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1 Rensselaer Polytechnic Institute, Department of Chemistry, Troy, New York 12180, USA
2 Department of Biological Sciences, State University of New York at Albany, Albany, New York 12222, USA
Reprint requests to: Wilfredo Colón, Rensselaer Polytechnic Institute, Department of Chemistry, 110 8th Street, Troy, NY 12180, USA; e-mail: colonw{at}rpi.edu; fax: (518) 276-4887.
(RECEIVED December 18, 2001; FINAL REVISION April 4, 2002; ACCEPTED April 10, 2002)
3 Present address: Institute for Organic Chemistry, Center for Biological Magnetic Resonance, Johann Wolfgang Goethe-University, Marie-Curie-Strasse 11, N120-5 011 49 69 7982 9573, D-60439 Frankfurt am Main, Germany. 
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.5050102.
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Abstract |
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2U). The 
GH2O for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole. At concentrations <9 µM, the FIS dimer began to dissociate, as noted by the change in CD signal and size-exclusion high-pressure liquid chromatography retention times and peak width. The estimated dimer dissociation constant based on the CD and size-exclusion chromatography data is in the micromolar range, resulting in a GH2O of at least 5 kcal/mole less than that calculated from the urea denaturation data. This discrepancy suggests a deviation from a two-state denaturation model, perhaps due to a marginally stable monomeric intermediate. These observations have implications for the stability and function of FIS in vivo. Keywords: Factor for inversion stimulation, FIS; equilibrium denaturation; dimer stability; transcription regulation; dissociation constant; DNA binding
Abbreviations: CD, circular dichroism • far-UV, far ultraviolet • FIS, factor for inversion stimulation • HPLC, high-pressure liquid chromatography • Kd, dissociation constant • MW, molecular weight • near-UV, near ultraviolet • PB, potassium phosphate buffer • PCR, polymerase chain reaction • Tris-HCl, Tris hydroxymethyl aminomethane hydrochloride • WT, wild type
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Introduction |
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The Factor for Inversion Stimulation (FIS) is a dimeric DNA-binding and -bending protein found in Escherichia coli (E. coli) and other enteric bacteria (Thompson et al. 1987; Finkel and Johnson 1992; Osuna et al. 1995; Beach and Osuna 1998). It is involved in a number of cellular processes, including stimulation of site-specific DNA inversion reactions catalyzed by the Hin-family of recombinases (Johnson et al. 1986; Koch and Kahmann 1986; Haffter and Bickle 1987), stimulation of lambda and HKO22 phage DNA recombination (Thompson et al. 1987; Ball and Johnson 1991; Dorgai et al. 1993), stimulation of transcription of ribosomal and tRNA operons and other genes (Bosch et al. 1990; Ross et al. 1990; Xu and Johnson 1995a; Gonzalez-Gil et al. 1996), repression of its own transcription and that of several other genes (Ball et al. 1992; Xu and Johnson 1995b), and modulation of DNA topology (Schneider et al. 1997). FIS is composed of two identical 98-residue subunits, and several crystal structures have been solved, all revealing an intertwined dimer of predominantly 
-helical structure (Fig. 1
) (Kostrewa et al. 1991; Yuan et al. 1991; Safo et al. 1997). The N-terminus of FIS consists of a ß-hairpin and helix A, and is required for the stimulation of Hin-mediated DNA inversion (Koch et al. 1991; Osuna et al. 1991; Safo et al. 1997). Helix B runs through the center of the protein, and is highly involved in monomer–monomer interactions, as it makes extensive contacts with helices A and B of the other subunit in the dimer. The C-terminal region contains the helix-turn-helix DNA binding motif, and is required for DNA-binding and -bending functions. It consists of helices C and D, which are also involved in intramolecular interactions with helix B of the same subunit.
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The large changes in FIS concentration can potentially affect its dimer stability and, consequently, its function and susceptibility to proteolytic enzymes. Thus, it was of interest to closely examine the stability of this protein at physiologically relevant concentrations. In this study, we used various methods to investigate the FIS dimer dissociation constant and its equilibrium denaturation at various concentrations. The results have implications for the regulation of the functions of FIS, and for the regulation and degradation of other transcription factors and repressors.
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Results |
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) loss of helical structure with increasing urea concentrations was monitored by far-ultraviolet (far-UV) circular dichroism (CD) over a wide range of FIS concentrations (Fig. 2A) to determine if the loss of secondary structure is dependent on the dissociation of the dimer. The data shown in Figure 2A indicates that the unfolding of FIS, as shown by the loss of helical signal, is a concerted and concentration-dependent event. To probe for the presence of a dimeric intermediate with native-like secondary structure or a largely unfolded monomeric intermediate, we also monitored FIS denaturation using near-ultraviolet (near-UV) CD, tyrosine fluorescence, and fluorescence anisotropy.
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) arises from the different contributions of the four tyrosines located (Fig. 1) at positions 38 (helix A), 51 (beginning of helix B), 69 (end of helix B), and 95 (helix D). The equilibrium unfolding studies monitored by near-UV CD (277 nm) at 36 and 107 µM FIS, also demonstrate a two-state, concentration-dependent unfolding transition (Fig. 2B). Because the error in the near-UV CD is larger due to the low signal, it was not practical to perform denaturation experiments at lower FIS concentrations. Although the relative contribution of the individual tyrosine residues to the native near-UV CD spectrum is not clear, preliminary results with a Y51F FIS mutant indicate that about 70% of the signal is due to the tyrosine at position 51 (unpublished data). Because this residue is located at the dimer interface, it appears that the loss of the near-UV CD signal arises predominantly from the loss of the quaternary structure.
The equilibrium unfolding mechanism of FIS was furthered explored by monitoring the changes in steady-state tyrosine fluorescence intensity and anisotropy (Fig. 3). The denaturation of FIS monitored by both methods displays concentration-dependent transitions, as did the CD data. The increase in fluorescence signal between native FIS and FIS unfolded in 5 M urea is 
1.25 fold (data not shown). This low signal prevented the collection of reliable data at FIS concentrations below 36 µM. Even at higher FIS concentrations, the steep baselines defining the dependence of the native and unfolded state signals on urea (Fig. 3A), made the analysis of this data less reliable. This baseline effect seen in relative fluorescence intensity was not an issue in steady-state anisotropy experiments (Fig. 3B). It is expected that the anisotropy will decrease as FIS dissociation occurs, largely, but not exclusively, due to a decrease in correlation time. Although changes in anisotropy cannot be directly correlated with fractional dissociation/unfolding because of the changes in fluorophore quantum yield upon unfolding (Eftink 1994), this effect is minimized here due to the similar fluorescence intensities of the native and unfolded FIS states. Anisotropy changes can also be caused by changes in the lifetime of the excited state or through increased mobility of the tyrosine side chains upon unfolding (Eftink 1994). However, these changes should also be minimal because the four tyrosines are all partially solvent exposed, and the most restricted tyrosines (51 and 95) are held within the dimer interface (Fig. 1); therefore, their rotational freedom should parallel dissociation. Overall, it can be inferred that the majority of the decrease in anisotropy arises from changes in quaternary structure upon addition of urea. Through this method, the dissociation/unfolding of the FIS dimers appear to proceed through a concerted concentration-dependent process (Fig. 3B).
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) against urea concentration obtained by all methods thus far discussed, it is evident that at each FIS concentration used, the denaturation mechanism is consistent with a two-state process, whereby quaternary, tertiary, and secondary structure are lost in parallel (Fig. 4). Although the fluorescence data appears to deviate from the other data presented in Figure 4B and C, all of the data is within error. This data is best fit to a two-state denaturation pathway, as described in Materials and Methods. The parameters obtained from the global fitting and from the individual fit of each transition are in excellent agreement, yielding a GH2O of around 14 kcal/mole (Table 1). The larger errors of the near-UV CD and fluorescence data, due to the low signal and the steep baseline slopes, respectively, are reflected in the thermodynamic parameters.
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Results from the CD and HPLC experiments show that changes in signal begin to occur below 9 µM FIS (Fig. 5). When FIS is run on the HPLC at the highest protein concentrations (26 µM), it runs at the expected molecular weight of the dimer, determined according to a calibration curve based on molecular weight standards. Upon dilution of FIS, the HPLC data (Fig. 5B) clearly show an increase in both the retention time and the peak width at half height. However, this data (Fig. 5B, inset) could not be analyzed to obtain an estimation of the Kd because the ending retention time for monomeric FIS in the absence of urea was unknown. In contrast, the CD data could be fit to estimate the Kd of FIS using results from urea denaturation experiments, which indicated that unfolded FIS elicits 
11% of the native CD signal at 222 nm. It is important to emphasize that for this Kd analysis we assumed that FIS is dissociating to an unfolded monomer (two-state assumption), as suggested by the urea denaturation experiments. If our assumption is correct, the resulting Kd would be a good approximation of the real value, and the corresponding G of dissociation would match the G obtained from the urea denaturation studies. Under this assumption, we fit the CD data to the same two-state denaturation mechanism used to fit the urea denaturation data (see Materials and Methods) and calculated the Kd of FIS to be about 0.4 µM, which yields a GH2O of 9 kcal/mole for dissociation/unfolding. This is approximately 5 kcal/mole lower than the GH2O calculated by urea denaturation. Although this is only an estimated Kd, the log relationship between Kd and G means that relatively rough approximations in Kd will give relatively accurate Gs. These results imply that in the absence of urea FIS does not dissociate into the globally unfolded monomer as one would expect for a two-state system, but dissociates into a compact monomer. Thus, the two-state assumption that we made in calculating the Kd of FIS is not justified, and the actual Kd of FIS should be larger than the value calculated. The HPLC data (Fig. 5B) supports the CD results, suggesting a FIS Kd higher than 0.4 µM, and the population of a monomeric intermediate upon dilution.
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Discussion |
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), resulting in larger errors in the calculated G, the consistency of all the denaturation data across a broad concentration range (9–107 µM) strongly supports the two-state model. Individual and global data fitting yields a GH2O of 14.2 kcal/mole, which is consistent with the stability of other dimeric proteins of similar size (Neet and Timm 1994). Most equilibrium denaturation studies of dimeric proteins have shown that their denaturation mechanism is described by either a concerted two-state process (N2 2U) involving a native dimer (N2) and an unfolded monomer (2U), or a three-state process, involving a dimeric (N2 I2 2U) or monomeric (N2 2I 2U) intermediate (Neet and Timm 1994). Small dimeric proteins usually exhibit a concerted two-state equilibrium unfolding mechanism (Neet and Timm 1994; Xu et al. 1998) similar to that of many small monomeric proteins. Thus, the observed two-state denaturation of FIS is not surprising, considering both its small size and intertwined structure (Fig. 1). Moreover, 29% of the total surface area calculated for the individual FIS monomers is buried in the dimer interface, whereas proteins known to exhibit a three-state denaturation mechanism usually only bury about 10% of their total surface area (Xu et al. 1998). Thus, it appears that most of the stability of FIS arises from the monomer–monomer interactions rather than the folded monomers.
Is there a monomeric intermediate at low FIS concentration?
The disagreement between the GH2O calculated from urea denaturation (14.2 kcal/mole) and dilution (9 kcal/mole) experiments suggest that, at very low concentrations, FIS denaturation deviates from a two-state process. If dissociation and unfolding were concerted processes, at low FIS concentrations the GH2O calculated from urea denaturation experiments would match that obtained from the estimated Kd (Neet and Timm 1994). The observed deviation could be explained by the existence of an unstable monomeric intermediate that becomes populated when FIS is highly diluted in the absence of urea. Both the increase of HPLC retention times and the band broadening seen upon successive dilution of a FIS sample (inset, Fig. 5B) are consistent with the presence of a monomeric species in rapid equilibrium with the dimer (Woodbury et al. 2002). At the lower protein concentrations, the monomer becomes more populated, which leads to a shift in retention time and a trailing in the peak. A globally unfolded monomer would be expected to have a higher hydrodynamic radius than the native dimer, and therefore, would elute with shorter retention times. Indeed, the HPLC retention times when FIS is denatured in urea are shorter than that of the native dimer in the absence of urea (data not shown). Taken together, these observations suggest that at low FIS concentrations, a marginally stable monomeric intermediate is present.
These observations have general implications for denaturation studies of oligomeric proteins, many of which are performed (due to instrument sensitivity) at concentrations significantly higher than those present in vivo. Dimeric proteins with unstable monomeric intermediates may denature via a two-state mechanism in vitro, when studied at protein concentrations much higher than their Kds, where the dimer is further stabilized (i.e., higher Cm) relative to the monomeric intermediate (Neet and Timm 1994). At lower and perhaps more physiologically relevant protein concentrations (approaching the Kd), it may be possible to observe monomeric intermediates. This is particularly pertinent for small single-domain dimeric proteins, which are unlikely to exhibit stable monomeric equilibrium intermediates by urea denaturation. Thus, structure–function correlations of dimeric proteins based on in vitro denaturation experiments carried out at concentrations much higher than the protein's Kd, may not always hold biological relevance, especially if the monomeric state has altered functions in vivo.
Potential impact of Kd on FIS function
Although we are not able to calculate an accurate Kd for FIS, our CD and HPLC data clearly show that it is in the micromolar range. The relatively high Kd of FIS suggests that the amount of FIS protein present as dimers in E. coli could be affected by the drastic changes in its expression levels observed during growth in rich medium. During early exponential growth in rich medium, FIS levels rapidly accumulate to about 45 µM and then rapidly decline to less than 0.4 µM (if one assumes an average cell volume of approximately 1 µm3) (Neidhardt 1987). Thus, it is likely that FIS exists exclusively as dimers during early logarithmic growth phase, when its levels are highest. However, as the FIS concentration decreases during mid and late logarithmic growth phase, an appreciable fraction of dissociated monomers can be expected to become populated. This could enhance the cell's ability to remove dimeric FIS during this growth phase. Moreover, because FIS monomers are likely to be significantly more susceptible to proteolytic degradation than the dimers, the rate of FIS turnover would also step up during this stage. Thus, it is conceivable that the high Kd for FIS is critical for its rapid removal during mid and late logarithmic growth phase. Timely removal of FIS is very important because cell viability significantly decreases if FIS is constitutively expressed during stationary phase (Osuna et al. 1995).
FIS-DNA binding studies have been performed in vitro using FIS concentrations in the 4–40 nM range (Bruist et al. 1987; Ball et al. 1992; Ninnemann et al. 1992; Gosink et al. 1993; Beach and Osuna 1998), suggesting that most of the FIS protein in these experiments was initially present as monomers, assuming a Kd in the micromolar range. Although it has been generally assumed that FIS dimer formation is a prerequisite for DNA binding, the possibility that monomeric FIS is capable of DNA binding should also be considered. DNA binding by monomeric FIS, albeit with reduced efficiency, has already been suggested based on DNA binding results using a FIS mutant thought to be defective in dimerization (Deufel et al. 1997). Therefore, it is possible that partially folded and weakly stable FIS monomers could retain weak DNA binding activity. In this case, two monomers bound to adjacent half sites may be able to dimerize (given their high local concentration) and produce a stable nucleoprotein complex. Such a binding mechanism, if it exists, would probably rely on a higher number of monomer–DNA contacts per half-site than that of a preexisting dimer in solution. The observation that at lower FIS concentrations where monomer may be populated, FIS preferentially binds a few select DNA sequences, and at higher concentrations it can bind additional DNA sequences, in some cases with apparent nonspecificity (Schneider et al. 1997), might suggest that pre-formation of a stable dimer may be required for binding to other less-specific DNA sequences. Thus, FIS concentration-dependent affinities for different DNA sequences might be affected by the monomer–dimer dynamics that are possible because of its Kd.
Modulation of protein levels as a way of regulating protein function
There are examples of DNA-binding proteins that appear to be partially regulated by their levels of expression and the resulting effect on their oligomeric state (Sevenich et al. 1998; Palena et al. 1999). Among them is the nitrogen regulatory protein (NtrC), which is a transcription activator of genes involved in nitrogen metabolism (McFarland et al. 1981), and has been suggested to be an evolutionary precursor of FIS (Morett and Bork 1998). It is a dimeric protein of 102 kD, which contains a 20-kD DNA-binding domain with high structural homology to FIS (Rippe et al. 1998; Pelton et al. 1999). When phosphorylated, NtrC binds to two adjacent DNA sites with high cooperativity (Porter et al. 1993), and there is evidence that an octamer comprised of phosphorylated NtrC dimers is required for the formation of an active NtrC complex (Su et al. 1990; Wyman et al. 1997; Rippe et al. 1998). The concentration of NtrC required for octamer formation is >10 nM. The intracellular concentration of NtrC is estimated to be 10 nM when inactive and rises to 100 nM under nitrogen limiting conditions. This rise in concentration is an important part of the regulatory mechanism of NtrC (Sevenich et al. 1998). Similar to NtrC, it is probable that many oligomeric proteins require a drastic increase in expression levels to form a functional oligomer or a higher order oligomer possessing a different function.
Thus, the oligomeric state of many DNA-binding proteins in vivo may be important, not only for DNA binding, but for providing additional means for rapid regulation. Perhaps it is not coincidental that many transcription regulatory proteins have dissociation constants in the µM range (Farez-Vidal et al. 1996; Abril et al. 1997; Jana et al. 1997; Palena et al. 1999; Sengchanthalangsy et al. 1999; Steinmetzer et al. 2000). Determinations of how the levels of DNA-binding proteins correlate with their Kds and with their turnover rates should shed light on how these parameters may impact their function in vivo.
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Materials and methods |
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Sample preparation
Protein concentration (in monomer units) was obtained in 1 M NaCl, 25 mM Tris-HCl, using an 
278 of 6340 M-1 cm-1, which was determined as described by Pace et al. (1995). All experiments were performed over a range of concentrations at 20°C in either 10 mM potassium phosphate buffer (PB) at pH 7.2 or 20 mM MOPS (pH 7.2), with 0.1 M NaCl, unless otherwise stated. Urea denaturation experiments using 107 µM FIS were carried out at 0.2 M NaCl to prevent the precipitation of FIS that occurs in low salt conditions.
Circular dichroism studies
CD spectra were recorded on an OLIS CD instrument equipped with a dual-beam optical system. All CD samples were prepared in 10 mM PB (pH 7.2). Unfolded structure was observed in 10 mM PB (pH 7.2), 6 M urea, at 20°C, and reversibility was verified by returning the sample to native state conditions through a six-fold dilution. The change in far-UV signal upon FIS dilution was measured by averaging three full wavelength scans at different concentrations, each derived from serial dilutions of a 71 µM stock. All FIS dilutions were in 10 mM PB (pH 7.2), 0.17 M NaCl, except the 71 µM stock, which was in 0.33 M NaCl. The use of varying protein concentration and cell sizes in the dilution experiments required that all scans be plotted in molar ellipticity:
 | ((1)) |
Equilibrium urea denaturations in the far-UV were monitored at 222 nm to observe the -helical content and subtracted from the baseline at 245 nm. Denaturation experiments in the near-UV region were monitored at 277 and 330 nm was used for a baseline. Scans were time-averaged over 3 min with each urea concentration used. Samples were prepared independently or by a codilution method, in which FIS samples of progressively higher urea concentrations were made by repetitive withdrawal of native FIS from the cell followed by addition of the corresponding volume from a FIS solution in 8 M urea. Urea concentrations for all experiments were determined from the refractive index measurement using an Abbe refractometer (Pace et al. 1990).
Fluorescence studies
Fluorescence measurements were performed on a Hitachi F-4500 fluorescence spectrophotometer at 20°C using 1-cm path-length cuvettes. Excitation and emission wavelengths were 276 and 305 nm, respectively, with an excitation slit of 5 nm and an emission slit of 10 nm. Samples were prepared in 20 mM MOPS (pH 7.2). Equilibrium urea denaturations were performed using the dilution method described for the CD experiments. Samples were equilibrated for 4 min between measurements, and time scans were measured over 40 sec. The fluorescence signal of FIS was internally normalized.
Steady-state anisotropy was performed using Hitachi polarization accessories and carried out in 1-cm path-length cuvettes. Samples were prepared in 10 mM PB (pH 7.2). For each anisotropy value at least five measurements were recorded and averaged. The integration time was 1 sec. Anisotropy was calculated from the formulas:
 | ((2)) |
 | ((3)) |
Size-exclusion HPLC
HPLC measurements were performed on a Beckman System Gold Nouveau with the 166NM single wavelength detector (1-cm path-length flow cell), using a 7.8 mm x 30 cm TOSOH BIOSEP TSK-GEL 2000SWXL column. Samples were prepared in 20 mM PB (pH 7.2), 0.5 M NaCl. The HPLC data had to be collected under higher salt conditions than our spectroscopic data to prevent interaction with the column and the collection of lower concentration data points. FIS samples injected onto the column were immediately diluted 18-fold and rapidly came to equilibrium at the diluted concentration. The extent of sample dilution in the column was determined by monitoring at 276 nm and calculating the concentration of FIS from the absorbance at the peak maximum. To investigate the oligomerization state of FIS at different concentrations, the column was preequilibrated to the above conditions, and samples at varying FIS concentrations, achieved by serial dilution from a more highly concentrated stock, were injected onto the column. The elution of FIS was monitored at 220 nm, with the column running at a flow rate of 1mL/min. The column was calibrated using the proteins: bovine serum albumin (67 kD), ovalbumin (43 kD), human Cu/Zn super oxide disumutase (32 kD), cytochrome C (17 kD), ribonucleaseA (13.7 kD), and myoglobin (12.3 kD). The resulting calibration curve indicated that dimeric FIS ran on the column roughly as expected for a protein of its molecular weight (data not shown).
Data modeling
For comparison of data collected at different protein concentrations or by different spectral methods, the data was normalized to Fu, the apparent fraction of unfolded protein,
 | ((4)) |
The data from the urea denaturation experiments were best fit to a two-state dimer model, with native dimer (N2) in equilibrium with unfolded monomers (2U):
 | ((5)) |
The equilibrium constant for concerted unfolding and dissociation, Ku, is defined as Ku = [U]2/[N2]. The total protein concentration, Pt, in terms of monomer is Pt = 2N2 + U, and the fraction of native dimers (Fn) = 1 - Fu, where Fu = [U]/Pt. Combining these equations and solving for Fu in terms of the equilibrium constant Ku and Pt, one obtains the following equation:
 | ((6)) |
Rearranging equation 4 yields the equation used for fitting the denaturation transitions:
 | ((7)) |
The thermodynamic parameters listed in Table 1 were obtained by fitting the denaturation curves to equation 7, upon substitution of equation 6, by defining Ku according to the linear free-energy model, which states that the changes in free-energy (Gu) that accompany protein unfolding are linearly dependent on the concentration of urea denaturant (Schellman 1978; Schellman 1987):
 | ((8)) |
Fits of individual data sets were performed with KaleidaGraph v 3.5 (Synergy Software). Global analysis of the data was done by simultaneously fitting all of the data at different FIS concentrations from the different methods to the two-state model described above. This analysis was carried out using the program SAVUKA version 5.1, a nonlinear least-squares fitting program (Zitzewitz et al. 1995; Bilsel et al. 1999).
The change in signal that occurs upon dilution of protein was also modeled to the two-state system shown in equation 5. In this case, we varied the FIS concentration and directly solved for the Kd (dimer dissociation/unfolding constant) instead of Ku, by using equations 6 and 7.
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Acknowledgments |
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References |
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), 35.6 µM (
), 8.9 µM (
), or 1.8 µM (•). The insets in (A) and (B) show the full wavelength scans in the far- and near-UV regions, respectively. The molar ellipticity, [
], has units of degree•cm2•dmol-1. The symbols in inset (A) represent the full spectrum of native (
), and anisotropy (+) at FIS concentrations of 8.9 µM (A), 35.6 µM (B), or 107 µM (C). The data was normalized to apparent fraction unfolded using equation 4