Release of the neocarzinostatin chromophore from the holoprotein does not require major conformational change of the tertiary and secondary structures induced by trifluoroethanol.

Neocarzinostatin is a potent enediyne antitumor antibiotic complex in which a chromophore is noncovalently bound to a carrier protein. The protein regulates availability of the drug by proper release of the biologically active chromophore. To understand the physiological mechanism of the drug delivery system, we have examined the trifluoroethanol (TFE)-induced conformational changes of the protein with special emphasis on their relation to the release of the chromophore from holoneocarzinostatin. The effect of the alpha helix-inducing agent, TFE, on all the beta-sheet neocarzinostatin proteins was studied by circular dichroism, fluorescence, and (1)H NMR studies. By using binding of anilinonaphthalene sulfonic acid as a probe, we observed that the protein exists in a stable, partially structured intermediate state around 45-50% TFE, which is consistent with the results from tryptophan fluorescence and circular dichroism studies. The native state is stable until 20% TFE and is half-converted into the intermediate state at 30% TFE, which starts to collapse beyond 50%. High pressure liquid chromatographic analysis of the release of the chromophore caused by TFE treatment at 0 degrees C suggests that the release process, which occurs below 20% TFE, does not result from an observable conformational change in the protein. Kinetic measurements of the release of chromophore at 25 degrees C reveal that TFE does stimulate the rate of release, which increases sharply at 15% and reaches a maximum at 20% TFE, although no major secondary or tertiary structural change of the carrier protein is observed under these same conditions. Our data suggest that chromophore release results from a fluctuation of the protein structure that is stimulated by TFE. Complete release of the chromophore occurs at TFE concentrations where no overall observable unfolding of the apoprotein is seen. Thus, the results suggest that denaturation of the protein by TFE is not a necessary step for release of the tightly bound chromophore.

The biological function of the carrier apoprotein of the enediyne chromoprotein, which is a recently discovered natu-rally occurring antitumor antibiotic, has gained interest lately (1)(2)(3). One important pharmacological function is that the protein regulates availability of the drug by proper release of its biologically potent chromophore. To understand the physiological mechanism of the natural drug delivery system, it is interesting and essential to study how the protein releases the tightly bound chromophore.
The release of some small ligands such as dinitrophenyl (4) or NADH (5,6) has been well studied. The results show that the physiological mechanism of releasing a noncovalently bound cofactor from a protein complex can be triggered by a specific contact with some agent in the cell, for example, the anion-mediated iron release mechanism of transferrins (7). Alternatively, dissociation can also be caused by a spontaneous fluctuation of the protein conformation. The release of heme from myoglobin provides a good example (8,9). Currently, the molecular mechanism by which holoNCS 1 releases its chromophore is unclear. Initially, we wished to determine whether the protein needs to be unfolded for the release process.
Neocarzinostatin (NCS) is the first enediyne chromoprotein; it consists of a labile chromophore (NCS-Chr) (M r ϭ 659, Fig. 1) (10,11) that is tightly and noncovalently bound to an apoprotein (apoNCS) (113 amino acids) (12). The chromophore, with an unusual bicyclic dienediyne structure, is very potent in causing DNA damage. Upon cycloaromatization to a reactive radical intermediate, it abstracts hydrogens from the sugar moiety of DNA. Although apoNCS itself is inactive in inducing cleavage of DNA, it plays an important role in the drug action by protecting and storing the labile chromophore and by releasing the chromophore to the target DNA. It is of particular interest that NCS protein does not bind to DNA (13,14). There is evidence showing that release of the chromophore occurs before NCS interacts with DNA (14); DNA cleavage is believed to occur when the chromophore becomes activated after it is already intercalated into the DNA (12). HoloNCS thus regulates the availability of the drug by properly controlling the release of its bound chromophore. The biologically active chromophore has a very high affinity for its apoNCS (K d ϳ10 Ϫ10 M) (12). How holoNCS regulates the release of the tightly bound chromophore is completely unknown. It has been reported earlier that DNA cleavage induced by native NCS can be greatly stimulated by organic solvents and denaturants in vitro (14 -16). This effect might well result from a denaturation process that releases the active chromophore.
The primary structure of apoNCS ( Fig. 1) was first proposed in the early 70s (17,18), and later was revised several times (19 -22). The three-dimensional structure is almost superimposable on that of holoprotein, as determined by x-ray crystallography (23)(24)(25) and NMR (26 -37) studies. The only noteworthy difference between holo-and apoprotein is the position of the aromatic ring of the side chain of certain aromatic residues such as Phe 78 . The protein has two disulfide bridges and is kidney-shaped with two domains that hold a well defined binding cavity. The larger domain forms a seven-stranded antiparallel ␤-barrel, and the smaller domain consists of two antiparallel strands of ␤-sheet that are perpendicular to each other. Early chemical studies indicate that the first 20 amino acids do not associate with the active chromophore (38). The amino acid residues that form the hydrophobic binding pocket were assigned later by three-dimensional structural studies (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)39). Owing to its labile nature, after the chromophore molecule is released it quickly becomes inactivated, and only a few percent reach and cleave the target DNA (40). Although adding apoNCS can prevent inactivation, it also inhibits the release of the active chromophore (16). Apparently, a proper physiological timing of the releasing process plays an important role in the drug efficacy. On the other hand, although NCS is very potent against tumor cells, it damages normal cells as well (41). Understanding the release process of NCS could help to find a more selective delivery system that would facilitate the release of NCS-Chr only in specified conditions, as suggested by Ehrlich's idea of turning potential drugs into "magic bullets" (42).
To perform its biological activity, the chromophore needs to be released by somehow perturbing its strong affinity for the binding cavity that is formed by the all-␤-sheet apoNCS. Hydrophobic molecules may play a perturbing role in regulating the release of NCS-Chr. The penetration of [ 14 C]NCS into cell nuclei based on autoradiography was demonstrated in 1975 (13), which is before the discovery of the chromophore. The active chromophore is probably released either inside the cell nucleus or on the nuclear membrane. Simple organic co-solvents such as alcohols might effectively mimic the nonpolar cellular environment of the nuclear membrane. The ␣-helicogenic solvent, 2,2,2-trifluoroethanol (TFE), is a useful hydrophobic solvent, which mixes readily with water, and it might be effective in inducing a conformational change of the all ␤-sheet apoNCS. Therefore, we determine whether TFE induces a conformational change in apoNCS, and if so, whether the conformational change is correlated with chromophore release.

MATERIALS AND METHODS
NCS powder was obtained from Kayaku Laboratories Ltd. (Tokyo, Japan). The stock solution in water (0.87 mM) was stored in aliquots at Ϫ80°C in the dark. The apoNCS stock solution (0.13 mM) was prepared in a mixed buffer of 2.67 mM ammonium acetate (pH 5) and 20 mM sodium acetate (pH 5). TFE and 1-anilino-8-naphthalene sulfonic acid (ANS) were obtained from Merck and Sigma, respectively. 2,2,2-Trifluoroethanol-d 3 was purchased from Cambridge Isotope Laboratories. All solutions were prepared in 18.2 ohm of Milli-Q water, and the concen-tration of TFE was expressed as percentage of volume to volume.
Circular Dichroism-All CD measurements were carried out on a Jasco J715 spectropolarimeter. The instrument was calibrated with ammonium d-10-camphor sulfonate. The results are expressed as mean residue ellipticity, [], which is defined as [] ϭ obs /(113 ϫ l ϫ c), where obs is the observed ellipticity in degrees; c is the concentration in dmol per liter; l is the length of the light path in cm, and the constant 113 is the number of residues in apoNCS. The scan speed was 200 nm/min. The CD spectra, which are averaged from more than three scans, were measured with 10 M (final concentration) apoNCS containing requisite amounts (v/v) of TFE. A 1-mm quartz cell (about 300 l) was used for far-UV region (190 -250 nm) and 10-mm micro cell (about 70 l) for near-UV (240 to 320 nm) measurements. All spectra were recorded at 25 Ϯ 2°C and corrected for solvent and buffer blanks.
Fluorescence Spectroscopy-A Hitachi model F-4500 fluorescence spectrofluorimeter was used for measuring the effect of TFE on the fluorescence of NCS. A set of solutions (total volume, 250 l) containing the requisite amounts (v/v) of TFE was mixed with 5-10 M (final concentration) apoNCS. A rectangular micro cell (4 ϫ 10 mm) was used for measurements. The fluorescent emission spectra were recorded at the light path of 10 mm between 290 and 500 nm with an excitation wavelength of 280 nm. All spectra were corrected with solvent and buffer backgrounds. The resolution was 1.0 nm, and wavelength accuracy was within Ϯ 2 nm.
Anilinonaphthalene Sulfonic Acid Binding Studies-ANS binding studies were also carried out by Hitachi F-4500 spectrofluorimeter. A set of solutions was prepared as described previously except that apoNCS concentration was increased to 10 -50 M due to lower sensitivity. ANS was added at 10:1 molar ratio to apoNCS, and the total volume was 300 l. The fluorescent emission spectra were recorded between 375 and 625 nm with an excitation wavelength of 355 nm. All spectra were corrected with ANS, solvent, and buffer backgrounds. 1 H NMR Spectroscopy-1 H NMR spectra were recorded at 25°C on a Varian 600 MHz NMR spectrometer. About 2 mg of apoNCS was dialyzed against pure water and lyophilized to dryness before dissolving it into a mixture of H 2 O/D 2 O (9:1) containing requisite amounts (v/v) of TFE-d 3 . The pH of the unbuffered solution was at 5.
HPLC Analyses-Cycloaromatization reactions were assayed by Waters Millennium multisystems including model 600E solvent delivery system, model 996 photodiode array detector, and a model 474 fluorescence detector. The rate of multiwavelength measurement of photodiode array detector was 0.25 spectrum/s with 1.2 nm of resolution. The fluorescent excitation was 340 nm, and emission was detected at 440 nm with bandwidth of 40 nm. Separations were performed by a Waters -Bondapak reverse phase C18 column (particle size, 10 m; pore size, 125 Å; 0.39 ϫ 30 cm) with a 0.4-cm long guard column. The gradient was composed of 5 mM ammonium acetate, pH 4, in H 2 O/CH 3 OH with increasing concentrations of methanol at a flow rate of 1 ml/min. The typical gradient started from a sharp increase 0 -48% of methanol in 3 min and then a shallow linear increase to 80% in 62 min. A set of 10 M (final concentration) holoNCS solutions in 5 mM (final concentration) of ammonium acetate (pH 4) was mixed with 5 mM (final concentration) of GSH and Tris-HCl (100 mM, pH 7.0). Requisite percentage of TFE was added (final volume, 100 l) to facilitate the release of the chromophore. After 5 min of incubation at 0°C, the remaining intact chromophore that had been bound to the protein was analyzed by HPLC. The elution of NCS-Chr was detected at 45 Ϯ 2 min. Injections were done manually to ensure that the full amount of each solution was completely loaded.
Kinetic with 5 mM (final concentration) of GSH. The release of NCS-Chr was initiated by adding the requisite amount of TFE to the mixture (total volume, 300 l) in a rectangular micro cell (4 ϫ 10 mm). The sample temperature was controlled by a thermostatic cell holder equipped with a thermostatic circulator. Excitation was set at 340 nm at the 4-mm light path, and emission was recorded at 440 nm at the 10-mm light path. Because Time Scan Mode does not provide light shutter control function, which can lead to the light-induced degradation of NCS-Chr, a repetitive Wavelength Scan Mode with the shutter closed between each run was used. The wavelength interval was set at the minimum (10 nm, from 434 to 444 nm) with a scan rate of 1000 nm per min. The total light exposure time to the light-sensitive NCS sample was thus minimized to 36 s for an experiment that recorded data per min and monitored in 1 h. Measurement continued until the change of emission at 440 nm reached a plateau, which indicates that the reaction of the released chromophore has gone to completion.
The change in [holoNCS] with time was determined by increases of the fluorescent emission resulting from the thiol-induced drug product. Because the calibration curve shows a linear relation in the drug concentration range studied, the measured fluorescent emission in mV was directly converted into the concentration of the remaining intact chromophore that was bound to the protein by Equation 1: where F t is the measured fluorescent emission in mV at 440 nm at time t; F o is the initial reading after TFE was added; and F ϱ is the plateau reading after the reaction was completed.

RESULTS
Circular Dichroism-CD spectra were recorded at both nearand far-UV to monitor the conformational changes induced by titration of TFE. Within reasonable experimental errors, the CD spectrum of the native apoNCS (data not shown) is essentially consistent with one published recently (3). It shows a broad negative signal from 300 to 250 nm ([] 280 Х Ϫ0.6 ϫ 10 3 ) followed by a positive peak around 225 nm ([] 225 Х 0.2 ϫ 10 3 ) and a negative one centered at 212 nm ([] 212 Х Ϫ1 ϫ 10 3 ), indicative of the secondary structure being predominantly ␤-sheet.
The near-UV CD signal in proteins is believed to stem from aromatic side chains in the protein that have restricted motions, because of interactions with neighboring residues in the tertiary structure of the protein. In the case of apoNCS, the near-UV CD signal is centered on 280 nm. Fig. 2a shows the change in [] at 280 nm with TFE. Below 20% TFE, very little change of the tertiary structure is observed. As the TFE concentration increases above 20%, the mean residue ellipticity gradually increases with increase of the TFE concentration. The signals beyond 50% TFE are randomly scattered, because of the inherent nature of the relatively low intensity. The results suggest that considerable loss of the tertiary structural interactions in the protein occurs only above 20% TFE.
For the CD spectra in the far-UV region, addition of TFE up to 30% (v/v) appears to have no significant effect on the characteristic line features of apoNCS, although both [] 225 and [] 212 start to drop beyond 25% TFE to become less positive and more negative. Beyond 30%, the CD spectrum in the far-UV region, which monitors secondary structural transitions in proteins, shows a steep decrease in negative ellipticity in the region around 206 -222 nm. A large negative signal centered at 206 nm becomes obvious ([] 206 Х Ϫ4 ϫ 10 3 ) in 35% of TFE. Its intensity increases sharply with the increase of TFE content. Whereas it becomes about doubly intense at 45% TFE (compared with 35%) ([] 206 Х Ϫ8 ϫ 10 3 ), a broad negative shoulder also appears around 222 nm ([] 222 Х Ϫ5 ϫ 10 3 ). Such TFEinduced changes in spectra, which are close to helical spectra, are rather typical. Fig. 2b shows the change in [] at 215 nm, which is correlated with the conformational change of ␤-pleated backbone structure of apoNCS. The ellipticity value shows no significant change up to 25% TFE. Comparing the change at 215-280 nm, this result implies that the loss of tertiary structure may precede the transformation of the secondary structure induced by TFE. It is noteworthy to point out that an interesting shoulder appears around 45-50% TFE, suggesting that a stable intermediate or partially structured state forms on the way to unfolding. Presumably it is a chiefly helical state, based on its CD spectrum.
Fluorescence Spectroscopy-The change in tryptophan emission of apoNCS upon addition of TFE was studied by fluorescence spectroscopy. When excited at 280 nm, the emission maximum occurs at 346 nm with a slight red shift at higher TFE concentration (348 nm in 60% of TFE). Fig. 3a illustrates the change in the intensity of the fluorescence emission maximum with TFE. It is evident that the TFE treatment of NCS results in a marked loss of its fluorescence properties. Unlike CD spectra, which show no significant change until the amount of TFE is greater than 20% (near-UV) or 25% (far-UV), the TFE-induced fluorescent change becomes obvious at low concentrations (Ͻ20%). This result implies that the steric orientation of the aromatic side chain of the tryptophan residues may have changed, possibly because of hydrophobic interaction with TFE even at low TFE concentrations, although both the global and backbone skeleton of apoNCS still remain intact, based on CD data. It is very interesting to observe marginal but very clear increases of the fluorescence, starting from 30% TFE and reaching a maximum around 45-50%. These changes correspond very well with the observed shoulder in the change of the ellipticity at 215 nm. This result further supports the possible formation of a partially structured state induced by TFE. ANS Binding Studies-It has been shown that ANS, a fluorescent hydrophobic probe, has a stronger affinity for the molten globule intermediate than for the native protein or fully unfolded state (43). We carried out ANS binding studies with TFE-treated and -untreated NCS protein. Fig. 3b illustrates the TFE-induced changes of the intensity and the wavelength at the maximum emission of the fluorescence from ANS. Both changes are closely correlated with each other. The wavelength of maximum emission ( max ) of ANS exhibits a significant blue shift beyond 20% TFE (ϳ10 nm apart from 20 to 80% TFE). Beyond 20% TFE, the fluorescence also markedly increases to its maximum value at around 45-50% TFE, which corresponds to the shoulder region in the change of the wavelength of maximum intensity. Above 50% TFE the fluorescence gradually drops back to the initial values beyond 80% TFE. This result suggests that the tightly folded hydrophobic binding site of apoNCS can remain inaccessible to ANS molecules until 20% TFE, which is consistent with the observed tertiary structural changes analyzed from the ellipticity changes at 280 nm. Higher concentrations of TFE (20 -50%) relax the closely packed binding pocket into a stable partial structure. The partially opened structure increases the accessibility of hydrophobic sites for ANS binding. Excess TFE (50 -80%) unfolds the hydrophobic binding cavity completely, and the protein loses the ability to bind ANS. The stable partial structure with high ANS binding affinity is a characteristic feature of the molten globule state. It is noteworthy that a molten globule-like state is observed from the study of ANS binding in 45-50% TFE, which is consistent with the stable intermediate observed from the ellipticity change at 215 nm, as well as from the fluorescence change of tryptophan emission.
1 H NMR Spectroscopy-ApoNCS has been studied by sequential one-dimensional NMR spectroscopy in solvents that contain requisite amounts of TFE-d 3 . Fig. 4 illustrates the spectra. For the native form of apoNCS, the NMR lines are relatively narrow and show relatively good dispersion, which reflects the specific inter-residue interactions within the compact folded protein structure. Some line broadening is observed as the TFE concentration increases. The chemical shift changes induced by TFE are particularly evident in the aromatic region (6.5-7.5 ppm). Compared with the native state, the change induced by 10% TFE is already evident. This result is consistent with the observation from a fluorescence study using tryptophan emission. Apparently, the hydrophobic effect from even a small quantity of TFE is powerful enough to disturb the interaction or steric orientation of the aromatic side chain of nonpolar residues, whereas no major effect is observed on the secondary or tertiary structure of the protein under the same condition. Up to 20% TFE, the changes in NMR resonance signals can be monitored. Beyond that, large changes are observed. The spectrum of apoNCS in 40% TFE is drastically different from the one in native state and in 80%, in which the NMR spectrum shows that the protein is totally denatured.
HPLC Analyses for the Release of NCS-Chr-Once NCS-Chr is released from its protector protein, it becomes very labile and can barely be detected by aqueous chromatographic analysis. Furthermore, because the carrier protein can easily be dena-

FIG. 4. One-dimensional NMR spectra of apoNCS at 20°C in native state in 90% H 2 O/D 2 O (a); 10% TFE-d 3 /H 2 O (v/v) (b); 20% TFE-d 3 /H 2 O (v/v) (c); 40% TFE-d 3 /H 2 O (v/v) (d); and 80% TFE-d 3 / H 2 O (v/v) (e).
tured by a hydrophobic mobile phase such as methanol or acetonitrile in conventional reverse phase HPLC, it is technically difficult to distinguish whether or not the detected NCS-Chr has been released form TFE treatment. We have previously used GSH to distinguish qualitatively the bound NCS-Chr from the released form (44,45). GSH fails to interact with the bound form because of negative charge repulsion from the NCS protein, but it can effectively induce cycloaromatization of the enediyne nucleus of the unbound NCS-Chr to form an adduct (40). By adding GSH, all released NCS-Chr induced by TFE is inactivated by GSH, whereas the intact bound NCS-Chr can be detected by the established reverse phase chromatographic and spectroscopic methods (46). Fig. 5 demonstrates the analyzed percentage of the remaining bound NCS-Chr by HPLC from 1 nmol of holoNCS that was incubated with 5 mM GSH at 0°C. The incubation time was set at 5 min to facilitate the chromophore release, which is about the required minimum time for running a single spectroscopic spectrum to observe any conformational change of apoNCS. The results reveal that the percentage of chromophore release increases with TFE concentration until about 40% TFE. The nonlinear behavior at higher TFE concentrations probably occurs because of hydrophobic protection of the labile NCS-Chr, which reduces the rate of inactivation by GSH. Comparing the efficiency of TFE in causing chromophore release with that in causing the protein conformational change, it is apparent that the effect of TFE on the release of the chromophore is not correlated with the change of the backbone or tertiary structures of the protein. At 20% TFE, about 40% NCS-Chr is released within 5 min at 0°C, whereas the tightly folded protein structure remains stable in the same amount of TFE at a higher temperature, 25°C. In general, HPLC analyses suggest that TFE acts more efficiently in releasing the chromophore than in denaturing the tightly packed protein structure.
Kinetic Study of the Release of NCS-Chr from Holoprotein Induced by TFE-Because the release of NCS-Chr from holoprotein induced by TFE is a rapid and dynamic process, it is difficult to obtain any precise and quantitative information at room temperature simply by HPLC analyses. We therefore used a more sensitive fluorescence spectroscopic method to carry out a kinetic study at 25°C, for the purpose of obtaining a more accurate and direct comparison. It is known that GSH quickly reacts with the unbound NCS-Chr to form a stable tetrahydroindacene type of adduct, which generates about 60fold stronger fluorescence signal than that of the native epoxide form of NCS-Chr (46). We utilize GSH in excess to transform the released labile NCS-Chr into this highly fluorescent stable product, and we monitor the increase of the fluorescence with time. To avoid the degradation of NCS-Chr by excessive exposure to the excitation light source, the recording method is specifically designed so that the shutter is closed between measurements.
The release of chromophore can be expressed by Equations 2 and 3, holoNCS L | ; where [holoNCS] 0 is the initial or total concentration of NCS, and k 1 is the rate of release. The released NCS-Chr is quickly and irreversibly converted by GSH into the highly fluorescent drug-thiol adduct as shown in Equation 4.
The second-order rate of the reaction of GSH with NCS-Chr in the presence of DNA has been reported to be 14 -28 M Ϫ1 s Ϫ1 (47). Although much less efficient than apoNCS, it is known that DNA can effectively protect the labile NCS-Chr as well.
The degradation of the unbound NCS-Chr has been estimated to be about 30 times slower under the protection of calf thymus DNA (48). We therefore estimate that the second-order rate where k obs and n are the observed rate constant and reaction order for the measured rate of disappearance of holoNCS. The change in [holoNCS] with time was experimentally determined by the increase of fluorescence emission resulting from the thiol-induced drug product. The calibration curve of fluorescence emission versus concentration shows a linear relation in the drug concentration range studied (0 -10 M) (data not shown). Fig. 6a shows the logarithmic plot of [holoNCS], measured by fluorescence, versus time in 15% TFE. The data from the progressive release of the chromophore induced by TFE can be described by a linear fit. This result suggests that first-order kinetics represent well the release process. Fig. 6b shows the change of the observed kinetic constant, k obs , with concentration of TFE measured at 25°C. The rate constant at 0% TFE, which is nearly zero, is estimated by the reported lifetime of the degradation of holoNCS in aqueous solution (48). The results demonstrate that the observed rate, which is approximately equivalent to the rate of chromophore release, increases sharply with the percentage of TFE and reaches a maximum at about 20% TFE. However, instead of keeping the maximum plateau rate at higher TFE concentrations, the ob- served rate actually drops, a result that is similar to the HPLC analyses of chromophore release. It is known that an organic solvent can stabilize the isolated NCS-Chr, possibly by means of hydrophobic protection. As little as 3 M isopropyl alcohol can slow down the degradation of NCS-Chr about 6-fold (48). The reaction rate for the GSH-induced inactivation of NCS-Chr is expected to be slower at higher TFE concentrations. The protective effect results in an increase of [NCS-Chr] and makes the second term of Equation 3 too significant to be ignored. Under those circumstances, k obs is no longer dominated by the rate of release, k 1 .
In addition to the main study at 25°C, we have also investigated the rate of TFE-induced release at different temperatures (5-25°C), to correlate the chromophore release with properties of the transition intermediate in the TFE-induced dissociation process. The results show that at lower temperatures the rate of release decreases, and the maximum release rate occurs at a slightly higher percentage of TFE. DISCUSSION Since direct binding of NCS protein to DNA has not been observed (13,14), release of the active chromophore has to be the first step in its pharmacological action of cleaving DNA. NCS chromoprotein can be considered as a natural model of a drug delivery system. To study the release of NCS-Chr from its natural carrier protein is useful not only as a model for other biologically important chromoproteins but also for clinical applications. We initially use TFE, an ␣ helix-inducing agent, as an effective inducer for conformational change of the all ␤-NCS. The purpose is to study whether release of the chromophore results from a conformational change in its carrier protein.
Conformational Changes of NCS Protein-Because both components of holoNCS, the chromophore and the apoprotein, exhibit noticeable UV, CD, fluorescence, and NMR spectroscopic signals that extensively overlap each other, spectroscopic changes of holoNCS are difficult to interpret. We therefore rely on studies of apoNCS, whose compact structure is almost indistinguishable from that of holoNCS, to acquire information about TFE-induced conformational change in the holoprotein.
Because NCS is basically an all-␤-sheet protein, the conformational change induced by TFE, which induces helix formation, is expected to be evident. The experimental results from CD, tryptophan fluorescence, ANS binding, and 1 H NMR studies, on the effect of TFE over a wide range of concentration (0 -90%), are in good agreement. The specific interaction of the aromatic side chains can be disturbed at a low percentage of TFE (Ͻ20%), before any significant change occurs in the secondary or tertiary structure of the tightly folded, hydrophobic binding site. The all-␤-sheet native state, N, remains in its compact folded state until about 20% TFE. The tertiary structure is disrupted before the secondary structure, which re-  6. a, the logarithmic plot of the concentration changes with time in a 5 M holoNCS that contained 5 mM GSH. The concentration change, which was monitored by fluorescence at 25°C, was initiated by an addition of 15% TFE. b, the changes of the observed kinetic constant, k obs , for the TFE induced chromophore release. The rate constants were measured at 25°C. mains unchanged until about 25% TFE. Beyond 20% TFE the native state, N, changes into the intermediate state, I. In 45-50% TFE, the molten globule-like intermediate (I) is a well populated species, as observed by ANS binding. The molten globule-like intermediate can also be observed by other spectroscopic methods. Beyond 50%, I starts to collapse and is transformed into the denatured state, D, in 80% TFE. Scheme I depicts the equilibrium changes in conformation determined from the spectroscopic observations, i.e. above 20% TFE, an N 7 I equilibrium is observed; in 45-50% TFE, the maximum concentration of I is observed, and all molecules are apparently transformed into the molten globule-like intermediate, and above 50% TFE, an I 7 D equilibrium is observed. Fig. 7 shows the free energy change of transforming the native protein, N, into the molten globule-like state, I. The free energy of transforming N into I, ⌬G I , is estimated from the ellipticity changes at 280 and 215 nm and from the fluorescence emission change of the hydrophobic probe, ANS, by assuming that in 50% TFE the equilibrium from N to I is nearly complete and the change from I to D is negligible. For a two-state process, N 7 I, the fraction of I, F I , can be estimated by F I ϭ (X C Ϫ X N )/(X I Ϫ X N ), where X C , X N , and X I are the spectroscopic data at specified TFE percentages, 0 and 50% TFE. The free energy, ⌬G I , is related to F I by the equilibrium constant K I , where K I ϭ F I /(1 Ϫ F I ) and ⌬G I ϭ ϪRTlnK I . It is clear that ⌬G I estimated by different spectroscopic methods is nearly the same within reasonable experimental error. The concentration of TFE needed to obtain 50:50 N:I is about 30% TFE, where ⌬G I ϭ 0.
Chromophore Release from NCS Protein-Our main focus here is not to characterize the structure of the molten globulelike state of apoNCS but rather to determine whether release of the chromophore requires a major conformational change in the NCS protein. The kinetic results at 25°C show that the release of chromophore is half-way to the maximum rate in 15% TFE and reaches the maximum at 20%. On the other hand, the CD spectroscopic studies show that the close-packed NCS protein structure remains almost unchanged in these conditions. Because there is no extensive change in ellipticity value and accessibility for ANS binding below 20% TFE, release of the chromophore can be induced before any significant backbone or global conformational change occurs. ANS does not bind to native apoNCS, but whether or not the channel to the hydrophobic binding cavity is open is not known. TFE-induced dislocation of the side chains of some aromatic residues, which is observed by tryptophan fluorescence and NMR studies, could be sufficient to facilitate the release of the chromophore.
It is obvious that the N 3 I reaction, which occurs beyond 20% TFE, does not correspond to the chromophore release process, which reaches a maximum rate around 20% TFE. The results suggest that formation of the molten globule-like intermediate is not related to the chromophore release. These results also show that, when the chromophore release path reaches its maximum activity, the carrier protein retains its compact folded state. The release of NCS-Chr apparently does not require any major permanent change of the secondary or tertiary structure of the protein. It is important that TFE stimulates the release of the chromophore, which is consistent with the earlier report that the DNA cleavage induced by holoNCS can be stimulated by an organic solvent such as isopropyl alcohol (14 -16). The TFE stimulation of release may indicate that chromophore release results from a fluctuation of the protein structure, even though no major secondary or tertiary structural change is required. In the cell, such a fluctuation of the protein structure (which occurs spontaneously in vitro) might be caused by a contact with some cellular agent, such as a membrane.