Fluorescence enhancement of warfarin induced by interaction with β‐cyclodextrin

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism (blood clots). In aqueous media, warfarin forms inclusion complexes with a family of cyclic oligosaccharides, α, β, γ‐cyclodextrins (CD). The formation of these complexes results in enhancement of the fluorescence of warfarin. Such spectroscopic changes offer a venue for the development of bioanalytical methodologies for warfarin quantification in biological liquids. We characterized the photophysical properties of warfarin in solvents with varying polarity and viscosity. The fluorescence quantum yield of warfarin correlated: (1) strongly with the solvent viscosity (R = 0.979) and (2) weakly with the solvent polarity (R = 0.118). These findings indicate that it is the change of the viscosity, rather than polarity, of the microenvironment that causes the fluorescence enhancement of warfarin upon binding to β‐CD. Utilizing the observed fluorescence enhancement in fluorescence titration measurements, the binding constants of warfarin to β‐CD were obtained (2.6 × 10² M^?1–3.7 × 10² M^?1). Using multivariable linear analysis, we extracted the stoichiometry of warfarin‐β‐CD interaction (1:1). © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Charge-shift probes of membrane potential: a probable electrochromic mechanism for p-aminostyrylpyridinium probes on a hemispherical lipid bilayer.

The characteristics of the spectroscopic responses to membrane potential are examined for a series of dyes based on the 4-(p-aminostyryl)-1-pyridinium chromophore. An apparatus using an oxidized cholesterol hemispherical bilayer and phase-sensitive detection provides response spectra in either transmission or fluorescence excitation modes. All the probes with good binding properties display biphasic response spectra that are similar in both shape and magnitude. Detailed analysis of the response spectra allows all the previously discovered mechanisms for extrinsic potential sensitive molecular probes, which require a change in the probe's chemical environment, to be ruled out. The data are consistent with an electrochromic mechanism. Polarized fluorescence intensities from the membrane-bound probes indicate that the chromophore is optimally oriented for an electrochromic response.     

Characterization of serotonin in protein and membrane mimetic environments: a spectroscopic study

As a first step toward using the photophysical properties of serotonin to probe its interactions with biological target sites, we have examined its interactions with human serum albumin (HSA), chosen as a surrogate for the actual receptor proteins in physiological systems, and with sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/heptane/water reverse micelles, chosen as a biomembrane mimetic environment for the transmembrane portion of the receptor protein. Although the emission maximum of serotonin is relatively insensitive to the polarity of the local environment, which is attributed to lack of solvent dipolar reorientation of the 5-hydroxyindole chromophore, the fluorescence anisotropy (r) served as a useful and sensitive parameter from which the binding constants (K) and Gibbs energy changes (deltaG) were estimated for serotonin-HSA and serotonin-AOT reverse micellar interactions. Fluorescence-decay studies of serotonin show double-exponential kinetics in homogeneous aqueous solvent due to the structural heterogeneity arising from different rotamers of serotonin. In contrast, upon binding to HSA, a single-exponential fluorescence-decay profile was observed indicating the occurrence of a single structural species of serotonin in the protein environment. Furthermore, far-UV-circular-dichroism (CD) spectroscopic data indicate that the secondary structural features of HSA remain essentially intact after binding to serotonin. This preliminary research can be expected to open the door to extensive future studies on interactions of serotonin with relevant target proteins and associated cell membranes involved in its diverse physiological functions.     

Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA,protein and lipid

Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein–ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2‐(2′‐hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein‐binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed. Copyright © 2016 John Wiley & Sons, Ltd.     

Optical spectroscopic characterization of single tryptophan mutants of chicken skeletal troponin C: evidence for interdomain interaction.

The effects of metal ion binding on the optical spectroscopic properties and temperature stability of two single tryptophan mutants of chicken skeletal TnC, F78W and F154W, have been examined. The absence of tyrosine and other tryptophan residues allowed the unambiguous assignment of the spectral signal from the introduced Trp residue. Changes in the molar ellipticity values in the far-UV CD spectra of the mutant proteins on metal ion binding were similar to those of wild-type TnC suggesting that the introduction of the Trp residue had no effect on the total secondary structure content. The fluorescence and near-UV absorbance data reveal that, in the apo state, Trp-78 is buried while Trp-154 is exposed to solvent. Additionally, the highly resolved (1)L(b) band of Trp-78 seen in the near-UV absorbance and CD spectra of the apo state of F78W suggest that this residue is likely in a rigid molecular environment. In the calcium-saturated state, Trp-154 becomes buried while the solvent accessibility of Trp-78 increases. The fluorescence emission and near-UV CD of Trp-78 in the N-terminal domain were sensitive to calcium binding at the C-terminal domain sites. Measurements of the temperature stability reveal that events occurring in the N-terminal domain affect the stability of the C-terminal domain and vice versa. This, coupled with the titration data, strongly suggests that there are interactions between the N- and C-terminal domains of TnC.     

Molecular interaction of manganese based carbon monoxide releasing molecule (MnCORM) with human serum albumin (HSA)

In the present study, the interaction between the HSA and MnCORM in vitro under physiological conditions, was investigated through ultraviolet-visible (UV-vis) absorption, fluorescence, time-resolved fluorescence, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopic techniques and in silico molecular docking methods. Binding parameters such as the binding constant, number of binding sites and binding force were obtained from the fluorescence data. Thermodynamic interaction revealed that the reaction was spontaneous (ΔG < 0) and hydrogen bond and van der Waals interaction were primarily involved in the binding. The changes induced in the secondary structure conformation due to the MnCORM interaction were monitored using CD and FT-IR spectroscopic techniques. The results showed reduction in α-helix conformation and corresponding increase in β-sheet and unordered structures due to slight unfolding. The time-resolved fluorescence decay confirmed the static quenching mechanism of the MnCORM. The molecular docking studies revealed that the MnCORM interacted at Sudlow’s site II of domain IIIA through hydrogen bond and van der Waals interactions. In order to understand the drug distribution and elimination, studies on the drug molecule interaction with HSA are vital. Therefore, it is evident that MnCORM interacts with HSA through ground state complex formation and thus suitable for in vivo delivery.     

The flavin environment in old yellow enzyme. An evaluation of insights from spectroscopic and artificial flavin studies

Spectroscopic and chemical modification studies of modified flavins bound to old yellow enzyme have led to predictions about the flavin environment of this enzyme. These studies analyzed solvent accessibility and hydrogen bonding patterns of particular flavin atoms, in addition to suggesting amino acid residues that are in close proximity to those atoms. Here, these studies are evaluated in the light of the crystal structure of old yellow enzyme to reveal that the spectroscopic and modified flavin results are generally consistent with the crystal structure. This highlights the fact that these are useful methods for studying flavin binding site structure. Although several of the inferred properties of the flavin environment are not consistent with the crystal structure, these discrepancies occurred in cases where an incorrect choice was made from among multiple plausible explanations for an experimental result. We conclude that modified flavin studies are powerful probes of flavin environment; however, it is risky to specify details of interactions, especially because of uncertainties due to induced charge delocalization in the flavin.     

Acrylodan-conjugated cysteine side chains reveal conformational state and ligand site locations of the acetylcholine-binding protein

We undertook cysteine substitution mutagenesis and fluorophore conjugation at selected residue positions to map sites of ligand binding and changes in solvent exposure of the acetylcholine-binding protein from Lymnaea stagnalis, a nicotinic receptor surrogate. Acrylodan fluorescence emission is highly sensitive to its local environment, and when bound to protein, exhibits changes in both intensity and emission wavelength that are reflected in the degree of solvent exclusion and the effective dielectric constant of the environment of the fluorophore. Hence, cysteine mutants were generated based on the acetylcholine-binding protein crystal structure and predicted ligand binding sites, and fluorescence parameters were assayed on the acrylodan-conjugated proteins. This approach allows one to analyze the environment around the conjugated fluorophore side chain and the changes induced by bound ligand. Introduction of an acrylodan-cysteine conjugate at position 178 yields a large blue shift with alpha-bungarotoxin association, whereas the agonists and alkaloid antagonists induce red shifts reflecting solvent exposure at this position. Such residue-selective changes in fluorescence parameters suggest that certain ligands can induce distinct conformational states of the binding protein, and that mutually exclusive binding results from disparate portals of entry to and orientations of the bound alpha-toxin and smaller acetylcholine congeners at the binding pocket. Labeling at other residue positions around the predicted binding pocket also reveals distinctive spectral changes for alpha-bungarotoxin, agonists, and alkaloid antagonists.     

Pressure effect on the stability and the conformational dynamics of the D-Galactose/D-Glucose-binding protein from Escherichia coli

The effect of the pressure on the structure and stability of the D-Galactose/D-Glucose binding protein (GGBP) from Escherichia coli was studied by steady-state and time-resolved fluorescence spectroscopy, and the ability of glucose ligand to stabilize the GGBP structure was also investigated. Steady-state fluorescence experiments showed a marked quenching of fluorescence emission of GGBP in the absence of glucose. Instead, the presence of glucose seems to stabilize the structure of GGBP at low and moderate pressure values. Time-resolved fluorescence measurements showed that the GGBP taumean in the absence of glucose varies significantly up to 600 bar, while in the presence of the ligand it is almost unaffected by pressure increase up to 600 bar. The effect of the pressure on GGBP was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results and confirm that the presence of glucose is able to contrast the negative effects of pressure on the protein structure. Taken together, the spectroscopic and computer simulation studies suggest that at pressure values up to 2000 bar the structure of GGBP in the absence of glucose remains folded, but a significant perturbation of the protein secondary structures can be detected. The binding of glucose reduces the negative effect of pressure on protein structure and confers protection from perturbation especially at moderate pressure values.     

Investigations of the effects of ethanol on warfarin binding to human serum albumin

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37 degrees C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 +/- 0.2 microM, 0.1% ethanol 5.86 +/- 0.1 microM, 0.3% ethanol 5.83 +/- 0.2 microM, 0.5% ethanol 6.76 +/- 0.1 microM, 1% ethanol 7.01 +/- 0.1 microM, 3% ethanol 9.9 +/- 0.7 microM, 5% ethanol 13.01 +/- 0.1 microM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6. 62 +/- 1.6 microM, 0.1% ethanol 6.81 +/- 1.1 microM, 0.3% ethanol 8. 26 +/- 2.5 microM, 0.5% ethanol 8.86 +/- 1.9 microM, 1% ethanol 11. 01 +/- 4.2 microM, 3% ethanol 20.75 +/- 2.4 microM, 5% ethanol 21.67 +/- 2.2 microM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.     

An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures

Aptamers can be highly specific for their targets, which implies precise molecular recognition between aptamer and target. However, as small polymers, their structures are more subject to environmental conditions than the more constrained longer RNAs such as those that constitute the ribosome. To understand the balance between structural and environmental factors in establishing ligand specificity of aptamers, we examined the RNA aptamer (NEO1A) previously reported as specific for neomycin-B. We show that NEO1A can recognize other aminoglycosides with similar affinities as for neomycin-B and its aminoglycoside specificity is strongly influenced by ionic strength and buffer composition. NMR and 2-aminopurine (2AP) fluorescence studies of the aptamer identified a flexible pentaloop and a stable binding pocket. Consistent with a well-structured binding pocket, docking analysis results correlated with experimental measures of the binding energy for most ligands. Steady state fluorescence studies of 2AP-substituted aptamers confirmed that A16 moves to a more solvent accessible position upon ligand binding while A14 moves to a less solvent accessible position, which is most likely a base stack. Analysis of binding affinities of NEO1A sequence variants showed that the base in position 16 interacts differently with each ligand and the interaction is a function of the buffer constituents. Our results show that the pentaloop provides NEO1A with the ability to adapt to external influences on its structure, with the critical base at position 16 adjusting to incorporate each ligand into a stable pocket by hydrophobic interactions and/or hydrogen bonds depending on the ligand and the ionic environment.     

Examination of EmrE conformational differences in various membrane mimetic environments.

Ethidium multidrug resistance protein (EmrE) is a member of the small multidrug resistance family of proteins and is responsible for resistance in Escherichia coli to a diverse group of lipophilic cations. Research is beginning to elucidate structural information as well as substrate binding and extrusion mechanisms for this protein. However, the choice of membrane mimetic environment to perform structural studies needs to be made. In this study EmrE was solubilized in different membrane mimetic environments to investigate the influence of environment on the structure and dynamics of the protein by comparing the fluorescence properties of emission maxima, peak shifts, relative intensities, acrylamide quenching constants, and polarization. Taken together, the different fluorescence observations on EmrE in the various membrane mimetic systems tested suggest that the tryptophan residues in EmrE are present in the most flexible and exposed state when solubilized in methanol, followed by sodium dodecyl sulfate and urea. The two detergents N-dodecyl-beta-D-maltoside (DM) and polyoxyethylene(8)dodecyl ether, for the most part, only display subtle differences between the spectral properties with DM best representing the lipid environment. The conformation of EmrE is clearly more open and dynamic in detergent relative to being reconstituted in small unilamellar vesicles. The fluorescence observations of EmrE solubilized in trifluoroethanol shows an environment that is similar to that of EmrE solubilized in detergents. Additionally, secondary structure was monitored by circular dichroism (CD). The CD spectra were similar among the different solubilizing conditions, suggesting little difference in alpha-helical content. This work establishes groundwork for the choice of solubilizing conditions for future structural, folding, and ligand binding studies.     

Solvent dependent photocyclization and photophysics of some 2-ethynylbiphenyls.

The spectroscopic properties and photochemical behavior of molecules having 2-ethynylbiphenyl or 2-phenyldiphenylacetylene structures are reported. These molecules undergo photocyclization reactions to yield phenanthrene and dihydrophenanthrene products via putative isophenanthrene (cyclic allene) intermediates. Phenanthrene formation from the isophenanthrene intermediates does not occur via a unimolecular sigmatropic hydrogen shift, but rather by protonation or hydrogen abstraction mechanisms involving the solvent. Cyclization efficiencies are much lower than is the case for previously-investigated 2-vinylbiphenyl systems. The 2-phenyldiphenylacetylenes have high fluorescence quantum yields and long singlet lifetimes when compared to diphenylacetylene. The 2-ethynylbiphenyls decay via a combination of fluorescence and intersystem crossing.     

Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity

While antimicrobial and cytolytic peptides exert their effects on cells largely by interacting with the lipid bilayers of their membranes, the influence of the cell membrane lipid composition on the specificity of these peptides towards a given organism is not yet understood. The lack of experimental model systems that mimic the complexity of natural cell membranes has hampered efforts to establish a direct correlation between the induced conformation of these peptides upon binding to cell membranes and their biological specificities. Nevertheless, studies using model membranes reconstituted from lipids and a few membrane-associated proteins, combined with spectroscopic techniques (i.e. circular dichroism, fluorescence spectroscopy, Fourier transform infra red spectroscopy, etc.), have provided information on specific structure-function relationships of peptide-membrane interactions at the molecular level. Reversed phase-high performance chromatography (RP-HPLC) and surface plasmon resonance (SPR) are emerging techniques for the study of the dynamics of the interactions between cytolytic and antimicrobial peptides and lipid surfaces. Thus, the immobilization of lipid moieties onto RP-HPLC sorbent now allows the investigation of peptide conformational transition upon interaction with membrane surfaces, while SPR allows the observation of the time course of peptide binding to membrane surfaces. Such studies have clearly demonstrated the complexity of peptide-membrane interactions in terms of the mutual changes in peptide binding, conformation, orientation, and lipid organization, and have, to a certain extent, allowed correlations to be drawn between peptide conformational properties and lytic activity.     

Binding of teicoplanin and vancomycin to bovine serum albumin in vitro: a multispectroscopic approach and molecular modeling

In this paper, the binding properties of teicoplanin and vancomycin to bovine serum albumin (BSA) were investigated using fluorescence quenching, synchronous fluorescence, Fourier transform infrared (FTIR), circular dichroism (CD) and UV–vis spectroscopic techniques and molecular docking under simulative physiological conditions. The results obtained from fluorescence quenching data revealed that the drug–BSA interaction altered the conformational structure of BSA. Meanwhile, the 3D fluorescence, CD, FTIR and UV–vis data demonstrated that the conformation of BSA was slightly altered in the presence of teicoplanin and vancomycin, with different reduced α‐helical contents. The binding distances for the drug–BSA system were provided by the efficiency of fluorescence resonance energy transfer (FRET). Furthermore, the thermodynamic analysis implied that hydrogen bond and van der Waals' forces were the main interaction for the drug–BSA systems, which agreed well with the results from the molecular modeling study. The results obtained herein will be of biological significance in future toxicological and pharmacological investigation. Copyright © 2013 John Wiley & Sons, Ltd.     

D-trehalose/D-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis: the binding of trehalose and maltose results in different protein conformational states

In this work, we used fluorescence spectroscopy, molecular dynamics simulation, and Fourier transform infrared spectroscopy for investigating the effect of trehalose binding and maltose binding on the structural properties and the physical parameters of the recombinant D-trehalose/D-maltose binding protein (TMBP) from the hyperthermophilic archaeon Thermococcus litoralis. The binding of the two sugars to TMBP was studied in the temperature range 20 degrees-100 degrees C. The results show that TMBP possesses remarkable temperature stability and its secondary structure does not melt up to 90 degrees C. Although both the secondary structure itself and the sequence of melting events were not significantly affected by the sugar binding, the protein assumes different conformations with different physical properties depending whether maltose or trehalose is bound to the protein. At low and moderate temperatures, TMBP possesses a structure that is highly compact both in the absence and in the presence of two sugars. At about 90 degrees C, the structure of the unliganded TMBP partially relaxes whereas both the TMBP/maltose and the TMBP/trehalose complexes remain in the compact state. In addition, Fourier transform infrared results show that the population of alpha-helices exposed to the solvent was smaller in the absence than in the presence of the two sugars. The spectroscopic results are supported by molecular dynamics simulations. Our data on dynamics and stability of TMBP can contribute to a better understanding of transport-related functions of TMBP and constitute ground for targeted modifications of this protein for potential biotechnological applications.     

Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin. A spectroscopic study.

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem-human serum albumin (ferric HSA-haem) and of ferrous nitrosylated haem-human serum albumin (ferrous HSA-haem-NO) is reported. Ferric HSA-haem is hexa-coordinated, the haem-iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules--buried in close proximity to the haem binding pocket--induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem-iron, and the quenching of the 1H-NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA-haem, corresponding to the ibuprofen secondary cleft, are 5.4 +/- 1.1 x 10(-4) m and 2.1 +/- 0.4 x 10(-5) m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA-haem is lower than that reported for drug binding to haem-free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 +/- 0.2 x 10(-8) m in the absence of drugs to 1.5 +/- 0.2 x 10(-7) m in the presence of ibuprofen and warfarin. Ferrous HSA-haem-NO is a five-coordinated haem-iron system. Drug binding to the warfarin primary site of ferrous HSA-haem-NO induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear-cut evidence for the drug-induced shift of allosteric equilibrium(a) of HSA.     

Binding of europium(III) ions to RNA stem loops: role of the primary hydration sphere in complex formation

Understanding the process by which RNA molecules fold into stable structures includes study of the role of site-bound metal ions. Because the alkaline earth metal ions typically associated with RNA structure [most often Mg(II)] do not provide convenient spectroscopic signals, replacement with metal ions having spectroscopically useful properties has been a valuable approach. The luminescence properties of the lanthanide(III) series, in particular europium(III), have made them useful in the study of complexation with biomolecules. We review the physical, chemical, and spectroscopic characteristics of Eu(III) that contribute to its value as a probe of RNA-metal ion interactions, and examples of information obtained from studies of Eu(III) bound to small RNA stem loops. Although Eu(III) has similar site preference to Mg(II), luminescence and isothermal titration calorimetry measurements indicate that Ln(III) loses water molecules from the inner hydration sphere more readily than does Mg(II), resulting in more direct coordination between RNA and the metal ion and very different energetics of binding. In some cases, e.g., a GAAA tetraloop, binding appears to occur by a lock and key process; in the same base sequence containing certain deoxynucleoside substitutions that alter loop structure, binding appears to occur by an induced fit process.     

Denaturation of replication protein A reveals an alternative conformation with intact domain structure and oligonucleotide binding activity

Replication protein A (RPA) is a heterotrimeric, multidomain, single-stranded DNA-binding protein. Using spectroscopic methods and methylene carbene-based chemical modification methods, we have identified conformational intermediates in the denaturation pathway of RPA. Intrinsic protein fluorescence studies reveal unfolding profiles composed of multiple transitions, with midpoints at 1.5, 2.7, 4.2, and 5.3 M urea. CD profiles of RPA unfolding are characterized by a single transition. RPA is stabilized with respect to the CD-monitored transition when bound to a dA15 oligonucleotide. However, oligonucleotide binding appears to exert little, if any, effect on the first fluorescence transition. Methylene carbene chemical modification, coupled with MALDI-TOF mass spectrometry analysis, was also used to monitor unfolding of several specific RPA folds of the protein. The unfolding profiles of the individual structures are characterized by single transitions similar to the CD-monitored transition. Each fold, however, unravels with different individual characteristics, suggesting significant autonomy. Based on results from chemical modification and spectroscopic analyses, we conclude the initial transition observed in fluorescence experiments represents a change in the juxtaposition of binding folds with little unraveling of the domain structures. The second transition represents the unfolding of the majority of fold structure, and the third transition observed by fluorescence correlates with the dissociation of the 70- and 32-kD subunits.