopy has been used to study the rotational motions of the labeled protein on the microsecond time scale. Effective rotational correlation times deduced from the diagnostic line-height ratios in the second-harmonic, 90 degrees out-of-phase (V2') spectra are much larger than those deduced from the spectral integrals, indicating the presence of large-scale segmental motions, in addition to rotation of the protein as a whole. Experiments involving controlled cross-linking of the protein by glutaraldehyde, as well as measurements of the line broadening of the conventional electron spin resonance spectra, support this interpretation. Both the spectral integrals and diagnostic line-height ratios are found to increase irreversibly with time on incubation at temperatures greater than 20 degrees C, corresponding to a decrease in the segmental motion of the protein and probably also in the overall protein rotation. The native enzyme displays a marked nonlinearity in the Arrhenius temperature dependence of the activity at temperatures above 20 degrees C, and the activity decreases with a half-life of ca. 70 min on incubation at 37 degrees C (but not on incubation at low temperature), paralleling the time- and temperature-dependent changes in the saturation-transfer spectra of the labeled protein. Both of these observations suggest that the changes observed in the molecular dynamics could correspond to functional properties of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)