Photon and fluorescence correlation spectroscopy and light scattering of eye-lens proteins at moderate concentrations

The bovine eye-lens protein, alpha L-crystallin, has been studied with photon correlation spectroscopy to obtain the mutual diffusion coefficient, Dm, with fluorescence correlation spectroscopy to determine the tracer diffusion coefficient, DT, and with light scattering to get the isothermal osmotic compressibility (delta pi/delta c) P,T. The concentration dependence of Dm, DT, and (delta pi/delta c) P,T up to a volume fraction phi of the protein of 2.5 x 10(-2) has been interpreted on the basis of four different interaction potentials: (a) an extended hard-sphere potential; (b) a shielded Coulomb potential; (c) a shielded Coulomb interaction where the effect of counterions is included; (d) a simple mixed potential. The three parameters Dm, DT, and (delta pi/delta c) P,T have also been combined in the generalized Stokes-Einstein equation, Dm = [(delta pi/delta c)P,T . (1--phi) . (DT)]/(kappa B . T). Our results indicate that, in the case that photon correlation spectroscopy gives the mutual diffusion coefficient Dm, the applicability of the Stokes-Einstein equation can be questioned; or that, when one assumes the Stokes-Einstein equation to be valid, there is significant discrepancy between the result of photon correlation spectroscopy and Dm.     

Inhibition of lipases by proteins. A kinetic study with dicaprin monolayers

We report further investigations on protein inhibition of pancreatic and microbial lipases carried out with the monolayer technique. When beta-lactoglobulin A, melittin, serum albumin, myoglobin, and a protein inhibiting lipase from soybean were preincubated with a dicaprin film at a surface pressure of 35 dynes/cm, no activity was detected with horse pancreatic or Rhizopus delemar lipases. By contrast, Rhizopus arrhizus and Geotrichum candidum lipase activities were not impaired under the same conditions. Experiments using mixed lipid-protein film transfer clearly show that the inhibition of pancreatic lipase is due to the protein associated with lipid and not caused by direct protein-enzyme interaction in the aqueous phase. Three parameters were used to determine the surface properties of the various proteins at the dicaprin/water interface; namely, the initial rate of surface pressure increase, (delta pi/delta t)t = 0, the maximal surface pressure increase, delta pi max, and the critical surface pressure, pi c. A positive correlation was observed between values of (delta pi/delta t)t = 0 of proteins and their respective capacity to inhibit pancreatic and R. delemar lipases. By contrast, there was no apparent correlation with the two other parameters, delta pi max or pi c.     

Effects of lipid composition and packing on the adsorption of apolipoprotein A-I to lipid monolayers

To better understand the factors controlling the binding of apolipoprotein molecules at the surfaces of serum lipoprotein particles, the adsorption of human apolipoprotein A-I to phospholipid monolayers has been studied. The influence of lipid packing was investigated by spreading the monolayers at various initial surface pressures (pi i) and by using various types of lipid. The adsorption of 14C-methylated apolipoprotein A-I was monitored by simultaneously following the surface radioactivity (which could be converted to the surface concentration of protein, gamma) and the change in surface pressure (delta pi). In general, increasing the pi i of lipid monolayers reduces the adsorption of apolipoprotein A-I; for expanded egg phosphatidylcholine (PC) monolayers at pi i greater than or equal to 32 dyn/cm, gamma and delta pi are zero. The degree of adsorption of the apolipoprotein is also influenced by the physical state of the lipid monolayers. Thus, at a given pi i, apolipoprotein A-I adsorbs more to expanded monolayers than to condensed monolayers so that, at a given subphase concentration of protein, gamma of apolipoprotein A-I with various phospholipid monolayers decreases in the order egg PC greater than egg sphingomyelin greater than distearoyl-PC. The plot of gamma against pi i for adsorption of apolipoprotein A-I to dipalmitoylphosphatidylcholine (DPPC) monolayers shows an inflection at pi i = 8 dyn/cm; at this pi, the DPPC monolayer undergoes a phase transition from liquid (expanded) to solid (condensed) state. Addition of cholesterol generally decreases the adsorption of apolipoprotein A-I to egg PC monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local anesthetics and pressure: a comparison of dibucaine binding to lipid monolayers and bilayers

The binding of the local anesthetic dibucaine to monolayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was studied with a Langmuir trough at pH 5.5 (22 degrees C, 0.1 M NaCl). At this pH value only the charged form of the local anesthetic exists in solution. Charged dibucaine was found to be surface active and to penetrate into the lipid monolayer, with the hydrophobic part of the molecule being accommodated between the fatty acyl chains of the lipid. The dibucaine intercalation could be quantitated by measuring the expansion of the film area, delta A, at constant surface pressure, pi. At a given surface pressure, delta A increased with increasing dibucaine in the buffer phase. On the other hand, keeping the dibucaine concentration constant, the area increase, delta A, was strongly dependent on the surface pressure. The area increase, delta A, was large at low surface pressure and decreased with increasing surface pressure. A plot of the relative change in surface area, delta A/A, versus the surface pressure yielded straight lines in the pressure range of 25-36 mN/m for five different concentrations. The delta A/A vs. pi isotherms intersected at pi = 39.5 +/- 1 mN/m with delta A = O, indicating that charged dibucaine apparently can no longer penetrate into the monolayer film. By making judicial assumptions about the area requirement of dibucaine the monolayer expansion curves could be transformed into true binding isotherms. Dibucaine binding isotherms were constructed for different monolayer pressures and were compared to a bilayer binding isotherm measured under similar conditions with ultraviolet spectroscopy. The best agreement between monolayer and bilayer binding data was obtained for a monolayer held at a pressure of 30.7 to 32.5 mN/m, which can thus be considered as the bilayer-monolayer equivalence pressure. It is further suggested from this analogy that the binding of dibucaine does not change the internal pressure in the bilayer phase, at least not in the concentration range of physiological interest (0-2 mM dibucaine) but induces a lateral expansion. At higher molar ratios of cationic dibucaine to lipid, chi b, in the monolayer (chi b greater than 0.20) the area increase is larger than would be expected from the molecular dimensions of dibucaine. This is probably due to charge repulsion effects, which at still higher molar ratios (chi b greater than 0.6) lead to a micellisation. The pressure dependence of the intercalation of cationic dibucaine into lipid membranes may also be of relevance for the phenomenon of pressure reversal in anesthesia.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative monolayer investigations of surface properties of negatively charged glycosphingolipids from vertebrates (gangliosides) and invertebrates (SGL-II, lipid IV)

The surface properties of four negatively charged glycosphingolipids from vertebrates, the sialo-glycosphingolipids (= gangliosides) GM1, GD1a, GT1b and a sulfo-glycosphingolipid (= sulfatide), and of the two negatively charged glycosphingolipids from lower invertebrates, the glucurono-glycosphingolipid Lipid IV and the aminophosphono-glycosphingolipid SGL-II were investigated in monolayers at the air/water interface. The molecular peculiarities under investigation were surface pressure (pi) and surface potential (delta V) which are described for Lipid IV and SGL-II for the first time. The surface pressure/area isotherms of all glycosphingolipids were typical of a liquid-expanded monolayer and, with the exception of SGL-II, exhibited a phase transition to a liquid-condensed state at surface pressures above 20 mN/m. The surface potential/molecular area data found for gangliosides in the closely packed state at pi = 30 mN/m (GM1: delta V = -17 mV; GD1a: delta V = -35 mV; GT1b: delta V = -39 mV) showed only a slight influence of the additional number of negatively charged residues. For Lipid IV, the surface behavior was very similar to GM1 both possessing one negative group per molecule, whereas in SGL-II also the surface potential data (delta V = +173 mV) were different compared with GD1a both possessing two negative groups per molecule. The addition of Ca2+ condensed the monolayers of all glycolipids and increased the potential in the direction to more positive values, but these findings were less effective in SGL-II films. On the basis of monolayer results presented here, in biological membranes of invertebrates especially Lipid IV might play a similar role as the ganglioside GM1 in vertebrate cells.     

Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence

The mechanism of the shifting of the bioluminescence spectrum from the reaction of bacterial luciferase by lumazine protein is investigated by methods of fluorescence dynamics. A metastable intermediate is produced on reaction of Vibrio harveyi luciferase with FMNH2 and O2. It has an absorption maximum at 374 nm and a rotational correlation time (phi) derived from the decay of its fluorescence (maximum 500 nm) anisotropy of 90 ns (2 degrees C). Lumazine protein from Photobacterium phosphoreum has an absorption maximum at 417 nm and a fluorescence maximum at 475 nm. Lumazine protein forms a protein-protein complex with luciferase, and the complex has a phi of approximately 100 ns. A mixture of lumazine protein and the intermediate would be expected to have an average correlation time (phi av) around 100 ns, but instead, the result is anomalous. The phi av is much lower and is also wavelength dependent. For excitation at 375 nm, which is mainly absorbed in the flavin chromophore of the intermediate, phi av = 25 ns, but at 415 nm, mainly absorbed by the lumazine derivative ligand of lumazine protein, phi av approximately 50 ns. It is proposed that protein-protein complexation occurs between lumazine protein and the luciferase intermediate and that in this complex energy transfer from the flavin to the lumazine is the predominant channel of anisotropy loss. A distance of 20 A between the donor and acceptor is calculated. In the bioluminescence reaction of intermediate with tetradecanal, a fluorescent transient species is produced which is the bioluminescence emitter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of the pulmonary microvascular reflection coefficient to protein in dogs

We used a new technique to estimate the pulmonary microvascular membrane reflection coefficient to plasma protein (sigma d) in anesthetized dogs. In five animals we continuously weighed the lower left lung lobe and used a left atrial balloon to increase the pulmonary microvascular pressure (Pc). We determined the relationship between the rate of edema formation (S) and Pc and estimated the fluid filtration coefficient (Kf) as delta S/delta Pc. From the S vs. Pc relationship and Kf, we estimated the Pc at which S/Kf = 10 mmHg for each dog. This pressure (P10) was 38.0 +/- 5.8 (SD) mmHg, and the plasma protein osmotic pressure (pi c) was 14.9 +/- 3.7 mmHg. In five additional dogs in which we decreased pi c to 2.9 +/- 1.7 mmHg, P10 = 27.2 +/- 2.6 mmHg. The P10 vs. pi c regression line fit to the data from all 10 dogs was P10 = 0.92 pi c +/- 24.4 mmHg (r = 0.88). We estimated sigma d from the slope of the regression line as sigma d = square root of delta P10/delta pi c. With this technique, we estimated that, with 95% probability, sigma d lies between 0.72 and unity. This is higher than most previous sigma d estimates.     

Sodium transport and oxygen consumption in toad bladder. A thermodynamic approach.

The relationship between active sodium transport and oxygen consumption was investigated in toad urinary bladder exposed to identical sodium-Ringer's solution at each surface, while controlling the transepithelial electrical potential difference delta phi. Rates of sodium transport and oxygen consumption were measured simultaneously, both in the short-circuited state (delta phi = 0) and when delta phi was varied. Under short-circuit conditions, when the rates of active sodium transport changed spontaneously or were depressed with amiloride, the ratio of active sodium transport to the estimated suprabasal oxygen consumption Na/O2 was constant for each tissue, but varied among different tissues. Only when delta phi was varied did the ratio Na+/O2 change with the rate of active sodium transport; under these circumstances dNa+/dO2 was constant but exceeded the ratio measured at short-circuit [(Na+/O2)delta phi = 0[. This suggests that coupling between transport and metabolism is incomplete. The results are analyzed according to the principles of nonequilibrium thermodynamics, and intepreted in terms of a simple model of the transepithelial sodium transport system.     

The effects of protein hydrophobic exposure on the slope of the line in the deltapi vs pi(i) plot

The deltapi vs pi(i) curve from monolayer surface pressure detection is a powerful method to characterize the membrane insertion. The deltapi vs pi(i) curve is fixed by two parameters, the pi(c) and the slope. The intersected point (pi(c)) of deltapi vs pi(i) curve with abscissa is generally used as a quantitative measure of the membrane insertion ability of a protein. In the current work we demonstrate a correlation between the variation in the slope of the deltapi vs pi(i) curve with protein hydrophobic exposure by performing monolayer experiments on three different proteins, human apolipoprotein H, trichosanthin, and mutant trichosanthin. The value of /slope/ increases gradually following the degree of hydrophobic exposure. These findings suggest that the two parameters, the pi(c) and the slope, will complement each other to interpret the lipid/protein interaction involved in membrane insertion.     

Adsorption of bovine prothrombin to spread phospholipid monolayers

The interaction of bovine prothrombin with phospholipids was measured, using as the lipid source monolayers spread at the air-buffer interface. Fluorescence spectroscopy was implemented to determine the equilibrium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The increase in surface pressure (pi) from the protein-monolayer adsorption was also measured and, with values of the adsorbed protein concentration (c[s]), was used to calculate dc(s)/d(pi). At a particular phosphatidylserine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/PS monolayers, dc(s)/d(pi) was independent of the initial surface pressure (pi[i]), when this latter value exceeded 30 mN/m. However, dc(s)/d(pi) varied significantly with the relative PS content of the monolayer. Values of the equilibrium dissociation constants calculated from the concentration dependence of delta(pi) indicated that the affinity of prothrombin for LE monolayers was higher at higher PS contents and lower packing densities. The affinity of prothrombin for liquid-condensed (LC) PC/PS monolayers was found to be much weaker relative to LE monolayers of similar phospholipid composition. This approach, employing spread monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentration in protein-monolayer suspensions, offers significant advantages for the investigation of protein-membrane interaction. The equilibrium characteristics that describe the interaction of prothrombin with the different phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interactions are involved in the adsorption of vitamin K-dependent coagulation and anticoagulation proteins to model membrane systems.     

Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop.

Escherichia coli transports Fe3+ as a ferrichrome complex through the outer membrane in an energy-dependent process mediated by the FhuA protein. A FhuA deletion derivative lacking residues 322 to 355 (FhuA delta322-355) forms a permanently open channel through which ferrichrome diffused. This finding led to the concept that the FhuA protein forms a closed channel that is opened by input of energy derived from the electrochemical potential across the cytoplasmic membrane, mediated by the Ton system. In this study, we constructed various FhuA derivatives containing deletions inside and outside the gating loop. FhuA delta322-336 bound ferrichrome and displayed a residual Ton-dependent ferrichrome transport activity. FhuA delta335-355 no longer bound ferrichrome but supported ferrichrome diffusion through the outer membrane in the absence of the Ton system. FhuA delta335-355 rendered cells sensitive to sodium dodecyl sulfate and supported diffusion of maltotetraose and maltopentaose in a lamB mutant lacking the maltodextrin-specific channel in the outer membrane. Cells expressing FhuA delta70-223, which has a large deletion outside the gating loop, were highly sensitive to sodium dodecyl sulfate and grew on maltodextrins but showed only weak ferrichrome uptake, suggesting formation of a nonspecific pore through the outer membrane. FhuA delta457-479 supported Ton-dependent uptake of ferrichrome. None of these FhuA deletion derivatives formed pores in black lipid membranes with a stable single-channel conductance. Rather, the conductance displayed a high degree of current noise, indicating a substantial influence of the deletions on the conformation of the FhuA protein. FhuA also supports infection by the phages T1, T5, and phi80 and renders cells sensitive to albomycin and colicin M. Cells expressing FhuA delta322-336 were sensitive to albomycin and colicin M but were only weakly sensitive to T5 and phi480 and insensitive to T1. Cells expressing FhuA delta335-355 were resistant to all FhuA ligands. These results indicate different structural requirements within the gating loop for the various FhuA ligands. Cells expressing FhuA delta457-479 displayed a strongly reduced sensitivity to all FhuA ligands, while cells expressing FhuA delta70-223 were rather sensitive to all FhuA ligands except albomycin, to which they were nearly resistant. It is concluded that residues 335 to 355 mainly determine the properties of the gate with regard to FhuA permeability and ligand binding.     

DCCD-sensitive, Na+-dependent H+-influx process coupled to membrane potential formation in membrane vesicles of Halobacterium halobium

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) on light-induced H+-transport and transmembrane electric potential (delta phi) formation were studied in the membrane vesicles of Halobacterium halobium R1M1. In accordance with our previous finding of the existence of two DCCD-binding components in vesicle membrane using 14C-DCCD (Konishi & Murakami FEBS Lett. 169, 283-286 (1984)), DCCD inhibited the H+-influx process biphasically; that is, the H+-influx process which is electrically silent was initially inhibited at concentrations below 30 nmol of DCCD/mg vesicle protein, while another H+-influx process which is coupled to delta phi formation was secondarily inhibited above this concentration of DCCD. The latter H+-influx process was highly dependent on the Na+ concentration. The extents of Na+-dependent recovery of delta phi formation and H+-influx were quantitatively correlated. From these results, it was concluded that the second DCCD-sensitive H+-influx process which is coupled to delta phi formation is due to the hypothetical Na+/H+-antiporter postulated by Lanyi and MacDonald (Biochemistry 15, 4608-4614 (1976)). It was also found that Li+ can be substituted for Na+ in this system, as is the case with Na+/H+-antiporters found in other organisms.     

Changes in the electrical and visco-elastic properties of bilayer lipid membranes during interaction with proteins and lipoproteins

A method for simultaneous registration of planar bilayer lipid membrane (BLM) DC conductance G, capacitance C, surface potential difference delta phi and transversal elasticity module E is developed. C, delta phi and E are proportional to the amplitude of the first, second and third harmonics of capacitance current respectively. A comparative study of the interaction of BLM with very low density lipoproteins (VLDL), influenza virus matrix protein (M-protein) and yeast invertase was carried out. The kinetics of delta phi, E and G changes at different concentrations of VLDL, and dependence of delta phi and G on M-protein and invertase concentration was investigated. It is shown for VLDL invertase and M-protein that the changes in delta phi and E occur before the change in G. The method used permits to study peculiarities of individual stages of interaction between charge particles, supramolecular structures and lipid membranes.     

Prediction of C alpha-H...O and C alpha-H...pi interactions in proteins using recurrent neural network

In this study, an attempt has been made to develop a method for predicting weak hydrogen bonding interactions, namely, C alpha-H...O and C alpha-H...pi interactions in proteins using artificial neural network. Both standard feed-forward neural network (FNN) and recurrent neural networks (RNN) have been trained and tested using five-fold cross-validation on a non-homologous dataset of 2298 protein chains where no pair of sequences has more than 25% sequence identity. It has been found that the prediction accuracy varies with the separation distance between donor and acceptor residues. The maximum sensitivity achieved with RNN for C alpha-H...O is 51.2% when donor and acceptor residues are four residues apart (i.e. at delta D-A = 4) and for C alpha-H...pi is 82.1% at delta D-A = 3. The performance of RNN is increased by 1-3% for both types of interactions when PSIPRED predicted protein secondary structure is used. Overall, RNN performs better than feed-forward networks at all separation distances between donor-acceptor pair for both types of interactions. Based on the observations, a web server CHpredict (available at http://www.imtech.res.in/raghava/chpredict/) has been developed for predicting donor and acceptor residues in C alpha-H...O and C alpha-H...pi interactions in proteins.     

Cholesterol in condensed and fluid phosphatidylcholine monolayers studied by epifluorescence microscopy

Epifluorescence microscopy was used to investigate the effect of cholesterol on monolayers of dipalmitoylphosphatidylcholine (DPPC) and 1 -palmitoyl-2-oleoyl phosphatidylcholine (POPC) at 21 +/- 2 degrees C using 1 mol% 1-palmitoyl-2-[12-[(7-nitro-2-1, 3-benzoxadizole-4-yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) as a fluorophore. Up to 30 mol% cholesterol in DPPC monolayers decreased the amounts of probe-excluded liquid-condensed (LC) phase at all surface pressures (pi), but did not effect the monolayers of POPC, which remained in the liquid-expanded (LE) phase at all pi. At low pi (2-5 mN/m), 10 mol% or more cholesterol in DPPC induced a lateral phase separation into dark probe-excluded and light probe-rich regions. In POPC monolayers, phase separation was observed at low pi when > or =40 mol% or more cholesterol was present. The lateral phase separation observed with increased cholesterol concentrations in these lipid monolayers may be a result of the segregation of cholesterol-rich domains in ordered fluid phases that preferentially exclude the fluorescent probe. With increasing pi, monolayers could be transformed from a heterogeneous dark and light appearance into a homogeneous fluorescent phase, in a manner that was dependent on pi and cholesterol content. The packing density of the acyl chains may be a determinant in the interaction of cholesterol with phosphatidylcholine (PC), because the transformations in monolayer surface texture were observed in phospholipid (PL)/sterol mixtures having similar molecular areas. At high pi (41 mN/m), elongated crystal-like structures were observed in monolayers containing 80-100 mol% cholesterol, and these structures grew in size when the monolayers were compressed after collapse. This observation could be associated with the segregation and crystallization of cholesterol after monolayer collapse.     

DCCD-sensitive Na+-transport in the membrane vesicles of Halobacterium halobium

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) and various ionophores on light-induced 22Na+-transport were studied in right-side-out membrane vesicles from Halobacterium halobium R1M1. The light-induced Na+ efflux was inhibited at the same DCCD concentration (greater than 40 nmol/mg protein) as required for inhibition of the Na+-dependent membrane potential (delta phi) formation. This supports our previous indication that the DCCD-sensitive, Na+-dependent transformation of pH-gradient (delta pH) into delta phi is mediated by Na+/H+-antiporter (Murakami, N. and Konishi, T. (1985) J. Biochem. 98, 897-907). FCCP or a combination of valinomycin and triphenyltin (TPT) inhibits the light-induced Na+ efflux in accordance with the notion of protonmotive force (delta mu H+)-driven antiporter. However, a marked lag in initiation of the Na+ efflux occurred in the presence of valinomycin, TPMP+, or a small amount of FCCP, suggesting that a gating step is involved in the Na+ efflux. On the other hand, the delta pH-dissipating ionophore TPT did not cause the lag. A simultaneous determination of delta phi, delta pH, and Na+ efflux rate at the initial stage of illumination revealed that the antiporter is gated by delta phi rather than delta mu H+.     

Crystal structure and conformation of a highly constrained linear tetrapeptide Boc-Leu-dehydro Phe-Ala-Leu-OCH3.

The conformation of a tetrapeptide containing a dehydro amino acid, delta ZPhe, in its sequence has been determined in the crystalline state using X-ray crystallographic techniques. The tetrapeptide, Boc-Leu-delta ZPhe-Ala-Leu-OCH3, crystallizes in the orthorhombic space group P2(1)2(1)2(1) with four molecules in a unit cell of dimensions a = 11.655(1) A, b = 15.698(6) A and c = 18.651(3) A V = 3414.9 A and Dcalc = 1.12 g/cm-3. The asymmetric unit contains one tetrapeptide molecule, C30H46N4O7, a total of 41 nonhydrogen atoms. The structure was determined using the direct methods program SHELXS86 and refined to an R-factor of 0.049 for 3347 reflections (I3.0(I). The linear tetrapeptide in the crystal exhibits a double bend of the Type III-I, with Leu1 (phi = -54.1 degrees, psi = -34.5 degrees) and delta ZPhe2 (phi = -59.9 degrees, psi = -17.1 degrees) as the corner residues of Type III turn and delta ZPhe2 (phi = -59.9 degrees, psi = -17.1 degrees) and Ala3 (phi = -80.4 degrees, psi = 0.5 degrees) residues occupying the corners of Type I turn, with delta ZPhe as the common residue in the double bend. The turn structures are further stabilized by two intramolecular 4----1 type hydrogen bonds.     

Swelling and de-swelling kinetics of gelatin hydrogels in ethanol-water marginal solvent

Controlled osmotic swelling and de-swelling measurements have been performed on gelatin, a polyampholyte, hydrogels suspended in water-ethanol marginal solvent at room temperature (20 degrees C) where the alcohol concentration was changed from 0 to 100% (v/v). The change in gel mass was monitored as function of time until osmotic equilibrium was established with the surrounding solvent. It was observed that osmotic pressure of polymer-solvent mixing, pi(m)相似文献   

Orientation and penetration depth of monolayer-bound p40phox-PX

X-ray reflectivity was used to study the interaction of the PX domain of p40(phox) protein (p40(phox)-PX) with a Langmuir monolayer of a mixture of SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), SOPS (1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine), and DPPtdIns(3)P (1,2-dipalmitoylphosphatidylinositol 3-phosphate) lipids supported on a buffered aqueous solution. The reflectivity is analyzed in terms of the known crystallographic structure of the p40(phox)-PX domain and a slab model that represents the lipid layer, yielding an electron density profile of the lipid layer and bound PX domains. This analysis determines the angular orientation and penetration depth of the p40(phox)-PX domain bound to the SOPC/SOPS/DPPtdIns(3)P monolayer. The best fit orientation is characterized by the following angles: theta = 30 +/- 10 degrees and phi = 140 +/- 30 degrees. These angles describe rotations, about axes in a coordinate system fixed to the domain, that are required to orient the domain with respect to the lipid layer at the interface. The protein penetrated into the lipid layer by 9 +/- 2 A, indicating that the protein penetrated into the headgroup region, but not deeply into the hydrocarbon region of the monolayer. In this analysis, polar Tyr(94) and hydrophobic Val(95) penetrated deepest into the lipid monolayer. The backbone of these residues was approximately 5 A above the headgroup-buffer interface, i.e., at the level of the SOPC/SOPS lipid phosphates. Positively charged Lys(92) and Lys(98) were also near the SOPC/SOPS lipid phosphates. This position of the protein allows for a favorable electrostatic contribution to binding.