Extrinsic Fluorescence Spectroscopy Research Articles

Spectroscopic Studies on the Comparative Interaction of Cationic Single-Chain and Gemini Surfactants with Human Serum Albumin

To gain insights into the comparative effect of single-chain/gemini surfactants on proteins and the possible implications, the interaction of human serum albumin (HSA) with cationic single-chain surfactant cetyltrimethylammonium bromide (CTAB) and its gemini counterpart bis(cetyldimethylammonium)butane dibromide with spacer -(CH(2))(4)- (designated as G4) using turbidity measurements, far-UV and near-UV circular dichroism (CD), intrinsic fluorescence and extrinsic fluorescence spectroscopy at pH 7.0 are reported in this contribution. A decrease of 33.5% alpha-helical content at 22.5 microM G4 was monitored compared to a 15% decrease at 2,250 microM CTAB. Against a 3.5% increase at 11,250 microM CTAB, a rise of 21.1% in the alpha-helical content was observed 375 microM G4. The result is related to the stronger electrostatic and hydrophobic forces in G4, owing to the presence of two charged headgroups and two hydrophobic hydrocarbon tails that make it to bind strongly to the protein compared to its single chain counterpart, CTAB, resulting in larger unfolding. The stabilization at higher concentrations is attributed to the highly hydrophobic microdomain of the G4 aggregates formed at such concentrations. The results of the multi-technique approach are consistent with the fact that the gemini surfactants are more efficient than their conventional single-chain counterparts and hence may be used more effectively in the renaturation of proteins produced in the genetically engineered cells via the artificial chaperone protocol, as solubilizing agents to recover proteins from insoluble inclusion bodies and in drug delivery.

Physical Characterization of Clostridium Difficile Toxins and Toxoids: Effect of the Formaldehyde Crosslinking on Thermal Stability

Nosocomial diarrhea and pseudomembranous colitis causing toxins A and B from Clostridium difficile were studied at pH 5-8 and over the temperature range of 10-85 degrees C. The proteins were crosslinked with formaldehyde to inactivate them to toxoid forms and permit their use as vaccines. Structural changes and aggregation behavior were monitored with circular dichroism, intrinsic and extrinsic (ANS) fluorescence spectroscopy, turbidity measurements, high-resolution UV absorbance spectroscopy and dynamic light scattering. The combined results were summarized in empirical phase diagrams. Toxins A and B had similar secondary structure with a combination of helical, beta-sheet and unordered character and were partially unfolded at pH 5-5.5. Upon heating, toxin A at all pH values partially unfolded at approximately 45 degrees C and formed insoluble aggregates at approximately 50 degrees C. Toxin B partially unfolded at approximately 40 degrees C, and upon heating to approximately 50 degrees C precipitated at pH 5.0-6.0 and formed soluble aggregates at pH 6.5-7.5. The thermal stability of the toxins was pH-dependent with the proteins more thermally stable at higher pH. Similar studies of formaldehyde crosslinked toxoids A and B revealed enhanced thermal stability, in which secondary and tertiary structure changes as well aggregation were delayed by about 10 degrees C. These studies reveal both similarities and differences between C. difficile toxins A and B and demonstrate the stabilizing effect of formaldehyde crosslinking on the thermal stability of their corresponding toxoids.

Formation of a molten globule like state in bovine serum albumin at alkaline pH

Little work has been done to understand the folding profiles of multi-domain proteins at alkaline conditions. We have found the formation of a molten globule-like state in bovine serum albumin at pH 11.2 with the help of spectroscopic techniques; like far and near ultra-violet circular dichroism, intrinsic and extrinsic fluorescence spectroscopy. Interestingly, this state has features similar to the acid-denatured state of human serum albumin at pH 2.0 reported by Muzammil et al. (Eur J Biochem 266:26-32, 1999). This state has also shown significant increase in 8-anilino-1-naphthalene-sulfonate (ANS) binding in compare to the native state. At pH 13.0, the protein seems to acquire a state very close to 6 M guanidinium hydrochloride (GuHCl) denatured one. But, reversibility study shows it can regain nearly 40% of its native secondary structure. On the contrary, tertiary contacts have disrupted irreversibly. It seems, withdrawal of electrostatic repulsion leave room for local interactions, but disrupted tertiary contacts fail to regain their original states.

Differential effects of a green tea derived polyphenol (−)‐epigallocatechin‐3‐gallate on the muscle‐specific decrease in calcium response to acidosis

(−)‐Epigallocatechin‐3‐gallate (EGCg), a green tea derived polyphenol, has received much attention as a protective agent against cardiovascular diseases. In this study, we determined the effects of EGCg on changes in pH sensitivity of myofilament response to Ca2+ in myofibrils prepared from porcine ventricular myocardium and chicken pectoral muscle. In comparison with skeletal muscle, lowering pH from 7.0 to 6.5 and/or 6.0 EGCg has significantly blunted the acidic effect on the reduction of Ca2+ sensitivity for myofibrillar ATPase activity in cardiac muscle. Studies with recombinant mouse cardiac (cTnC) and chicken fast skeletal troponin C (sTnC) by circular dichroism (CD), intrinsic and extrinsic fluorescence spectroscopy showed that EGCg specifically binds to cTnC with a dissociation constant ~ 3–4 μM, but no effect on sTnC. Presumably binding to the cTnC C‐lobe, EGCg acted to alter the Ca2+‐binding to cTnC and compromised the interference of proton effect on the Ca2+ binding at acidic pH. To demonstrate the isoform‐specific effects on the action of EGCg, the pH‐sensitivity of Ca2+ response was examined in cardiac myofibrils replaced with either sTnC or cTnC and skeletal myofibrils exchanged with whole cardiac Tn complex (cTn). The data obtained suggested that EGCg interactions with the cardiac isoform specific TnC or Tn complex could alter the pH effect on myofilament Ca2+ sensitivity in striated muscle.

Differential effects of a green tea-derived polyphenol (−)-epigallocatechin-3-gallate on the acidosis-induced decrease in the Ca2+ sensitivity of cardiac and skeletal muscle

(-)-Epigallocatechin-3-gallate (EGCg), a green tea-derived polyphenol, has received much attention as a protective agent against cardiovascular diseases. In this study, we determined its effects on the acidosis-induced change in the Ca(2+) sensitivity of myofilaments in myofibrils prepared from porcine ventricular myocardium and chicken pectoral muscle. EGCg (0.1 mM) significantly inhibited the decrease caused by lowering the pH from 7.0 to 6.0 in the Ca(2+) sensitivity of myofibrillar ATPase activity in cardiac muscle, but not in skeletal muscle. Studies on recombinant mouse cardiac troponin C (cTnC) and chicken fast skeletal troponin C (sTnC) using circular dichroism and intrinsic and extrinsic fluorescence spectroscopy showed that EGCg bound to cTnC with a dissociation constant of approximately 3-4 muM, but did not bind to sTnC. By presumably binding to the cTnC C-lobe, EGCg decreased Ca(2+) binding to cTnC and overcame the depressant effect of protons on the Ca(2+) sensitivity of the cardiac contractile response. To demonstrate isoform-specific effects of the action of EGCg, the pH sensitivity of the Ca(2+) response was examined in cardiac myofibrils in which endogenous cTnC was replaced with exogenous sTnC or cTnC and in skeletal myofibrils in which the endogenous sTn complex was replaced with whole cardiac Tn complex (cTn). The results suggest that the binding of EGCg to the cardiac isoform-specific TnC or Tn complex alters the effect of pH on myofilament Ca(2+) sensitivity in striated muscle.

Parathyroid hormone is a heparin/polyanion binding protein: binding energetics and structure modification.

The interaction of four representative polyanions with parathyroid hormone (PTH) residues 1-84 has been investigated utilizing a variety of spectroscopic and calorimetric techniques. Each of the polyanions employed demonstrate enthalpically driven binding to PTH (1-84) with significant affinity. The polyanions heparin, dextran sulfate, phytic acid, and sucrose octasulfate induce alpha-helical structure in PTH to varying extents depending on the ratio of polyanion to protein employed. Intrinsic and extrinsic fluorescence spectroscopy suggests significant protein tertiary structure alteration upon polyanion binding. Although structural modification occurred upon polyanion binding, PTH colloidal stability was increased depending on the ratio of polyanion to protein used. Nevertheless, the bioactivity of PTH in the presence of various ratios of heparin was not altered. The potential biological significance of PTH/polyanion interactions is discussed.

Partially Folded Conformations of Bovine Liver Glutamate Dehydrogenase Induced by Mild Acidic Conditions

The acid-induced unfolding of bovine liver glutamate dehydrogenase (GDH) was studied using various spectroscopic methods such as far- and near-UV circular dichroism (CD), intrinsic and 1-anilino naphthalene-8-sulphonate (ANS) extrinsic fluorescence spectroscopy, light scattering and fluorescence quenching in 20 mM mixed buffer at various pHs. CD spectra show that at pH 3.5, GDH retains its secondary structure substantially, whereas its tertiary structure content is reduced considerably. Intrinsic fluorescence of GDH and ANS binding suggest that, at pH 3.5, the hydrophobic surface of enzyme is more exposed in comparison to the native form. Acrylamide quenching indicates more exposure of tryptophan residues of enzyme at pH 3.5 in comparison to pH 7.5. Another partially unfolded intermediate was detected at pH 5.0, which with its ANS binding capacity lies between the pH 3.5 intermediate and the native form of the enzyme. Gel filtration results revealed that the enzyme at pH 3.5 is dissociated into trimeric species whereas it exists as hexamer at pH 7.5 and 5.0. All the data taken together suggest the existence of two partially unfolded states of GDH at moderate acidic pHs which may be considered as molten and pre-molten globule-like states.

Stepwise modification of lysine residues of glucose oxidase with citraconic anhydride

Structural properties of modified forms of glucose oxidase made by stepwise specific modification have been investigated. By a single step modification, one of the modified forms resulted in the conversion of native structure of glucose oxidase to molten globule like form [S. Hosseinkhani, B. Ranjbar, H. Naderi-Manesh, M. Nemat-Gorgani, FEBS Lett. 561 (2004) 213–216]. Chemical modification of lysine residues in glucose oxidase was carried out using different concentration of citraconic anhydride. Modification brought about changes in the tertiary structure with some degree of alteration in secondary structure. FTIR, far and near-UV CD spectropolarimetry, intrinsic and extrinsic fluorescence spectroscopy showed structural changes of glucose oxidase in a concentration dependent manner. This was supported by comparative study of secondary and tertiary structure.

Effect of pH and ionic strength on the physical stability of adenovirus type 5

The thermal stability of adenovirus type 5 (Ad5) was investigated over the pH range 3−8 employing a variety of biophysical techniques under conditions of low and high ionic strength. Analysis of the structural stability of Ad5 by dynamic light scattering, intrinsic and extrinsic fluorescence, and second derivative UV absorption spectroscopies suggest that the capsid stability of Ad5 increases with decreasing pH under both ionic strength conditions. Significant aggregation, however, was observed at pH≤5 under conditions of low ionic strength. These studies also suggest that the physical stability of Ad5 is significantly enhanced under acidic conditions in the presence of 1M NaCl. Evaluation of the quaternary structural stability of Ad5 by dynamic light scattering and extrinsic fluorescence spectroscopy suggest that the Ad5 capsid undergoes a two-step dismantling process wherein the viral particles initially expand in size near 50°C and the DNA core is at least partially exposed to the surrounding solvent. Complete capsid disassembly and total exposure of the DNA core follows at higher temperatures. Data generated during these studies were combined employing a multidimensional eigenvector approach that combines data from numerous techniques into a colored representation. This picture, or “empirical phase diagram,” provides an intuitive representation of the physical stability of Ad5 over the pH range 4−8 from 10°C to 85°C. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Structural modulation of calmodulin and calmodulin-dependent protein kinase II by pea protein hydrolysates

The effects of two fractions of pea protein hydrolysate with high levels of positively charged amino acids on the structural conformations of calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) were determined using fluorescence and circular dichroism methods. In the presence of Ca2 + , addition of the protein hydrolysates to CaM and CaM/CaMKII complex led to increased exposure of aromatic groups as measured by intrinsic and extrinsic fluorescence spectroscopy. Near-UV circular dichroism data revealed an increase in the tertiary structure of CaM in the presence of pea protein hydrolysates. Effect of the protein hydrolysates on the CaM structure was greater with the fraction that contained higher contents of arginine and lysine when compared with the fraction with lower levels of these two amino acids. We concluded that the presence of the pea protein hydrolysates led to rearrangement of the native protein structure and exposure of buried hydrophobic groups of CaM and/or CaMKII.

Hydrostatic Pressure Induces the Fusion-active State of Enveloped Viruses

Enveloped animal viruses must undergo membrane fusion to deliver their genome into the host cell. We demonstrate that high pressure inactivates two membrane-enveloped viruses, influenza and Sindbis, by trapping the particles in a fusion-intermediate state. The pressure-induced conformational changes in Sindbis and influenza viruses were followed using intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, and fusion, plaque, and hemagglutination assays. Influenza virus subjected to pressure exposes hydrophobic domains as determined by tryptophan fluorescence and by the binding of bis-8-anilino-1-naphthalenesulfonate, a well established marker of the fusogenic state in influenza virus. Pressure also produced an increase in the fusion activity at neutral pH as monitored by fluorescence resonance energy transfer using lipid vesicles labeled with fluorescence probes. Sindbis virus also underwent conformational changes induced by pressure similar to those in influenza virus, and the increase in fusion activity was followed by pyrene excimer fluorescence of the metabolically labeled virus particles. Overall we show that pressure elicits subtle changes in the whole structure of the enveloped viruses triggering a conformational change that is similar to the change triggered by low pH. Our data strengthen the hypothesis that the native conformation of fusion proteins is metastable, and a cycle of pressure leads to a final state, the fusion-active state, of smaller volume.

Fluorescence characterization of structural transitions at the strong actin binding motif in skeletal myosin affinity labeled at cysteine 540 with novel spectroscopic cysteaminyl mixed disulfides.

We have synthesized the luminescent and fluorescent lanthanide chelate S-(2-nitro-5-thiobenzoic acid)cysteaminyldiethylenetriaminepentaacetate-5-[(2-aminoethyl)am ino ]naphthalene-1-sulfonic acid as well as the fluorescent analogue S-(2-nitro-5-thiobenzoic acid)cysteaminyl-5-carboxyfluorescein using the procedure we recently described [Bertrand, R., Capony, J.-P., Derancourt, J., and Kassab, R. (1999) Biochemistry 38, 11914-11925]. Both mixed disulfides react with the skeletal myosin motor domain (S-1) as actin site-directed agents and label exclusively and stoichiometrically Cys 540 in the hydrophobic strong actin binding helix-loop-helix motif, causing only a 1.9-2.4-fold decrease in the V(max) for acto-S-1 ATPase. The covalently attached cysteaminyl probe side chain spans maximally 17 and 8 A, respectively, and the fluorophores have different polarity, volume, and flexibility. Thus, they may provide complementary spectroscopic information on the environmental properties of this critical actin binding region. Here, we have analyzed by extrinsic fluorescence spectroscopy S-1 derivatized with the fluorescein label or with the Tb(3+) or Eu(3+) chelate of the other label to assess the conformational transitions precisely occurring at this site upon interaction with F-actin, nucleotides, or phosphate analogues. For either label, specific spectral changes of significant amplitude were obtained, identifying at least two major structural states. One was mediated by rigor binding of F-actin in the absence or presence of MgADP. It was abolished by MgATP, and it was not produced by the binding of nonpolymerizable G-actin. A modeling of the corresponding changes in the intensity and lambda(max) of the fluorescence emission spectra, achieved using the fluorescent adducts of 2-mercaptoethanol in varying concentrations of dimethylformamide, illustrates the predicted apolar nature of the strong acto-S-1 interface. A second state was promoted by the binding of ATP, AMP-PNP, ADP.AlF4, ADP. BeFx, or PP(i). It should be prevalent in the weak acto-S-1 binding complexes. The accompanying fluorescence intensity reduction, observed with each label, in both the absence and presence of F-actin, would result from a specific modification by these ligands of the probe orientation and/or solvent accessibility as suggested by acrylamide quenching experiments. It could represent the spectral manifestation of the predicted allosteric linkage from the ATPase site to the strong actin binding site of S-1 that modulates the acto-S-1 affinity. Our study offers the basis necessary for further detailed spectroscopic investigations on the conformational dynamics in solution of the stereospecific and hydrophobic actin binding motif during the skeletal cross-bridge cycle.

Deconstructing the interaction of glu-plasminogen with its receptor α-enolase

Objective: Plasminogen binds with apparent low affinity to cell-surface receptors via its lysine binding sites. This enhances/stabilizes the activation-susceptible conformation. However, it is not known whether this lysine-mediated conformational change of plasminogen may affect its subsequent dissociation rate and hence its stability at the cell surface. Therefore, we sought to determine the relationship between the lysine-dependent conformation of plasminogen and its dissociation rate from its receptor.Design: BIACORE experiments were used to determine the kinetics of the interaction of glu-plasminogen with its receptor α-enolase. Intrinsic and extrinsic fluorescence spectroscopy were utilized to confirm if α-enolase induced a conformational change to glu-plasminogen as predicted by analyses of the BIACORE data.Results: The dissociation of glu-plasminogen from α-enolase was mediated by at least two components with apparent dissociation rate constants of kd1= 4.7 × 10−2s−1and kd2= 1.6 × 10−3s−1. This second slower dissociation event reflects an increase in the stability of the complex. Global analysis of the interaction suggested a two-state conformational change reaction, mediated by a concentration-dependent increase in the initial association rate constant. The apparent Kdpredicted by this analysis was 1μM. Fluorescence spectroscopy confirmed that α-enolase induced a more open conformation of glu-plasminogen.Conclusions: These results provide direct evidence that the binding of glu-plasminogen to α-enolase is not simply a low-affinity interaction, but involves a multivalent, competition binding reaction that is associated with a glu-plasminogen conformational change. This mechanism is compatible with the structure of glu-plasminogen. This has implications for the stability of binding and activation of glu-plasminogen at the cell surface.

Extrinsic fluorescence probe study of human serum albumin using Nile red

Nile red bound to human serum albumin (HSA) shows an order of magnitude increase in the probe's fluorescence intensity. Here, we report on the fluorescence characteristics of the probe-protein complex in Trizma buffer (pH 7.1), urea, guanidine hydrochloride, and AOT/isooctane/buffer reverse micelles using both steady-state and time-resolved fluorescence techniques. With a view to illustrating the use of extrinsic probe fluorescence spectroscopy in protein research, we demonstrate that protein unfolding can be observed through measurements of the probe's time-resolved anisotropy and steady-state fluorescence spectrum. Moreover, this shows that thermal unfolding is fundamentally different from using denaturant, with respect to changes in both the nanosecond diffusional rotation of the probe at intermediate stages and in the denatured protein's structure. Also, the large Stokes shift of Nile red allows the changes in the environment of the probe-protein complex in reverse micelles of varying waterpool size to be easily identified in the steady-state fluorescence. This was not seen in earlier work exploiting the intrinsic tryptophan fluorescence of HSA and further demonstrates the complementary information that extrinsic fluorescence probe studies can offer protein science. We discuss the complex acrylamide quenching characteristics of Nile red bound to HSA in terms of the possibility of at least two binding sites for the probe and the effect of acrylamide on the probe-protein structure at very high quencher concentrations.