Multilamellarity, structure and hydration of extruded POPC vesicles by SANS

The small-angle neutron scattering (SANS) data of 12 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) dispersions at low lipid concentration (1 mg per 100-mg heavy water) prepared by 5, 9 and 29 extrusions through filters of pores with 50, 100, 200 and 400 nm diameter are presented. They were analyzed within a theory that permits the determination of both structural and hydration parameters of the bilayers as well as the portions of multilamellar vesicles in dispersions with negligible long-range order between the vesicles. The scattering length density profile across the bilayers is approximated by assuming a central hydrocarbon core surrounded by a water-accessible coat. It is modeled by two different forms of functions. In the boat model, the scattering length density of the coat changes linearly from core to water, whereas in the strip model it is constant across the water-accessible coat. It was found that the boat model reflects the reality better than the strip model. The decrease of the multilamellar vesicle portions, either with increasing the number of extrusions at same filter size and with decreasing the filter size, was characterized quantitatively.     

Hydration effects of heparin on antithrombin probed by osmotic stress.

Antithrombin is a key inhibitor of blood coagulation proteases and a prototype metastable protein. Heparin binding to antithrombin induces conformational transitions distal to the binding site. We applied osmotic stress techniques and rate measurements in the stopped flow fluorometer to investigate the possibility that hydration changes are associated with these transitions. Water transfer was identified from changes in the free energy of activation, Delta G(++), with osmotic pressure pi. The Delta G(++) was determined from the rate of fluorescence enhancement/decrease associated with heparin binding/release. The volume of water transferred, Delta V, was determined from the relationship, Delta G/pi = Delta V. With an osmotic probe of 4 A radius, the volumes transferred correspond to 158 +/- 11 water molecules from reactants to bulk during association and 162 +/- 22 from bulk to reactants during dissociation. Analytical characterization of water-permeable volumes in x-ray-derived bound and free antithrombin structures were correlated with the volumes measured in solution. Volume changes in water permeable pockets were identified at the loop-insertion and heparin-binding regions. Analyses of the pockets' atomic composition indicate that residues Ser-79, Ala-86, Val-214, Leu-215, Asn-217, Ile-219, and Thr-218 contribute atoms to both the heparin-binding pockets and to the loop-insertion region. These results demonstrate that the increases and decreases in the intrinsic fluorescence of antithrombin during heparin binding and release are linked to dehydration and hydration reactions, respectively. Together with the structural analyses, results also suggest a direct mechanism linking heparin binding/release to loop expulsion/insertion.     

Protein folding transition states probed by loop extension

We propose a new way to characterize protein folding transition states by (1) insertion of one or more residues into an unstructured protein loop, (2) measurement of the effect on protein folding kinetics and thermodynamics, and (3) analysis of the results in terms of a rate-equilibrium free energy relationship, alpha(Loop). alpha(Loop) reports on the fraction of molecules that form the perturbed loop in the transition state. Interpretation of the changes in equilibrium free energy using standard polymer theory can help detect residual structure in the unfolded state. We illustrate our approach with data for the model proteins CI2 and the alpha spectrin SH3 domain.     

Effect of osmotic stress on live cell plasma membranes,probed via Laurdan general polarization measurements

Here we seek to gain insight into changes in the plasma membrane of live cells upon the application of osmotic stress using Laurdan, a fluorescent probe that reports on membrane organization, hydration, and dynamics. It is known that the application of osmotic stress to lipid vesicles causes a decrease in Laurdan’s generalized polarization (GP), which has been interpreted as an indication of membrane stretching. In cells, we see the opposite effects, as GP increases when the osmolarity of the solution is decreased. This increase in GP is associated with the presence of caveolae, which are known to disassemble and flatten in response to osmotic stress.     

Protein and chlorophyll in photosystem II probed by infrared spectroscopy

The infrared spectra of photosystem II (PS II) enriched submembrane fractions isolated from spinach are obtained in water and in heavy water suspension Other spectra are obtained after a photooxidation reaction was performed on PS II to bleach the pigments. The water bands are removed by computer subtraction and the amide bands (A, B, I, II, and III) of the protein are identified. Computer enhancement techniques are used to narrow the bandwidth of the bands that the weak chlorophyll bands, buried in the much stronger protein bands, can be observed. Comparing the spectra of native and photooxidized PS II pr in water and in heavy water, we determine that three polypeptide domains are present in the native material. The first domain, which contains 22% of th is situated in the peripheral region of the PS II system. The polypeptides in this region are unfolded and devoid of chlorophyll. The second domain con of the polypeptides, is more organized, and contains the chlorophylls. The third domain has an alpha-helix configuration, does not contain chlorophyll, a affected by the photooxidation reaction or by the proton/deuteron exchange. Three different types of chlorophyll organisation are identified: two have carbonyl groups non-bonded, differing from one another only in their hydrophobic milieux; the third is weakly bonded to another unidentified group. Other forms of chlorophyll organisation are present but could not be observed because their absorption is buried in the protein amide I band.     

Protein structure probed by polarization spectroscopy. I. Evidence for fibrinogen rigidity from stationary fluorescence

Two fluorescent derivatives of human fibrinogen have been synthesized, by the covalent bonding of 1-dimethylaminoaphthalene-5-sulphonyl and methylpyrene chromophores, to investigate the internal molecular dynamics of the protein in solution. The stationary fluorescence depolarization of these derivatives under isothermal conditions is described here while in an accompanying paper (part II) a time-resolved study is reported. From the static fluorescence data it is concluded that reorientational processes in the subnanosecond and microsecond time ranges account for all the observed depolarization. The faster motion was assigned to the restricted, localized oscillations of the label while the slow motion was ascribed to the overall rotation of the protein molecule. Consequently, the protein in solution appears considerably rigid in the 10-1000 ns range, in contrast with a previous conception of a flexible fibrinogen based on non-isothermal depolarization experiments. These previous experiments are, in fact, concordant with the rigid fibrinogen proposed here if they are reinterpreted using Weber's early ideas on thermally activated depolarization (G. Weber, J. Biochem. 51 (1952) 145).     

Protein mobility and diffusive barriers in Escherichia coli: consequences of osmotic stress

The effect of osmotic stress on the intracellular diffusion of proteins in Escherichia coli was studied, using a pulsed version of fluorescence recovery after photo-bleaching, pulsed-FRAP. This method employs sequences of laser pulses which only partly bleach the fluorophores in a cell. Because the cell size and geometry are taken into account, pulsed-FRAP enables to measure diffusion in very small cells of different shapes. We found that upon an osmotic upshock from 0.15 to 0.6 Osm, imposed by NaCl or sorbitol, the apparent intracellular diffusion (D) of mobile green fluorescent protein (GFP) decreased from 3.2 to 0.4 microm(2) s(-1), whereas the membrane permeable glycerol had no effect. Exposing E. coli cells to higher osmolalities (> 0.6 Osm) led to compartmentalization of the GFP into discrete pools, from where the GFP could not escape. Although free diffusion through the cell was hindered, the mobility of GFP in these pools was still relatively high (D approximately 0.4 microm(2) s(-1)). The presence of osmoprotectants restored the effect of osmotic stress on the protein mobility and apparent compartmentalization. Also, lowering the osmolality from 0.6 Osm back to 0.15 Osm restored the mobility of GFP. The implications of these findings in terms of heterogeneities and diffusive barriers inside the cell are discussed.     

Effect of osmotic stress on thylakoid fine structure inPorphyra umbilicalis

Summary The fine structural organization of thylakoid membranes in intact cells ofPorphyra umbilicalis, an intertidal red alga, was studied using the freeze-fracture method with special emphasis on changes induced by hypo- and hyperosmotic stresses. In osmotically adapted plants the density of intramembraneous particles on the PF-face increases considerably in the osmotic range from 5-fold diluted to 6-fold concentrated artifical seawater medium ASP₁₂, while that on the EF-face remains constant. The size of the particles on both fracture faces decreases strongly from extreme hypoosmotic to extreme hyperosmotic stress. These findings are discussed with relation to their biological significance.The author is member of the Arbeitsgemeinschaft für Elektronenmikroskopie an der Tierärztlichen Hochschule Hannover.     

Protein structure probed by polarization spectroscopy. II. A time-resolved fluorescence study of human fibrinogen

Human fibrinogen in solution was studied by monitoring the time-resolved depolarization of the fluorescence emitted by two spectroscopic labels of which the fluorescence lifetimes differ by an order of magnitude. Contrary to a long-held view, no evidence of molecular flexibility was found in the 10-1000 ns range. In addition, from the rate of the overall rotation, it is proposed that a prolate and symmetric ellipsoid of 47 X 10.5 nm may represent the time-averaged hydrodynamic size and shape of the protein in solution. This rigid and highly hydrated structure (4 g water/g protein) accommodates the latest nodular models obtained from electron microscopy, explains the singular hydrodynamics of fibrinogen and, apparently, it would perform the two main functions of the protein in haemostasis, blood coagulation and platelet aggregation, more efficiently than the flexible molecule.     

Protein kinases involved in mating and osmotic stress in the yeast Kluyveromyces lactis

Systematic disruption of genes encoding kinases and mitogen-activated protein kinases (MAPKs) was performed in Kluyveromyces lactis haploid cells. The mutated strains were assayed by their capacity to mate and to respond to hyperosmotic stress. The K. lactis Ste11p (KlSte11p) MAPK kinase kinase (MAPKKK) was found to act in both mating and osmoresponse pathways while the scaffold KlSte5p and the MAPK KlFus3p appeared to be specific for mating. The p21-activated kinase KlSte20p and the kinase KlSte50p participated in both pathways. Protein association experiments showed interaction of KlSte50p and KlSte20p with Gα and Gβ, respectively, the G protein subunits involved in the mating pathway. Both KlSte50p and KlSte20p also showed interaction with KlSte11p. Disruption mutants of the K. lactis PBS2 (KlPBS2) and KlHOG1 genes of the canonical osmotic response pathway resulted in mutations sensitive to high salt and high sorbitol but dispensable for mating. Mutations that eliminate the MAPKK KlSte7p activity had a strong effect on mating and also showed sensitivity to osmotic stress. Finally, we found evidence of physical interaction between KlSte7p and KlHog1p, in addition to diminished Hog1p phosphorylation after a hyperosmotic shock in cells lacking KlSte7p. This study reveals novel roles for components of transduction systems in yeast.     

Hydrostatic and osmotic pressure study of the RNA hydration

The tertiary structure of nucleic acids results from an equilibrium between electrostatic interactions of phosphates, stacking interactions of bases, hydrogen bonds between polar atoms and water molecules. Water interactions with ribonucleic acid play a key role in its structure formation, stabilization and dynamics. We used high hydrostatic pressure and osmotic pressure to analyze changes in RNA hydration. We analyzed the lead catalyzed hydrolysis of tRNA^Phe from S. cerevisiae as well as hydrolytic activity of leadzyme. Pb(II) induced hydrolysis of the single phosphodiester bond in tRNA^Phe is accompanied by release of 98 water molecules, while other molecule, leadzyme releases 86.     

Protein diffusion in the periplasm of E. coli under osmotic stress

The physical and mechanical properties of the cell envelope of Escherichia coli are poorly understood. We use fluorescence recovery after photobleaching to measure diffusion of periplasmic green fluorescent protein and probe the fluidity of the periplasm as a function of external osmotic conditions. For cells adapted to growth in complete medium at 0.14–1.02 Osm, the mean diffusion coefficient <D_peri> increases from 3.4 μm² s⁻¹ to 6.6 μm² s⁻¹ and the distribution of D_peri broadens as growth osmolality increases. This is consistent with a net gain of water by the periplasm, decreasing its biopolymer volume fraction. This supports a model in which the turgor pressure drops primarily across the thin peptidoglycan layer while the cell actively maintains osmotic balance between periplasm and cytoplasm, thus avoiding a substantial pressure differential across the cytoplasmic membrane. After sudden hyperosmotic shock (plasmolysis), the cytoplasm loses water as the periplasm gains water. Accordingly, <D_peri> increases threefold. The fluorescence recovery after photobleaching is complete and homogeneous in all cases, but in minimal medium, the periplasm is evidently thicker at the cell tips. For the relevant geometries, Brownian dynamics simulations in model cytoplasmic and periplasmic volumes provide analytical formulae for extraction of accurate diffusion coefficients from readily measurable quantities.     

Oligomerization and aggregation of bovine pancreatic ribonuclease A: backbone hydration probed by infrared band-shift

Protein hydration plays a crucial role in almost all aspects of biomolecular processes. In this research, we studied the hydration/dehydration-induced infrared amide I band-shift by using poly-L-lysine and bovine pancreas ribonuclease A as model polypeptides. It was found that a 1-4 cm(-1) shift could be clearly distinguished for all regular secondary structures during protein thermal unfolding. This shift was proven to be due to backbone hydration but not from experimental error, temperature effect or possible incomplete hydrogen/deuterium exchange of the samples. Moreover, we also found that protein aggregation was closely associated with the backbone hydration/dehydration status of proteins. In conditions favoring aggregation, a significant shift to a higher wavenumber of the band from the intermolecular beta-sheet structures in aggregates was observed. The present study suggested that the changes of the amounts of regular secondary structures could be monitored by the intensity changes, while the changes of the hydration status could be monitored by the shift of the infrared bands.     

Glucocorticoid receptor structure as probed by endogenous proteases

Transformation of the glucocorticoid-receptor complex by heating the cytosol in the presence of calcium is accompanied by formation of a series of truncated complexes, of which DI and DIIc are the major members. Formation of DIIc (but not of DI) is inhibited by leupeptin, and the intact transformed complex DIIa appears instead. Estimation of the molecular weights and Stokes' radii of all major complexes revealed that forms DI and DIIc have the same Mr, 48 kDa, but differ in shape, and appear to be digestion products generated by cleavage at the same site. Proteolysis of glucocorticoid receptor, covalently labelled with [3H]dexamethasone mesylate in rat thymus and brain cytosol, corroborated these findings and further implied that DI is the product of digestion of the non-transformed form of the receptor. Covalently labelled receptor fragments, related to the products formed when cytosol is heated, are detected in the nuclei of thymocytes, implying that the same proteolytic cleavages sites are involved in receptor turnover. Cleavage sites in the non-transformed covalently labelled receptor were identified in the "stepladder" of fragments of Mr, 85, 65, 49, 35, 27-30 kDa, generated in the absence of calcium, with an additional 78 kDa fragment in its presence. In the transformed conformation, two of the cleavage sites giving rise to the 65 and 35 kDa fragments, appear to be protected. It is speculated that the change in the proteolytic susceptibility of the cleavage site for the 35 kDa fragment relates to the "unmasking" of enhancer-activating and/or DNA-binding receptor functions previously postulated.     

Regulation of PIKfyve phosphorylation by insulin and osmotic stress

PIKfyve is a protein and lipid kinase that plays an important role in membrane trafficking, including TGN to endosome retrograde sorting and in insulin-stimulated translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the plasma membrane. We have previously demonstrated that PIKfyve is phosphorylated in response to insulin in a PI3-kinase and protein kinase B (PKB)-dependent manner. However, it has been implied that this was not due to direct phosphorylation of PIKfyve by PKB, but as a result of an insulin-induced PIKfyve autophosphorylation event. Here we demonstrate that purified PIKfyve is phosphorylated in vitro by a recombinant active PKB on two separate serine residues, S318 and S105, which flank the N-terminal FYVE domain of the protein. Only S318, however, becomes phosphorylated in intact cells stimulated with insulin. We further demonstrate that S318 is phosphorylated in response to hyperosmotic stress in a PI3-kinase- and PKB-independent manner. Importantly, the effects of insulin and sorbitol were not prevented by the presence of an ATP-competitive PIKfyve inhibitor (YM20163) or in a mutant PIKfyve lacking both lipid and protein kinase activity. Our results confirm, therefore, that PIKfyve is directly phosphorylated by PKB on a single serine residue in response to insulin and are not due to autophosphorylation of the enzyme. We further reveal that two stimuli known to promote glucose uptake in cells, both stimulate phosphorylation of PIKfyve on S318 but via distinct signal transduction pathways.     

Domain structure and function of dynamin probed by limited proteolysis

Dynamin is a 100-kDa GTPase with multiple domains. Some of these have known functions, namely, the N-terminal GTPase domain, the PH domain that binds phosphatidylinositol lipids, and the C-terminal proline-arginine-rich domain (PRD) that binds to several SH3 domain-containing dynamin partners. Others, for example, the "middle" located between the GTPase domain and the PH domain and a predicted alpha-helical domain located between the PH domain and PRD, have unknown functions. Dynamin exists as a homotetramer in solution and self-assembles into higher-order structures resembling rings and helical stacks of rings. Dynamin self-assembly stimulates its GTPase activity. We used limited proteolysis to dissect dynamin's domain structure and to gain insight into intradomain interactions that regulate dynamin self-assembly and stimulate GTPase activity. We found that the PH domain functions as a negative regulator of dynamin self-assembly and stimulates GTPase activity and that the alpha-helical domain, termed GED for GTPase effector domain, is required for stimulated GTPase activity.     

Protein hydration and the huge electrostriction

Recent experiments indicated the existence of hydration shells about biomolecules with densities markedly higher than that of bulk water. The compression is due to the pull of the dipoles of H(2)O molecules, necessary to achieve the thermodynamic equilibrium, from bulk water into the high field (approx. 10(9) V/m) region at the surface of the protein molecule. The electric field values at the surfaces of the biomolecules are calculated on the basis of the known densities. The reverse calculation of the limiting density values on the basis of known electric field distributions is performed, too. The results compare favourably with experiment.     

Photosynthesis under osmotic stress

The reversibility of the inhibition of photosynthetic reactions by water stress was examined with four systems of increasing complexity—stromal enzymes, intact chloroplasts, mesophyll protoplasts, and leaf slices. The inhibition of soluble chloroplast enzymes by high solute concentrations was instantly relieved when solutes were properly diluted. In contrast, photosynthesis was not restored but actually more inhibited when isolated chloroplasts exposed to hypertonic stress were transferred to conditions optimal for photosynthesis of unstressed chloroplasts. Upon transfer, chloroplast volumes increased beyond the volumes of unstressed chloroplasts, and partial envelope rupture occurred. In protoplasts and leaf slices, considerable and rapid, but incomplete restoration of photosynthesis was observed during transfer from hypertonic to isotonic conditions. Chloroplast envelopes did not rupture in situ during water uptake. It is concluded that inhibition of photosynthesis by severe water stress is at the biochemical level brought about in part by reversible inhibition of chloroplast enzymes and in part by membrane damage which requires repair mechanisms for reversibility. Both soluble enzymes and membranes appear to be affected by the increased concentration of internal solutes, which is caused by dehydration.