Mechanisms of human plasma protein adsorption on the surface of perfluorocarbon emulsion stabilized with proxanol 268

It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion stabilized with proxanol 268 is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to an increase in the total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and fibrinogen is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been set that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicate considerable conformational rearrangements of this protein during adsorption. It was shown that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction is completely or partially quenched.     

Mechanisms of human plasma proteins adsorption on the surface of perfluorocarbon emulsion stabilized with proxanol 268

It has been shown that sorption of most proteins with the molecular weight lower than 200 kDa from human blood plasma on the surface of perfluorocarbon emulsion, stabilized with proxanol 268, is mainly based on hydrophobic interaction, whereas sorption of immunoglobulin G is mainly the result of electrostatic interaction. The removal of lipidic components from plasma leads to the increase of a total amount of adsorbed proteins by 35%. Particularly, when lipidic components are removed, sorption of apolipoprotein AI and immunoglobulin G is considerably bettered as well as sorption of other proteins with the molecular weight of about 50 and 60 kDa occurs. It has been out that apolipoprotein AI in the adsorbed condition loses its capability of tryptophan fluorescence, which might be probably determined by the quenching influence of the perfluorocarbon core of nanoparticle. We think that the findings obtained also indicates considerable conformational rearrangements of this protein during adsorption. It was shown, that the fluorescence of proteins with sorption on nanoparticles in emulsion based on the hydrophobic interaction, is completely or partially quenched.     

Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The binding constants of BCPT with BSA or HSA were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained and the binding force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented. The linear range is 5.36-67.0 microg mL(-1) with recovery of 101.1% for BSA, and the linear range is 8.28-144.9 microg mL(-1) with recovery of 102.6% for HSA. Determination of the proteins in bovine serum or in human serum by this method gives results which are very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of BCPT in human serum samples.     

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.     

A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands.

Ca2+ binding to calmodulin in the pCa range 5.5-7.0 exposes hydrophobic sites that bind hydrophobic inhibitory ligands, including calmodulin antagonists, some Ca2+-antagonists and calmodulin-binding proteins. The binding of these hydrophobic ligands to calmodulin can be followed by the approx. 80% fluorescence increase they produce in dansylated (5-dimethylaminonaphthalene-1-sulphonylated) calmodulin (CDRDANS). In the presence of Ca2+, calmodulin binds the calmodulin inhibitor, R24571, with an affinity of approx. 2-3 nM and hydrophobic ligands, including trifluoperazine (TFP), W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide], fendiline, felodipine and prenylamine, with affinities in the micromolar range. This binding is strongly Ca2+-dependent and Mg2+-independent. Calmodulin shows a reasonably high degree of specificity in its binding of these ligands over other ligands tested. CDRDANS, therefore, provides a convenient and simple means of monitoring the interaction of a variety of hydrophobic ligands with the Ca2+-dependent regulatory protein, calmodulin. CDRDANS binds to phospholipid vesicles made of (dimyristoyl)phosphatidylcholine (DMPC) or (dipalmitoyl)phosphatidylcholine (DPPC) and produces fluorescence increases only in the presence of Ca2+ and at temperatures above their gel-to-liquid crystalline phase transition. Although the fluorescence changes in CDRDANS accurately report phase transitions in these liposomes, its binding to these vesicles is weak. Calmodulin probably requires a high-affinity lipid-bound receptor protein for its high-affinity binding to natural membranes.     

Steady-state and time resolved fluorescence of albumins interacting with N-oleylethanolamine, a component of the endogenous N-acylethanolamines

The functions of N-acylethanolamines, minor constituents of mammalian cells, are poorly understood. It was suggested that NAEs might have some pharmacological actions and might serve as a cytoprotective response, whether mediated by physical interactions with membranes or enzymes or mediated by activation of cannabinoid receptors. Albumins are identified as the major transport proteins in blood plasma for many compounds including fatty acids, hormones, bilirubin, ions, and many drugs. Moreover, albumin has been used as a model protein in many areas, because of its multifunctional binding properties. Bovine (BSA) and human (HSA) serum albumin are similar in sequence and conformation, but differ for the number of tryptophan residues. This difference can be used to monitor unlike protein domains. Our data suggest that NOEA binds with high affinity to both albumins, modifying their conformational features. In both proteins, NOEA molecules are linked with higher affinity to hydrophobic sites near Trp-214 in HSA or Trp-212 in BSA. Moreover, fluorescence data support the hypothesis of the presence of other NOEA binding sites on BSA, likely affecting Trp-134 environment. The presence of similar binding sites is not measurable on HSA, because it lacks of the second Trp residue.     

Selectivity in lipid binding to the bacterial outer membrane protein OmpF

The outer membrane porin OmpF from Escherichia coli has been reconstituted into lipid bilayers of defined composition, and fluorescence spectroscopy is used to characterize its interaction with the surrounding lipid. OmpF is a trimer within the membrane. It contains two Trp residues per monomer, Trp(214) at the lipid-protein interface and Trp(61) at the trimer interface. The fluorescence of Trp-214 in the mutant W61F is quenched by dibromostearoylphosphatidylcholine (di(Br(2)C18:0)PC), whereas the fluorescence of Trp(61) in the mutant W214F is not quenched by di(Br(2)C18:0)PC when fluorescence is excited directly through the Trp rather than through the Tyr residues. Measurements of relative fluorescence quenching for OmpF reconstituted into mixtures of lipid X and di(Br(2)C18:0)PC have been analyzed to give the binding constant of lipid X for OmpF, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). The phosphatidylcholine showing the strongest binding to OmpF is dimyristoyloleoylphosphatidylcholine (di(C14:1)PC) with binding constants decreasing with either increasing or decreasing fatty acyl chain length. Comparison with various theories for hydrophobic matching between lipids and proteins suggests that in the chain length range from C14 to C20, hydrophobic matching is achieved largely by distortion of the lipid bilayer around the OmpF, whereas for chains longer than C20, distortion of both the lipid bilayer and of the protein is required to achieve hydrophobic matching. Phosphatidylcholine and phosphatidylethanolamine bind with equal affinity to OmpF, but the affinity for phosphatidylglycerol is about half that for phosphatidylcholine.     

Fluorescent probes for measuring the binding constants and distances between the metal ions bound to Escherichia coli glutamine synthetase.

TNS, 2-p-toluidinylnaphthalene-6-sulfonate, has been used as a fluorescent probe to determine the binding constants of metal ions to the two binding sites of Escherichia coli glutamine synthetase (GS). TNS fluorescence is enhanced dramatically when bound to proteins due to its high quantum yield resulting from its interactions with hydrophobic regions in proteins. The fluorescence energy transfer from a hydrophobic tryptophan residue of GS to TNS has been detected as an excitation band centered at 280 nm. Therefore, TNS is believed to be bound to a hydrophobic site on the GS surface other than the active site and is located near a hydrophobic Trp residue of GS. GS binds lanthanide ions [Ln(III)] more tightly than either Mn(II) or Mg(II), and the binding constants of several lanthanide ions were determined to be in the range (2.1-4.6) x 10(10) and (1.4-3.0) x 10(8) M-1 to the two metal binding sites of GS, respectively. The intermetal distances between the two metal binding sites of GS were also determined by measuring the efficiencies of energy transfer from Tb(III) to other Ln(III) ions. The intermetal distances of Tb(III)-Ho(III) and Tb(III)-Nd(III) were 7.9 and 6.8 A, respectively.     

How to evaluate the distribution of an "invisible" amphiphile between biological membranes and water

To evaluate the distribution of an amphiphile or its binding to membranes whose properties are affected by such binding, it is only necessary to establish to what extent the dose-response to the amphiphile depends on the membrane concentration. The measured response only needs to reflect local events. This method of evaluation does not depend on the precise shape of the dose-response curve and is particularly useful for amphiphiles devoid of properties like fluorescence or radioactivity which would allow their direct assay. In this work, we establish the validity of this approach by comparing it with direct conventional determinations. Two parameters are especially suitable for such evaluation: the perturbation of an enzyme's activity, produced by many amphiphiles, and the fluorescence quenching of membrane-embedded proteins by chromophoric amphiphiles through long-range F?rster transfer. We illustrate this approach in sarcoplasmic reticulum membranes containing Ca2(+)-ATPase as the main protein constituent. The equilibrium distribution of the antioxidant 4-nonylphenol was deduced from its inhibition of ATPase activity, whereas the equilibrium distribution of the calcium ionophore calcimycin (A23187) and of its brominated analog 4-bromo-A23187 were determined from their quenching of ATPase fluorescence. Apparent partition coefficients K* in the range of 10(5) (expressed as (moles of lipid/liter)-1) were obtained for these highly hydrophobic molecules.     

Correctly folded proteins make twice as many hydrophobic contacts

A novel statistical analysis of non-bonded contacts in a set of known protein structures shows that the natural residue types fall into five or six groups distinguishable by nearest neighbor preference. The observed pattern of contact specificities clearly reflects residue hydrophobicity and charge. Its most striking feature is that residues in the hydrophobic group make about twice as many contacts with one another as would be expected on a random basis. A similar increase in hydrophobic contact frequency can be observed at the level of individual proteins. Native proteins make, on average, about twice as many hydrophobic contacts as corresponding misfolded proteins, generated by computer. On the basis of these observations increased hydrophobic contact frequency is proposed as a simple model of the hydrophobic effect.     

Structural stability and reversible unfolding of recombinant porcine S100A12

Porcine S100A12 is a member of the S100 proteins, family of small acidic calcium-binding proteins characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events such as the regulation of protein phosphorylation, enzymatic activity, protein-protein interaction, Ca2+ homeostasis, inflammatory processes and intermediate filament polymerization. In addition, members of this family bind Zn2+ or Ca2+ with cooperative effect on binding. In this study, the gene sequence encoding porcine S100A12 was obtained by the synthetic gene approach using E. coli codon bias. Additionally, we report a thermodynamic study of the recombinant S100A12 using circular dichroism, fluorescence and isothermal titration calorimetry. The results of urea and temperature induced unfolding and refolding processes indicated a reversible two-state process. Also, the ANS fluorescence studies showed that in presence of divalent ions the protein exposes hydrophobic sites which could facilitate the interaction with other proteins and trigger the physiological responses.     

X-ray structure and ligand binding study of a moth chemosensory protein

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. Such proteins, of M(r) 13,000, have been isolated from several sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. One of them, CSPMbraA6, a 112-amino acid antennal protein, has been expressed in large quantities and is soluble in the Escherichia coli periplasm. X-ray structure determination has been performed in parallel with ligand binding assays using tryptophan fluorescence quenching. The protein has overall dimensions of 25 x 30 x 32 A and exhibits a novel type of alpha-helical fold with six helices connected by alpha-alpha loops. A narrow channel extends within the protein hydrophobic core. Fluorescence quenching with brominated alkyl alcohols or fatty acids and modeling studies indicates that CSPMbraA6 is able to bind such compounds with C12-18 alkyl chains. These ubiquitous proteins might have the role of extracting hydrophobic linear compounds (pheromones, odors, or fatty acids) dispersed in the phospholipid membrane and transporting them to their receptor.     

Positions of polar amino acids alter interactions between transmembrane segments and detergents

α-Helical transmembrane (TM) segments in membrane proteins are comprised primarily of hydrophobic amino acids that accommodate insertion from water into the nonpolar membrane bilayer. In many such segments, however, polar residues are also present for structural or functional reasons. These latter residues impair the local favorable acyl interactions required for solvation by hydrophobic media such as phospholipids in native bilayers or detergents used for in vitro characterization. Using a series of Lys-tagged designed TM-like peptides (typified by KK-YAAAIAAIAWAIAAIAAAIAA-KKK) in which single-Asn residue substitutions (from Ile or Ala) were made successively from the center of the hydrophobic region toward the C-terminus, we demonstrate that polar residues strongly alter the nature of the interaction between TM segments and the solvating detergent. Through the application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, and tryptophan fluorescence, we observed drastic differences in the structures of the detergent-peptide complexes that contain relatively minor sequence differences. For example, the blue shift of the Trp fluorescence (indicating local detergent solvation at this location) differs by as much as ~10 nm depending upon the position of a single Asn substitution in an otherwise identical segment. The overall results suggest that polar point mutations occurring in a biological membrane will elicit comparable effects, placing a significant refolding burden on the local protein structure and potentially leading to disease states through altered protein--lipid interactions in membrane proteins.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

4,4'-Dianilino-1,1'-binaphthyl-5,5'-sulfonate, a novel molecule having chaperone-like activity

4,4'-Dianilino-1,1'-binaphthyl-5,5'-sulfonate (bis-ANS) and 1-anilinonaphthalene-8-sulfonate (ANS) are hydrophobic probes that are widely used in protein folding studies, using their capacity to bind to hydrophobic regions of partially unfolded proteins and in turn leading to an increase in fluorescence. Here we reveal a novel chaperone-like activity for bis-ANS, which acted as a highly effective inhibitor for the thermal- or chemical-induced aggregation of alcohol dehydrogenase, insulin or the whole cell extract of Escherichia coli, with ANS showing a much weaker effect. The studies to elucidate the mechanism underlying this activity show that bis-ANS is able to form stable soluble aggregates with the denaturing proteins and dramatically increase its fluorescence intensity upon incubation with aggregation-prone proteins. Moreover, we found that bis-ANS is able to prevent the heat inactivation of citrate synthase. These observations suggest that bis-ANS is able to block the exposed hydrophobic surfaces to suppress protein aggregation, acting in a way similar to what small heat shock proteins (one sub-class of molecular chaperones) do. The data presented here, together with the report that bis-ANS was able to suppress the amyloid formation of the prion peptide [J. Biol. Chem. 279 (2004) 5346], suggest that this molecule may be used as a potential protein stabilizer in addition to its current application as a hydrophobic probe.     

Interaction of beta-lactoglobulin with resveratrol and its biological implications

Beta-lactoglobulin (beta-LG), the major whey protein in the milk of ruminants, has a high affinity for a wide range of compounds. Resveratrol (3,5,4'-trihydroxystilbene), a natural polyphenolic compound found in grapes and red wine, exhibits many physiological effects associated with health benefits. In this study, the interaction of resveratrol with beta-LG was investigated using circular dichroism, fluorescence and UV-vis absorbance. Self-association of resveratrol possibly occurs at high concentrations. Resveratrol interacts with beta-LG to form 1:1 complexes. Resveratrol is bound to the surface of the protein because beta-LG-bound polyphenol is in a weaker hydrophobic environment relative to 75% ethanol. The binding constant for the resveratrol-beta-LG interaction is between 10(4) and 10(6) M (-1), as determined by protein or polyphenol fluorescence. The beta-LG-resveratrol interaction may compete with self-association of both the polyphenol and the protein. It has no apparent influence on beta-LG secondary structure but partially disrupts tertiary structure. Complexing with beta-LG provides a slight increase in the photostability of resveratrol and a significant increase in its hydrosolubility.     

Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, we developed a reagentless, regenerable and rapid immunosensing system to determine immunoglobulin G (IgG). Fluorescence intensity of the immobilized FITC depends on IgG concentration, ranging from 10 to 50 microg/ml, specifically, even with co-existing proteins. The response time is 30 min during steady-state measurement and is less than a minute during transient measurement. When the FITC-labeled protein A binds to IgG, the surrounding atmosphere of FITC becomes hydrophobic. Since the fluorescence intensity of fluorescent substances generally increases at a hydrophobic environment, FITC fluorescence intensity increases with the concentration of protein A bonding to IgG. This system is regenerable because the fluorescence enhancement repeatedly occurs every time the immobilized fluorescent reagent is immersed in sample solutions.     

Calcium-induced conformational changes of the recombinant CBP3 protein from Dictyostelium discoideum

Calcium-binding proteins play various and significant roles in biological systems. Conformational changes in their structures are closely related to their physiological functions. To understand the role of calcium-binding protein 3 (CBP3) in Dictyostelium discoideum, its recombinant proteins were analyzed using circular dichroism (CD) and fluorescence spectroscopy. Gel mobility shift analysis showed that Ca2+ induced a mobility shift of the recombinant CBP3. Far ultra-violet CD spectra and intrinsic fluorescence spectra on CBP3 and its N- and C-terminal domains exhibited that they underwent a conformational rearrangement depending upon Ca2+ binding. Measurement of Ca2+ dissociation constants demonstrated that CBP3 had high affinity toward Ca2+ in the sub-micromolar range and N-terminal domain had higher affinity than C-terminal domain. The changes of fluorescence spectra by an addition of 8-anilino-1-naphthalene sulfonic acid indicated that the hydrophobic patches of CBP3 and its C-terminal domain are likely to be more exposed in the presence of Ca2+. Since the exposure of hydrophobic patches is thermodynamically unfavorable, Ca2+-bound CBP3 may interact with other proteins in vivo. All these data suggest that Ca2+ induces CBP3 to be more favorable conformation to interact with target proteins.     

Fluorescence studies of rat cellular retinol binding protein II produced in Escherichia coli: an analysis of four tryptophan substitution mutants.

Rat intestinal cellular retinol binding protein II (CRBP II) is an abundant 134-residue protein that binds all-trans-retinol which contains 4 tryptophans in positions 9, 89, 107, and 110. Our ability to express CRBP II in Escherichia coli and to construct individual tryptophan substitution mutants by site-directed mutagenesis has provided a useful model system for studying the fluorescence of a multi-tryptophan protein. Each of the four mutant proteins binds all-trans-retinol with high affinity, although their affinities are less than that of the wild-type protein. Steady-state and time-resolved fluorescence analyses of these proteins indicate that W107 is at the hydrophobic binding site, W110 is in a polar environment, and the remaining two tryptophans are in a hydrophobic environment. Time-resolved fluorescence study indicates that excited-state energy transfer occurs from the hydrophobic tryptophans to W110. The Stern-Volmer analysis with acrylamide of these proteins reveals that static quenching occurs in the W9F mutant protein while others do not. The fluorescence of rat intestinal fatty acid binding protein (I-FABP), a related protein of known X-ray structure, was also studied for comparison. The results of these findings, coupled with those derived from NMR studies and molecular graphics, suggest that CRBP II undergoes minor structural changes in all of the mutant proteins. Since these effects may be cumulative on the protein structure and function, any conclusions derived from higher mutants in this family of proteins must be treated with caution.