Slow internal dynamics of membrane proteins in mechanisms of protease-induced aggregation of platelets

Room temperature tryptophan phosphorescence (RTTP) of suspensions of human platelets was studied. RTTP spectra and decay kinetics of both intact platelets and those after short-term incubation with low concentrations of thrombin or trypsin (0.3-50 microg/ml) were investigated. Protease-induced changes in the RTTP lifetime of platelets were observed, and interpreted in terms of the slow internal dynamics of membrane protein modification. The functional role of membrane protein internal dynamics is discussed in the context of platelet aggregation and signal transduction processes.     

The effect of oxidative stress induced by t-butyl hydroperoxide on the structural dynamics of membrane proteins of Chinese hamster fibroblasts

A method based on the measurement at room temperature of tryptophan phosphorescence (RTTP) gives the unique possibility to investigate the dynamic structure of membrane proteins without their isolation from cells. This method was used to study the influence of tert- butyl hydroperoxide (t -BHP) on Chinese hamster fibroblasts. The treatment of fibroblasts with t -BHP in a concentration range of 0. 5-2 m m for 60 min caused an increase of frequency and amplitude of membrane protein motions with lifetimes of hundreds miliseconds (a decrease of RTTP tau(2)). In parallel, cell viability was studied by trypan blue exclusion test and the content of thiobarbituric acid reactive substances was measured in cells. The dependences of the RTTP tau(2)and cell viability on t -BHP concentration were similar. Contrary to this, t -BHP did not induce the activation of lipid peroxidation processes in cells. This indicates that cell death is connected with the excessive increase of intramolecular dynamics of membrane proteins during t -BHP action.     

Study of the equilibrium dynamics of cell protein structure using the tryptophan fluorescence method at room temperature

Room-temperature tryptophane phosphorescence (RTTP) of liver tissue cells has been studied. It is shown that over a millisecond range RTTP is absent in soluble proteins of the cytoplasm, karyoplasm, mitochondrial matrix, and the phosphorescent signal is controlled only by proteins of the subcellular structures incorporated into the membranes. It is concluded that, unlike the membrane proteins, the cytoplasm and organelle matrix-soluble proteins are characterized by a high level of intramolecular equilibrium mobility, which causes RTTP quenching following a dynamic mechanism. In membrane proteins, which fluoresce in a millisecond range the level of equilibrium conformation motions is limited, probably, due to protein-protein and protein-lipid interactions.     

Kinetics of inactivation of lectin: analysis of a method of tryptophan phosphorescence at room temperature

The internal dynamics of muscle actin during inactivation induced by guanidine hydrochloride (0.5-1.8 M) was studied by the method of room-temperature tryptophan phosphorescence (RTTP). It was shown that the essentially unfolded actin intermediate, which appears within the first minutes of incubation with guanidine hydrochloride, exhibits no RTTP, suggesting a high lability of its structure. Subsequent accumulation of associates of inactivated actin is accompanied by a significant increase in the intensity and decay time of RTTP, which is caused by the rigidity of the structure of inactivated actin. The kinetic dependencies of the intensity and lifetime of RTTP of actin during its inactivation depended on the concentration of the protein and guanidine hydrochloride.     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at approximately 14 degrees C, whereas, freeze-dried platelets exhibited a main phase transition approximately 12 degrees C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42 degrees C, whereas proteins in the rehydrated platelets denatured at 48 degrees C.     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at ~14°C, whereas, freeze-dried platelets exhibited a main phase transition ~12°C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42°C, whereas proteins in the rehydrated platelets denatured at 48°C.     

Effect of the antioxidant ionol on proteolytic apparatus of aging coleoptiles of wheat seedlings grown in light

The dynamics of changes in total proteolytic activity and activities of various groups of proteases in the coleoptiles of 3- to 12-day-old wheat seedlings grown in light with and without antioxidant BHT (2,6-di-tert-butyl-4-methylphenol) was studied. It was established that the specialized proteases that easily hydrolyze specific synthetic substrates and the enzymes actively hydrolyzing histone H1 dominate in young coleoptiles of 3- to 4-day-old seedlings. Proteases that degrade equally well the majority of the studied substrates are accumulated in the cells of old coleoptiles of 11- to 12-day-old seedlings. Under the effect of BHT, the plants grown in light (in comparison with etiolated seedlings) demonstrated a somewhat changed dynamics of proteolytic activity in young coleoptiles and the disappearance of proteases active toward histone H1. An inhibitory analysis revealed a relative domination of cysteine proteases in young coleoptiles at the initial development stage of seedlings, whereas the fraction of serine proteases markedly increased in old coleoptiles. We presume that the revealed quantitative and qualitative changes in the proteolytic apparatus of the coleoptile cells induced by BHT may be largely responsible for the retardant and geroprotective effect of this antioxidant in plants.     

Effect of Antioxidant BHT on the Proteolytic Apparatus of Aging Coleoptiles of Wheat Seedlings Grown in Light

The dynamics of changes in total proteolytic activity and activities of various groups of proteases in the coleoptiles of 3- to 12-day-old wheat seedlings grown in light with and without antioxidant BHT (2,6-di-tert-butyl-4-methylphenol) was studied. It was established that the specialized proteases that easily hydrolyze specific synthetic substrates and the enzymes actively hydrolyzing histone H1 dominate in young coleoptiles of 3- to 4-day-old seedlings. Proteases that degrade equally well the majority of the studied substrates are accumulated in the cells of old coleoptiles of 11- to 12-day-old seedlings. Under the effect of BHT, the plants grown in the light (in comparison with etiolated seedlings) demonstrated a somewhat changed dynamics of proteolytic activity in young coleoptiles and the disappearance of proteases active toward histone H1. An inhibitory analysis revealed a relative domination of cysteine proteases in young coleoptiles at the initial development stage of seedlings, whereas the fraction of serine proteases markedly increased in old coleoptiles. We presume that the revealed quantitative and qualitative changes in the proteolytic apparatus of the coleoptile cells induced by BHT may be largely responsible for the retardant and geroprotective effect of this antioxidant in plants.     

Phosphorescent analysis of the action of detergents on the internal dynamics of membrane proteins of human erythrocytes

The slow (millisecond) internal dynamics of proteins isolated from human erythrocyte membranes under the action of ionic and nonionic detergents: sodium dodecyl sulfate (0.1-6 mM), sodium deoxycholate (0.16-6 MM), N-Lauroylsarcosine Na+-salt (Sarkosyl) (0.17-6 mM), digitonin (0.025-6 MM), and Tween-20 (0.01-6 mM) has been studied by the method of room-temperature tryptophan phosphorescence. It has been established that the destruction of protein ensembles, the disturbance of protein-lipid interactions, and the unfolding of proteins in membrane result in a considerable increase of slow intramolecular dynamics of proteins. The effects of detergents on the structural and dynamical state of membrane proteins differ depending on their chemical features. On the bases of the results obtained, it has been concluded that the low internal dynamics of membrane proteins in situ, compared with most soluble proteins, is due to the presence of protein ensembles in membrane, the isolation of macromolecules from the aqueous surroundings by the lipid bilayer, and a high content of alpha-helices and beta-sheets in macromolecules.     

Changes in the distribution of platelet membrane proteins revealed by energy transfer

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.     

Na+-Ca2+ exchange in the axolemma-rich membrane vesicle preparations from the walking-leg nerves of the American lobster

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.     

Tryptophan phosphorescence study of the influence of detergents on the internal dynamics of human erythrocyte membrane proteins

Room-temperature tryptophan phosphorescence was used to assess the slow (millisecond) internal dynamics of proteins in isolated human erythrocyte membranes under the action of detergents: dodecylsulfate, lauroyl sarcosinate, deoxycholate, digitonin, and Tween 20 (concentrations varied from 0.01 to 6 mM). All detergents markedly enhanced the slow internal dynamics, but the dose-response patterns were specific for each agent. The aggregate data support the idea that the slow internal dynamics is restricted in membrane proteins relative to soluble proteins mostly because of intramembrane protein association and isolation from the aqueous milieu, with a possible contribution of a more rigid secondary structure.     

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.     

Intramolecular dynamics of human erythrocyte membrane proteins upon modification of spectrin by tryptophan phosphorescence

The slow (millisecond) protein internal dynamics of isolated human erythrocyte membranes in suspension without treatment, after deleting 95% of spectrin, after spectrin thermal denaturation upon acidification of medium in the pH range 6.0-4.0, and spectrin extracted in solution from membranes has been studied by room-temperature tryptophan phosphorescence. It has been established that integral proteins and spectrin differ in structural and dynamic state. Millisecond movements of structural elements of integral proteins are more restricted compared with those of spectrin. The removal of spectrin from the membrane led to an increase in slow fluctuations of integral protein structure. This indicates that spectrin participates in the control of the structural and dynamic state of erythrocyte membrane proteins. As medium was acidified in the pH range 6.0-4.0, the protein slow internal dynamics of membranes in native state decreased, which was explained by spectrin pH aggregation. After thermal denaturation of spectrin, no pH-induced increase of membrane protein structure rigidity was observed.     

Exploring the role of structure and dynamics in the function of chymotrypsin inhibitor 2

Increasing awareness of the possible role of internal dynamics in protein function has led to the development of new methods for experimentally characterizing protein dynamics across multiple time scales, especially using NMR spectroscopy. A few analyses of the conformational dynamics of proteins ranging from nonallosteric single domains to multidomain allosteric enzymes are now available; however, demonstrating a connection between dynamics and function remains difficult on account of the comparative lack of studies examining both changes in dynamics and changes in function in response to the same perturbations. In previous work, we characterized changes in structure and dynamics on the ps–ns time scale resulting from hydrophobic core mutations in chymotrypsin inhibitor 2 and found that there are moderate, persistent global changes in dynamics in the absence of gross structural changes (Whitley et al., Biochemistry 2008;47:8566–8576). Here, we assay those and additional mutants for inhibitory ability toward the serine proteases elastase and chymotrypsin to determine the effects of mutation on function. Results indicate that core mutation has only a subtle effect on CI2 function. Using chemical shifts, we also studied the effect of complex formation on CI2 structure and found that perturbations are greatest at the complex interface but also propagate toward CI2's hydrophobic core. The structure–dynamics–function data set completed here suggests that dynamics plays a limited role in the function of this small model system, although we do observe a correlation between nanosecond-scale reactive loop motions and inhibitory ability for mutations at one key position in the hydrophobic core.     

Influence of dietary fatty acids on membrane fluidity and activation of rat platelets

The apparent steady-state fluorescence anisotropy of DPH- or TMA-DPH-labeled washed rat platelets is strongly affected by factors that also influence the turbidity by these platelet suspensions. Sonicated preparations from platelet lipids have a low turbidity and give anisotropy values which are hardly affected by the experimental conditions. We studied the effect of four high-fat diets on membrane fluidity, lipid composition and activation tendency of washed platelets. The diets contained 50 energy% of oils with different levels of saturated and (poly)unsaturated fatty acids. Only small diet-induced differences in DPH fluorescence anisotropy were found, which were comparable for intact platelets and platelet lipids. These differences were unrelated to the degree of saturation of the dietary fatty acids. Platelets from rats fed mainly saturated fatty acids differed significantly from other diet groups in a higher unsaturation degree of phospholipids and a lower cholesterol/phospholipid ratio, but this was not detected by DPH in terms of decreased anisotropy. These platelets aggregated less than other platelets in response to thrombin or collagen. The lower response to collagen persisted in indomethacin-treated platelets activated with the thromboxane A2 mimetic U46619, indicating a different sensitivity of these platelets for thromboxane A2. We conclude that in rat platelets: (a) the overall membrane fluidity and phospholipid unsaturation degree are subject to strong homeostatic control; (b) steady-state anisotropy with DPH or TMA-DPH label is inadequate to reveal subtile changes in lipid profile; (c) changes in platelet responsiveness to thrombin and thromboxane A2, rather than (plasma) membrane fluidity, determine the effect of dietary fatty acids on platelet aggregation.     

Mobility modulation by local concanavalin A binding. Selectivity toward different membrane proteins

We have employed fluorescence photobleaching recovery to demonstrate selective immobilization of lymphocyte membrane proteins by localized concanavalin A (ConA) binding to the cell surface. Localized ConA binding was achieved by the binding of ConA coupled to paraformaldehyde-fixed platelets to mouse spleen lymphocytes. The effect of the localized cross-linking of ConA receptors on the lateral mobility of specific membrane proteins at regions distal to the ConA platelets was investigated. The diffusion of surface immunoglobulins and ConA receptors was inhibited above a threshold coverage (12%) of the upper lymphocyte surface by ConA platelets. In contrast, no effect was observed on the diffusion and aggregation of mouse histocompatibility antigens (H-2Kk) labeled with a fluorescent monoclonal antibody. Since the ConA modulation was shown to propagate through the cytoskeleton, these results indicate specificity in the interactions of membrane proteins with the cytoskeleton. This specificity enables a selective response of different membrane proteins to the ConA anchorage modulation.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

Plasma-coagulating and fibrinolytic activities of fungi in the genus Aspergillus

The exocellular plasmocoagulating and fibrinolytic activities were studied in 100 cultures of the Aspergillus genus belonging to 29 species during their submerged cultivation in three media. It has been found that 38 cultures can coagulate human plasma, 75 cultures can cause lysis of fibrin platelets, and 22 cultures are capable of dissolving standard plasma clots within 6 hours. The cultures synthesize three types of proteolytic enzymes according to the specificity toward blood proteins: (1) proteases with the predominant fibrinolytic action; (2) proteases which possess both the fibrinolytic and plasmocoagulating activities; (3) proteases manifesting only the plasmocoagulating action. A. ochraceus 19 producing individual plasmocoagulases and fibrinolytic enzymes at a high rate was isolated. The composition of the enzyme complex synthesized by the culture depended on the composition of the medium and on the cultivation conditions.     

A study of the Interaction Between Cetirizine and Plasma Membrane of Eosinophils, Neutrophils, Platelets and Lymphocytes using A fluorescence Technique

The effect of cetirizine on plasma membrane fluidity and heterogeneity of human eosinophils, neutrophils, platelets and lymphocytes was investigated using a fluorescence technique. Membrane fluidity and heterogeneity were studied by measuring the steady-state fluorescence anisotropy and fluorescence decay of 1-(4- trimethylammonium-phenyl)-6-phenyl-1, 3, 5-hexatriene (TMA-DPH) incorporated in the membrane. The results demonstrate that cetirizine (1 mug/ml) induced a significant increase in the Hpid order in the exterior part of the membrane and a decrease in membrane heterogeneity in eosinophils, neutrophils and platelets. Moreover, cetirizine blocked the PAF induced changes in membrane fluidity in these cells. Cetirizine did not influence significantly the plasma membrane of lymphocytes. These data may partially explain the effect ofcetirizine on inflammatory cell activities.