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Denaturation and Urea Gradient Gel Electrophoresis of Arginine Kinase: Evidence for a Collapsed-State Conformation
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 326, No. 1, February 1, pp. 93–99, 1996 Article No. 0051
Denaturation and Urea Gradient Gel Electrophoresis of Arginine Kinase: Evidence for a Collapsed-State Conformation Richard M. France and Steven H. Grossman1 Department of Chemistry, University of South Florida, Tampa, Florida 33620
Received July 24, 1995, and in revised form October 23, 1995
The unfolding transition of monomeric arginine kinase from shrimp was examined in a multiparameter equilibrium approach. Parameters investigated included catalytic activity, circular dichroism, intrinsic fluorescence characteristics including acrylamide quenching, and steady-state anisotropy of arginine kinase derivatized at the reactive cysteine with fluorescent dye 5-[[[(iodoacetyl)amine]ethyl]amino]naphthalene-1-sulfonic acid. The time course of electrophoretic patterns in urea gradient gels was also determined. Midpoints of the transitions varied considerably depending upon the parameter, indicating the presence of populated intermediates. Significant unfolding began after 2 M urea with most secondary and tertiary structure eliminated in 5 M urea. In dilute denaturant, arginine kinase exhibited a small increase in specific activity and physical properties characteristic of a protein with collapsed structure, including an increase in a-helical content, a decrease in intrinsic fluorescence (without a shift in the emission maximum), an increase in anisotropy, and a decrease in fractional accessibility by tryptophan to acrylamide quenching. The electrophoretic pattern of arginine kinase in urea gradient gels is consistent with the presence of a compact conformation in dilute denaturant. The results indicate the existence of a contracted overall conformation in dilute urea. The persistence of catalytic activity suggests this structure may be a functional molecular isoform, but the obvious differences in structure between the native state and the conformation of arginine kinase in 0.5 M urea raise the question of whether such isoforms may also be a type of folding intermediate. q 1996 Academic Press, Inc.
1
To whom correspondence should be addressed. Fax: (813) 9741733.
Detection and characterization of structured intermediates is an important goal of studies of protein folding. Simple two-state models consisting of native and fully denatured states are often adequate for describing folding mechanisms of small, monomeric proteins (1). However, from the application of a wide range of spectroscopic and electrophoretic techniques (2), investigators have described the occurrence of well-populated unfolding/folding intermediates. One such intermediate, the molten globule state, is conceptualized as possessing a compact, native-like secondary structure and fluctuating, more denatured-like, tertiary structure (3). Arginine kinase (AK)2 is a monomeric protein in the phosphagen kinase family which also contains the homologous, dimeric creatine kinase (4). The assembly mechanisms of the isozymes of creatine kinase have been characterized (5, 6) and evidence suggests the occurrence of a folding intermediate, which has some characteristics of a compact state (7). This finding prompted an examination of the structurally less complex, single-subunit AK to determine if a stable folding intermediate was detectable and, if so, to more fully characterize its conformational features. In this work, we have used purified monomeric AK from shrimp in a multiparameter approach, including electrophoretic mobilities in urea gradient gels (8) to describe the equilibrium unfolding/folding transitions. The results reveal the existence of a compact structure in dilute denaturant, clearly different from the native state, with some features resembling a molten globule, but with retention of catalytic activity. MATERIALS AND METHODS Arginine kinase activity was measured by a procedure modified from Kuby et al. (9) and Virden and Watts (10). The reaction mixture 2 Abbreviations used: AK, arginine kinase; UG–PAGE, urea gradient polyacrylamide gel electrophoresis; AEDANS-AK, arginine kinase derivatized at the reactive thiol with 5-[[[(iodoacetyl)amine]-
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contained 10 mM arginine, 2 mM ATP, and 3 mM magnesium acetate, in 0.1 M Tris/acetate, pH 8.4, with 10 mM mercaptoethanol. Enzyme (0.025 ml) was added to 0.175 ml of assay mixture and incubated for 90 s at 257C, and the reaction was stopped by the addition of 0.250 ml of 2.5% trichloroacetic acid. The mixture was heated for 1 min at 1007C to hydrolyze the phosphoarginine, then immediately cooled in an ice bath for 1 min and equilibrated at room temperature for 5 min. The inorganic phosphate liberated was determined by the Fisk– Subbarow method (11) using 0.5 ml ammonium molybdate and 0.050 ml reducing reagent (Sigma Chemical Co., St. Louis, MO). After 15 min, the absorbance was measured at 650 nm. Arginine was omitted from the assay mixture to correct for possible ATPase activity or nonenzymatic hydrolysis. Protein concentration was determined either by the Coomassie blue dye binding assay (12) or by measurement of the absorbance at 280 nm using the value of 0.67 for a 0.1% solution of AK (13). For assay in urea, AK in 0.1 M Tris/acetate and 10 mM mercaptoethanol, pH 8.4, was incubated in varying urea concentrations (in 0.1 M Tris/acetate and 10 mM mercaptoethanol, pH 8.4) at 247C for 60 min. The assay for these samples was then the same as that described above, except the reagent also contained the same concentration of urea as used to treat the protein. The specific activity of AK using this colorimetric assay was 18.0 mmol phosphate/min/mg. Details of the purification and characterization of AK from shrimp will be presented elsewhere. The enzyme was shown to be homogeneous according to SDS–polyacrylamide gel electrophoresis (14) and monomeric (41 kDa) by comparing the molecular weight obtained by gel filtration with the molecular weight determined from SDS– polyacrylamide gel electrophoresis. Dynamic light scattering analysis of the purified AK preparation with a DynaPro 801 molecular sizing instrument (Proteins Solutions, Inc., Charlottesville, VA) provided an estimated molecular weight of 39.9 kDa. The fluorescent dye 5-[[[(iodoacetyl)amine]ethyl]amino]naphthalene-1-sulfonic acid (Molecular Probes, Junction City, OR) was conjugated to the reactive thiol of AK by incubating a fivefold molar excess of dye to protein in 0.05 M potassium Hepes, pH 8.0 (15). After 18 h at 47C, the reaction mixture was filtered through Sephadex G-25 and dialyzed against reaction buffer containing 2 mM DTT. Spectral analysis revealed 0.85 mol of bound AEDANS per mole of AK. Fluorescence measurements were made with an SLM Model 48000 spectrofluorometer (SLM Instruments, Urbana, IL). Components of this instrument and data acquisition procedures for intensity and anisotropy have been described previously (15). All fluorescence measurements were made in the ratiometric mode with rhodamine as the quantum counter. In spectral analysis, from which single point intensities were digitized, fluorescence was detected with an emission monochromator using a 4-nm bandpass. The dependence of the anisotropy of AEDANS-AK on viscosity was determined using increasing concentrations of sucrose under isothermal (257C) conditions as described previously (17). Viscosities of combinations of buffer, sucrose, and urea were measured directly using a Gilmont Type V-2100 falling ball viscosimeter. Circular dichroism measurements were made with a Jasco J-500 spectropolarimeter using a 0.1-cm pathlength cell and repetitive scans from 350 to 205 nm. Protein samples (0.11 to 0.18 mg/ml) in urea and 0.02 M sodium phosphate buffer, pH 8.0, were equilibrated for 1 h at 257C before scanning. Parallel spectra of urea without protein were also obtained. Mean residue ellipticities, [U]222 , were obtained from U Å 3300De, where U is the ellipticity in degrees, De is the measured absorbance, and [U]222 Å UMMRW/10dc where MMRW is the mean residue weight, d is the cell path in cm, and c is the concentration in g/ml. The mean residue weight was calculated from the primary structure of AK from lobster (18) and the fractional ahelical content fh was calculated from the expression (19) ethyl]amino]naphthalene-1-sulfonic acid; SDS, sodium dodecyl sulfate; DTT, dithiothreitol.
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fh Å ([U]222 / 2340)/(30,300).
[1]
Polyacrylamide gels containing a 0 to 8 M urea concentration gradient were prepared as described by Creighton (2) with an inverse 15 to 11% acrylamide gradient to ensure uniformity of migration with respect to urea viscosity. Initial experiments using the prescribed buffer concentrations exhibited limited mobility of AK. The protocol of Laemmli (14) (without SDS), including 0.225 M Tris/HCl (pH 8.8) in the casting procedure and a running buffer containing 0.025 M Tris and 0.198 M glycine adjusted to pH 8.3, resulted in significant migration of AK after 4 h of electrophoresis. Four gels were cast concurrently and used in pairs for electrophoresis. The concentration of urea across the gel was determined after cutting the gel into 1cm strips which were incubated overnight in 0.05 M potassium phosphate buffer, pH 8.0. Aliquots of the gel extract were diluted 1:150 in potassium phosphate buffer. The urea concentration was measured by adapting a coupled enzyme assay (20) used for the analysis of blood urea nitrogen. The urea assay solution (Abbott Laboratories, North Chicago, IL) was prepared by mixing 0.1 ml of NADPH / H/ solution, 0.1 ml of enzyme/substrate solution containing urease, glutamate dehydrogenase, a-ketoglutarate, ADP, and 9.80 ml of 0.05 M potassium phosphate buffer, pH 8.0. The assays were performed by adding 0.01 ml of the urea gel extract to 0.99 ml of the assay solution, then determining the decrease in the absorbance at 340 nm. A calibration curve was used to correlate the urea concentration and urease activity.
RESULTS
Purity and molecular weight. Arginine kinase purified to homogeneity exhibits a single protein staining band after electrophoresis in SDS/polyacrylamide gels. The molecular weight, estimated from a comparison of the mobility of AK with several calibration proteins, is 41 kDa. The molecular weight determined by gel filtration using Sephadex G-100 was calculated to be 40 kDa, which agrees with the value of 39.9 kDa obtained by dynamic light scattering. These results indicate that AK is monomeric. Light scattering indicated the presence of less than 2% aggregated protein. Activity in urea. The activity of AK is increased by 20% following incubation in 0.5 M urea for 1 h (Fig. 1). Above 1 M urea, AK activity declines rapidly. Arginine kinase loses about 50% of its activity in 1.8 to 2.0 M urea. No activity is detected after incubating AK in 2.5 M urea for 1 h. Circular dichroism in urea. The circular dichroic spectrum of native AK was obtained by averaging five normalized scans at four protein concentrations. Native AK possesses 16% helical structure (19). In 1.5 M urea, AK molar ellipticity at 222 nm increases 35% (Fig. 1) which corresponds to a considerable increase in a-helix. Further increases in denaturant concentration result in a decrease in a-helical content in 4 M urea, and essentially none of the residual structures is retained in the a-helical conformation. These observations were made after 1 h of exposure to denaturant. Further incubation periods do not lead to additional changes at several urea concentrations tested. Fluorescence. The intrinsic fluorescence maximum of native AK is 337 nm. In denaturant up to 2.75 M
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linear response with extrapolation to 0.1501 for the limiting anisotropy (Fig. 3). In the presence of 0.5 M urea, the anisotropy remains higher across the range of viscosity when compared to the protein in the absence of urea. However, a distinct biphasic response is evident, which is more apparent at higher urea concentrations. The biphasic dependence of 1/rss on viscosity for AEDANS-AK in urea precluded determination of the limiting anisotropies and, therefore, evaluation of rotational correlation times. Quenching. The accessibility of the tryptophans to quenching was examined using the nonpolar fluorescence quencher acrylamide. The Stern–Volmer plots displayed downward curvatures indicating the presence of multiple quenching sites (22) which were instead analyzed according to the modified Stern– Volmer equation (23) F0/DF Å 1/([Q]faKQ) / (1/fa), FIG. 1. Activity and circular dichroism of AK as a function of urea concentration. The activity of AK (l) was determined colorimetrically. One unit of activity is 1 mmol of inorganic phosphate released per minute. The enzyme samples (AK, 0.040 mg/ml) were treated with denaturant for 1 h at the designated urea concentration, then transferred to the assay mixture containing the identical concentration of urea. Assays were performed for 2.0 min at 247C. Circular dichroism measurements (s): Samples of AK (0.11 to 0.21 mg/ml) were treated for 1 h with urea, then transferred to a 0.1-cm cuvette maintained at 247C and scanned from 350 to 205 nm in a Jasco 500 spectropolarimeter. The mean residue ellipticities are relative to the native state (U222 Å 07200).
urea, the emission maximum increases only 2 nm (Fig. 2), after which a sharp red shift begins, culminating in an emission maximum of 356 nm in 5 M urea. The relative intrinsic fluorescence intensity of AK at 356 nm declines 10% in 2.5 M urea, increases 20% between 2.5 and 3.5 M urea, and remains at approximately the intensity level of the native state from 4 to 8 M urea (Fig. 2). Intensity values were modified to correct for the direct effect of urea on tryptophan fluorescence, using N-acetyltryptophanamide as a model compound. The steady-state anisotropy of AEDANS-AK increases 4% between 0.25 and 1.0 M urea (Fig. 2). The anisotropy of AEDANS-AK is maximum at 0.0933 { 0.0003 in 0.5 M urea, significantly higher than 0.0897 { 0.0004 observed in buffer only. This change could be the result of several types of conformational change, including decreased macromolecular rotational diffusion, decreased segmental flexibility within the protein, or more limited motion of the probe-associated domain (21). The anisotropy decreases to 0.0246 { 0.0002 in 4 M urea and ultimately to 0.0236 { 0.0007 in 6 M urea. The dependence of the reciprocal of the anisotropy, 1/rss , of AEDANS-AK in buffer on viscosity exhibited a
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[2]
where F0 is the fluorescence intensity in the absence of quencher, DF is the change in fluorescence when the quencher is introduced, [Q] is the molar concentration of quencher, fa is the fractional accessibility of tryptophan to quencher, and KQ is the effective quenching constant, the product of the collisional rate constant and the fluorescence decay time. Modified Stern– Volmer plots for AK in urea are shown in Fig. 4. The linear relationship between F0/DF and 1/[Q] indicates suitability of analysis by the modified Stern–Volmer relationship. A substantial increase in KQ for AK begins after 2 M urea (Fig. 5), which is consistent with unfolding of the protein. Arginine kinase in the absence of denaturant exhibits nearly complete accessibility to acrylamide ( fa Å 0.99 { 0.03), while in 0.5 M urea this decreases to fa Å 0.89 { 0.02. Urea gel electrophoresis. The linearity of the urea concentration gradient in the polyacrylamide gel was confirmed by the urea gel calibration assay procedure. Initially, native homogeneous AK exhibits a faster migrating band in the region of 0.5 to 3 M urea after 100 min of UG–PAGE (Fig. 6). At 150 and 180 min, this transition is more evident with formation of three distinct regions. By 260 min, the gel exhibits an almost complete separation of a distinctly more mobile band in the 0 to 2 M urea region, followed by a faint, diffuse band from 2 to 3.5 M urea and then a slower, homogeneous band from 3.5 to 8 M urea. The time course of UG–PAGE, starting with denatured AK, exhibits essentially the same pattern of migration (gels not shown). DISCUSSION
Previous equilibrium and kinetic studies of creatine kinase (7, 24), a dimeric phosphagen kinase, revealed
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FIG. 2. Intrinsic fluorescence intensity and emission wavelength maximum of AK and anisotropy of AEDANS-AK as a function of urea concentration. Samples of AK (0.37 mg/ml) in buffer (0.05 potassium Hepes, pH 8.0, with 2 mM DTT) were incubated for 1 h in urea solutions at 247C. Samples were then diluted 1 to 5 in buffer containing the same urea concentrations. For the intrinsic fluorescence, excitation was at 295 nm and emission maxima (l) were determined from spectral scans between 300 and 400 nm. Relative fluorescence was measured at 356 nm and corrected for the presence of urea in the solvent and the direct effect of urea on the emission of tryptophan (s) (see text). For anisotropy measurements, samples contained 0.30 mg/ml AEDANS-AK (h) in 0.05 M potassium Hepes, pH 8.0, containing 2 mM DTT. After treatment with urea for 1 h, anisotropy was measured with excitation at 325 nm and emission was collected through two T-formatted Schott-type 470 cuton filters.
the occurrence a folding intermediate. Owing to the complexities of analyzing protein folding in the presence of subunit association in a multimeric protein, we examined AK, a simpler, albeit functionally analogous and structurally homologous (4), monomeric protein to search for and describe the structural features of a potentially analogous folding intermediate. During the course of this work, it was observed that transfer of native AK into dilute urea produced an apparently more compact protein structure which retained catalytic activity. Examination of a summary of the degree of parameter changes investigated as a function of urea concentration (Fig. 7) is consistent with a multiphasic model for denaturation. Focusing on the changes occurring in dilute denaturant reveals features characteristic of a structure more compact than the native state. The increase in activity in dilute denaturant has been observed for several other enzymes (25–27). This is generally attributed to a change in polypeptide flexibility in the domain of the active site. For AK, other results suggest a decrease in polypeptide flexibility in the active site (an increase in anisotropy of AEDANSderivatized AK), but as noted below, the steady-state anisotropy may be reflecting both localized and/or overall macromolecular rotation. Above 0.5 M urea, the cat-
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alytic activity of AK is the parameter most sensitive to denaturation. For purposes of comparison with other parameters only, assumption of a two-state model reveals a DGH2O of inactivation of 3.5 kcal/mol (28). Native AK possesses 16% a-helix, determined from the molar ellipticity at 222 nm by the method of Chen et al. (19). The 35% increase in molar ellipticity at 222 nm of AK between 0.25 and 2.5 M urea indicates not only resistance to denaturation of secondary structure but, apparently, the formation of more a-helix. The increase in secondary structure correlates with the urea gradient gel demonstrating a more compact protein state of AK in this range of denaturant concentration (see below). The stability of the secondary structure is revealed by a value of DGH2O Å 10.7 kcal/mol for the transition in circular dichroism and indicates that the structural feature most resistant to denaturation is the a-helix. The decrease in intrinsic fluorescence of AK in dilute urea may be due to more quenching within the protein or to exposure of the tryptophans to the aqueous media. That the decrease in intensity is not accompanied by a red shift in the emission maximum is consistent with internal quenching and the occurrence of a more compact conformation in dilute denaturant. The transition
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FIG. 3. Perrin plots for AK-AEDANS in urea. Samples of AK-AEDANS (2.0 ml of 0.25 mg/ml in 0.05 M potassium Hepes buffer, pH 8.0, and 2 mM DTT) were treated with increasing concentrations of sucrose at 257C. Steady-state anisotropy was measured as described for Fig. 4. Urea concentrations: (l), 0; (j), 0.5 M; (.) 3.5 M; (m) 4.5 M; (l) 6 M.
FIG. 4. Modified Stern–Volmer plots for quenching of the intrinsic fluorescence of AK. Samples (0.3 mg/ml) in 0.05 M potassium Hepes, pH 8.0, containing 2 mM DTT were titrated with aliquots from a 6 M stock solution of acrylamide. Fluorescence excitation wavelength was 295 nm and emission wavelength was 348 nm. Inner filter corrections were generally negligible. (s) 0 M urea; (l) 1 M urea; (,) 2 M urea; (.) 3 M urea; (h) 4 M urea; (j) 6 M urea; (n) 8 M urea.
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FIG. 5. Quenching constants (KQ) and fractional accessibility for AK with acrylamide as a function of the urea concentration. Values for KQ were determined from the modified Stern–Volmer plots as illustrated in Fig. 6. Fractional accessibility (l) and KQ (M01) (s).
FIG. 6. Time course of AK electrophoresis in urea gradient polyacrylamide gels. Native AK samples (0.50 mg/ml) in 0.01 Tris/HCl, pH 8.0, and 10 mM mercaptoethanol were subjected to electrophoresis (87C) for the indicated times and then gels were removed and stained with 0.1% Coomassie blue. Additional details are given in the text.
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FIG. 7. Summary of parameter changes in properties of AK as a function of urea concentration. fapp Å (pi 0 pD)/(pN 0 pD) 1 100, where pN , pD , and pi are the parameter values for the native state (zero urea), the value at the maximum change in parameter, and the intermediate parameter values, respectively. (,), Fluorescence emission maximum; (.), circular dichroism; (s), activity; (l) anisotropy.
to a higher intensity between 2 and 4 M urea signifies release of this quenching upon denaturation (5, 24) and possibly increased exposure of ionized tyrosines (29). When acrylamide is used as an extrinsic quencher, the quench constant for AK doubles between 2 and 4 M urea, implying a significant degree of unfolding, in agreement with the circular dichroism results. However, AK exhibits an anomalous decrease in fractional accessibility in dilute denaturant which is consistent with formation of a more compact conformation. Treatment of AK with urea greater than 1.5 M urea is clearly accompanied by a significant decrease in anisotropy, as expected for a protein undergoing denaturation. The small but experimentally significant increase in AEDANS-AK anisotropy (r) in low concentrations of urea indicates restriction of some depolarizing rotation, either locally or globally, although changes in fluorescence lifetime (t) or the limiting anisotropy (r0) could also account for the change in steady-state anisotropy, as described by the Perrin equation rss Å r0/ (1 / t/u), where u is the rotational correlation time. The biphasic dependence of the reciprocal of the anisotropy on viscosity in different concentrations of urea suggests the presence of multiple conformational states, which complicates the evaluation of the limiting anisotropy. Data not shown also suggest considerable heterogeneity in the fluorescence decay time of AE-
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DANS-AK in intermediate concentrations of urea. Consequently, time-resolved decay of the anisotropy could not be used to determine the structural origins of the increase in anisotropy observed in dilute urea. Urea gradient gel electrophoresis directly displays protein folding intermediates (2) and is important for confirming that intermediates detected by parameter changes under equilibrium conditions are not summation effects due to the simultaneous presence of native and denatured states. For AK, the distinct descending band observed early in electrophoresis may indicate a more compact conformation for the protein between 0.25 and 1.5 M urea, although this does not appear to be precisely the same range of denaturant in which other parameters point to a compact structure. The broad, diffuse band between 1.5 and 3 M urea corresponds to a folding/unfolding transition at a slower rate than the rate of electrophoresis (2). This is also the range for the midpoints in the unfolding transitions of AK, revealed by the changes in anisotropy (2.0 M urea), intrinsic fluorescence (3.0 M urea), and molar ellipticity at 222 nm (2.5 M urea). The slowest migrating band between 3 and 8 M urea represents the unfolded protein. Since the identical gel pattern is obtained when beginning with denatured AK, the refolding pathway may be the rapid reverse of the unfolding pathway (30). Compared with native protein, the structure of AK which exists in 0.5 M urea has (a) more circular dichroic absorbance in the far ultraviolet indicative of more helical content, (b) less fractional accessibility to tryptophans by acrylamide, (c) a significant decrease in intrinsic fluorescence, suggesting greater quenching between tryptophans, and (d) an increase in the anisotropy. From these data, it seems reasonable to suggest that the overall shape of AK in dilute urea is more compact than in the native solvent. Without meaningful time-resolved anisotropy data, it is not possible to evaluate the degree of tertiary flexibility, which may, in any event, be too slow for detection by timeresolved fluorescence analysis. However, the retention of catalytic activity suggests the absence of a largescale disruption of overall structure in dilute denaturant. The structure described does not appear to correspond to any of the four classes of protein folding intermediates found in acid solution (31). The persistence of catalytic activity, still accompanied by clear, if not dramatic, changes in conformation, suggests that this structure may be a thermodynamically accessible state even in the absence of urea. We have previously demonstrated that conformational isoforms of lobster muscle AK occur in the native solvent environment (15). The presence of dilute urea may shift the equilibrium to highly populate an isoform that is
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still active but with the apparently collapsed conformation described. Whether this would still be considered a folding intermediate on the path to the more populated native conformation in benign solvent is uncertain. ACKNOWLEDGMENTS Supported in part by grants (S.H.G.) from the American Heart Association, Florida Affiliate, and the National Science Foundation (9120260).
REFERENCES 1. Kim, P. S., and Baldwin, R. L. (1990) Annu. Rev. Biochem. 59, 631–660. 2. Creighton, T. E. (1979) J. Mol. Biol. 129, 235–264. 3. Ptitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., and Razgulyaev, O. I. (1990) FEBS Lett. 262, 20–24. 4. Anosike, E. O., Moreland, B. H., and Watts, D. C. (1975) Biochem. J. 145, 535–543. 5. Grossman, S. H., Pyle, J., and Steiner, R. J. (1981) Biochemistry 21, 6122–6128. 6. Grossman, S. H., Gray, K. A., and Lense, J. J. (1986) Biochim. Biophys. Acta 248, 234–242. 7. Grossman, S. H. (1994) Biochim. Biophys. Acta 1209, 19–23. 8. Creighton, T. E. (1984) Adv. Biophys. 18, 1–20. 9. Kuby, S. A., Nody, L., and Lardy, H. A. (1954) J. Biol. Chem. 210, 65–82. 10. Virden, R., and Watts, D. C. (1964) Comp. Biochem. Physiol. 13, 161–177. 11. Fiske, C. H., and Subbarow, Y. (1925) J. Biol. Chem. 56, 375– 400.
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12. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. 13. Virden, R., Watts, D. C., and Baldwin, E. (1965) Biochem. J. 94, 536–544. 14. Laemmli, U. K. (1970) Nature 227, 680–685. 15. Grossman, S. H. (1991) Biophys. J. 59, 590–597. 16. Lakowicz, J. R., Cherek, H., and Balter, A. (1981) J. Biochem. Biophys. Methods 5, 131–146. 17. Grossman, S. H. (1989) Biochemistry 28, 4894–4902. 18. Dumas, C., and Camonis, J. (1993) J. Biol. Chem. 268, 21599– 21605. 19. Chen, Y. H., Yang, J. T., and Martinez, H. M. (1972) Biochemistry 11, 4120–4131. 20. Talke, H., and Schubert, G. E. (1965) Klin. Wochenschr. 43, 174– 175. 21. Lakowicz, J. R. (1983) Principles of Fluorescence Spectroscopy, Plenum, New York. 22. Eftink, M. R., and Ghiron, C. A. (1981) Anal. Biochem. 114, 199– 227. 23. Lehrer, S. S. (1971) Biochemistry 10, 3254–3263. 24. Grossman, S. H., and Mixon, D. (1985) Arch. Biochem. Biophys. 236, 797–806. 25. Johnson, C. M., and Price, N. C. (1987) Biochem. J. 245, 525– 530. 26. Ma, Y. Z., and Tsou, C. L. (1991) Biochem. J. 277, 207–211. 27. Liang, S. J., Lin, Y. X., Zhou, J. M., Tsou, C. L., Wu, P., and Zhou, Z. (1990) Biochim. Biophys. Acta 1038, 240–246. 28. Pace, C. N. (1975) Crit. Rev. Biochem. 3, 1–43. 29. Yao, Q. Z., Tian, M., and Tsou, C. L. (1984) Biochemistry 23, 2740–2744. 30. Creighton, T. E. (1980) J. Mol. Biol. 137, 62–80. 31. Fink, A. L., Calciano, L. J., Goto, Y., Kurotsu, T., and Palleros, D. R. (1994) Biochemistry 33, 12504–12511.
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Vol. 326, No. 1, February 1, pp. 93–99, 1996 Article No. 0051
Denaturation and Urea Gradient Gel Electrophoresis of Arginine Kinase: Evidence for a Collapsed-State Conformation Richard M. France and Steven H. Grossman1 Department of Chemistry, University of South Florida, Tampa, Florida 33620
Received July 24, 1995, and in revised form October 23, 1995
The unfolding transition of monomeric arginine kinase from shrimp was examined in a multiparameter equilibrium approach. Parameters investigated included catalytic activity, circular dichroism, intrinsic fluorescence characteristics including acrylamide quenching, and steady-state anisotropy of arginine kinase derivatized at the reactive cysteine with fluorescent dye 5-[[[(iodoacetyl)amine]ethyl]amino]naphthalene-1-sulfonic acid. The time course of electrophoretic patterns in urea gradient gels was also determined. Midpoints of the transitions varied considerably depending upon the parameter, indicating the presence of populated intermediates. Significant unfolding began after 2 M urea with most secondary and tertiary structure eliminated in 5 M urea. In dilute denaturant, arginine kinase exhibited a small increase in specific activity and physical properties characteristic of a protein with collapsed structure, including an increase in a-helical content, a decrease in intrinsic fluorescence (without a shift in the emission maximum), an increase in anisotropy, and a decrease in fractional accessibility by tryptophan to acrylamide quenching. The electrophoretic pattern of arginine kinase in urea gradient gels is consistent with the presence of a compact conformation in dilute denaturant. The results indicate the existence of a contracted overall conformation in dilute urea. The persistence of catalytic activity suggests this structure may be a functional molecular isoform, but the obvious differences in structure between the native state and the conformation of arginine kinase in 0.5 M urea raise the question of whether such isoforms may also be a type of folding intermediate. q 1996 Academic Press, Inc.
1
To whom correspondence should be addressed. Fax: (813) 9741733.
Detection and characterization of structured intermediates is an important goal of studies of protein folding. Simple two-state models consisting of native and fully denatured states are often adequate for describing folding mechanisms of small, monomeric proteins (1). However, from the application of a wide range of spectroscopic and electrophoretic techniques (2), investigators have described the occurrence of well-populated unfolding/folding intermediates. One such intermediate, the molten globule state, is conceptualized as possessing a compact, native-like secondary structure and fluctuating, more denatured-like, tertiary structure (3). Arginine kinase (AK)2 is a monomeric protein in the phosphagen kinase family which also contains the homologous, dimeric creatine kinase (4). The assembly mechanisms of the isozymes of creatine kinase have been characterized (5, 6) and evidence suggests the occurrence of a folding intermediate, which has some characteristics of a compact state (7). This finding prompted an examination of the structurally less complex, single-subunit AK to determine if a stable folding intermediate was detectable and, if so, to more fully characterize its conformational features. In this work, we have used purified monomeric AK from shrimp in a multiparameter approach, including electrophoretic mobilities in urea gradient gels (8) to describe the equilibrium unfolding/folding transitions. The results reveal the existence of a compact structure in dilute denaturant, clearly different from the native state, with some features resembling a molten globule, but with retention of catalytic activity. MATERIALS AND METHODS Arginine kinase activity was measured by a procedure modified from Kuby et al. (9) and Virden and Watts (10). The reaction mixture 2 Abbreviations used: AK, arginine kinase; UG–PAGE, urea gradient polyacrylamide gel electrophoresis; AEDANS-AK, arginine kinase derivatized at the reactive thiol with 5-[[[(iodoacetyl)amine]-
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contained 10 mM arginine, 2 mM ATP, and 3 mM magnesium acetate, in 0.1 M Tris/acetate, pH 8.4, with 10 mM mercaptoethanol. Enzyme (0.025 ml) was added to 0.175 ml of assay mixture and incubated for 90 s at 257C, and the reaction was stopped by the addition of 0.250 ml of 2.5% trichloroacetic acid. The mixture was heated for 1 min at 1007C to hydrolyze the phosphoarginine, then immediately cooled in an ice bath for 1 min and equilibrated at room temperature for 5 min. The inorganic phosphate liberated was determined by the Fisk– Subbarow method (11) using 0.5 ml ammonium molybdate and 0.050 ml reducing reagent (Sigma Chemical Co., St. Louis, MO). After 15 min, the absorbance was measured at 650 nm. Arginine was omitted from the assay mixture to correct for possible ATPase activity or nonenzymatic hydrolysis. Protein concentration was determined either by the Coomassie blue dye binding assay (12) or by measurement of the absorbance at 280 nm using the value of 0.67 for a 0.1% solution of AK (13). For assay in urea, AK in 0.1 M Tris/acetate and 10 mM mercaptoethanol, pH 8.4, was incubated in varying urea concentrations (in 0.1 M Tris/acetate and 10 mM mercaptoethanol, pH 8.4) at 247C for 60 min. The assay for these samples was then the same as that described above, except the reagent also contained the same concentration of urea as used to treat the protein. The specific activity of AK using this colorimetric assay was 18.0 mmol phosphate/min/mg. Details of the purification and characterization of AK from shrimp will be presented elsewhere. The enzyme was shown to be homogeneous according to SDS–polyacrylamide gel electrophoresis (14) and monomeric (41 kDa) by comparing the molecular weight obtained by gel filtration with the molecular weight determined from SDS– polyacrylamide gel electrophoresis. Dynamic light scattering analysis of the purified AK preparation with a DynaPro 801 molecular sizing instrument (Proteins Solutions, Inc., Charlottesville, VA) provided an estimated molecular weight of 39.9 kDa. The fluorescent dye 5-[[[(iodoacetyl)amine]ethyl]amino]naphthalene-1-sulfonic acid (Molecular Probes, Junction City, OR) was conjugated to the reactive thiol of AK by incubating a fivefold molar excess of dye to protein in 0.05 M potassium Hepes, pH 8.0 (15). After 18 h at 47C, the reaction mixture was filtered through Sephadex G-25 and dialyzed against reaction buffer containing 2 mM DTT. Spectral analysis revealed 0.85 mol of bound AEDANS per mole of AK. Fluorescence measurements were made with an SLM Model 48000 spectrofluorometer (SLM Instruments, Urbana, IL). Components of this instrument and data acquisition procedures for intensity and anisotropy have been described previously (15). All fluorescence measurements were made in the ratiometric mode with rhodamine as the quantum counter. In spectral analysis, from which single point intensities were digitized, fluorescence was detected with an emission monochromator using a 4-nm bandpass. The dependence of the anisotropy of AEDANS-AK on viscosity was determined using increasing concentrations of sucrose under isothermal (257C) conditions as described previously (17). Viscosities of combinations of buffer, sucrose, and urea were measured directly using a Gilmont Type V-2100 falling ball viscosimeter. Circular dichroism measurements were made with a Jasco J-500 spectropolarimeter using a 0.1-cm pathlength cell and repetitive scans from 350 to 205 nm. Protein samples (0.11 to 0.18 mg/ml) in urea and 0.02 M sodium phosphate buffer, pH 8.0, were equilibrated for 1 h at 257C before scanning. Parallel spectra of urea without protein were also obtained. Mean residue ellipticities, [U]222 , were obtained from U Å 3300De, where U is the ellipticity in degrees, De is the measured absorbance, and [U]222 Å UMMRW/10dc where MMRW is the mean residue weight, d is the cell path in cm, and c is the concentration in g/ml. The mean residue weight was calculated from the primary structure of AK from lobster (18) and the fractional ahelical content fh was calculated from the expression (19) ethyl]amino]naphthalene-1-sulfonic acid; SDS, sodium dodecyl sulfate; DTT, dithiothreitol.
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fh Å ([U]222 / 2340)/(30,300).
[1]
Polyacrylamide gels containing a 0 to 8 M urea concentration gradient were prepared as described by Creighton (2) with an inverse 15 to 11% acrylamide gradient to ensure uniformity of migration with respect to urea viscosity. Initial experiments using the prescribed buffer concentrations exhibited limited mobility of AK. The protocol of Laemmli (14) (without SDS), including 0.225 M Tris/HCl (pH 8.8) in the casting procedure and a running buffer containing 0.025 M Tris and 0.198 M glycine adjusted to pH 8.3, resulted in significant migration of AK after 4 h of electrophoresis. Four gels were cast concurrently and used in pairs for electrophoresis. The concentration of urea across the gel was determined after cutting the gel into 1cm strips which were incubated overnight in 0.05 M potassium phosphate buffer, pH 8.0. Aliquots of the gel extract were diluted 1:150 in potassium phosphate buffer. The urea concentration was measured by adapting a coupled enzyme assay (20) used for the analysis of blood urea nitrogen. The urea assay solution (Abbott Laboratories, North Chicago, IL) was prepared by mixing 0.1 ml of NADPH / H/ solution, 0.1 ml of enzyme/substrate solution containing urease, glutamate dehydrogenase, a-ketoglutarate, ADP, and 9.80 ml of 0.05 M potassium phosphate buffer, pH 8.0. The assays were performed by adding 0.01 ml of the urea gel extract to 0.99 ml of the assay solution, then determining the decrease in the absorbance at 340 nm. A calibration curve was used to correlate the urea concentration and urease activity.
RESULTS
Purity and molecular weight. Arginine kinase purified to homogeneity exhibits a single protein staining band after electrophoresis in SDS/polyacrylamide gels. The molecular weight, estimated from a comparison of the mobility of AK with several calibration proteins, is 41 kDa. The molecular weight determined by gel filtration using Sephadex G-100 was calculated to be 40 kDa, which agrees with the value of 39.9 kDa obtained by dynamic light scattering. These results indicate that AK is monomeric. Light scattering indicated the presence of less than 2% aggregated protein. Activity in urea. The activity of AK is increased by 20% following incubation in 0.5 M urea for 1 h (Fig. 1). Above 1 M urea, AK activity declines rapidly. Arginine kinase loses about 50% of its activity in 1.8 to 2.0 M urea. No activity is detected after incubating AK in 2.5 M urea for 1 h. Circular dichroism in urea. The circular dichroic spectrum of native AK was obtained by averaging five normalized scans at four protein concentrations. Native AK possesses 16% helical structure (19). In 1.5 M urea, AK molar ellipticity at 222 nm increases 35% (Fig. 1) which corresponds to a considerable increase in a-helix. Further increases in denaturant concentration result in a decrease in a-helical content in 4 M urea, and essentially none of the residual structures is retained in the a-helical conformation. These observations were made after 1 h of exposure to denaturant. Further incubation periods do not lead to additional changes at several urea concentrations tested. Fluorescence. The intrinsic fluorescence maximum of native AK is 337 nm. In denaturant up to 2.75 M
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linear response with extrapolation to 0.1501 for the limiting anisotropy (Fig. 3). In the presence of 0.5 M urea, the anisotropy remains higher across the range of viscosity when compared to the protein in the absence of urea. However, a distinct biphasic response is evident, which is more apparent at higher urea concentrations. The biphasic dependence of 1/rss on viscosity for AEDANS-AK in urea precluded determination of the limiting anisotropies and, therefore, evaluation of rotational correlation times. Quenching. The accessibility of the tryptophans to quenching was examined using the nonpolar fluorescence quencher acrylamide. The Stern–Volmer plots displayed downward curvatures indicating the presence of multiple quenching sites (22) which were instead analyzed according to the modified Stern– Volmer equation (23) F0/DF Å 1/([Q]faKQ) / (1/fa), FIG. 1. Activity and circular dichroism of AK as a function of urea concentration. The activity of AK (l) was determined colorimetrically. One unit of activity is 1 mmol of inorganic phosphate released per minute. The enzyme samples (AK, 0.040 mg/ml) were treated with denaturant for 1 h at the designated urea concentration, then transferred to the assay mixture containing the identical concentration of urea. Assays were performed for 2.0 min at 247C. Circular dichroism measurements (s): Samples of AK (0.11 to 0.21 mg/ml) were treated for 1 h with urea, then transferred to a 0.1-cm cuvette maintained at 247C and scanned from 350 to 205 nm in a Jasco 500 spectropolarimeter. The mean residue ellipticities are relative to the native state (U222 Å 07200).
urea, the emission maximum increases only 2 nm (Fig. 2), after which a sharp red shift begins, culminating in an emission maximum of 356 nm in 5 M urea. The relative intrinsic fluorescence intensity of AK at 356 nm declines 10% in 2.5 M urea, increases 20% between 2.5 and 3.5 M urea, and remains at approximately the intensity level of the native state from 4 to 8 M urea (Fig. 2). Intensity values were modified to correct for the direct effect of urea on tryptophan fluorescence, using N-acetyltryptophanamide as a model compound. The steady-state anisotropy of AEDANS-AK increases 4% between 0.25 and 1.0 M urea (Fig. 2). The anisotropy of AEDANS-AK is maximum at 0.0933 { 0.0003 in 0.5 M urea, significantly higher than 0.0897 { 0.0004 observed in buffer only. This change could be the result of several types of conformational change, including decreased macromolecular rotational diffusion, decreased segmental flexibility within the protein, or more limited motion of the probe-associated domain (21). The anisotropy decreases to 0.0246 { 0.0002 in 4 M urea and ultimately to 0.0236 { 0.0007 in 6 M urea. The dependence of the reciprocal of the anisotropy, 1/rss , of AEDANS-AK in buffer on viscosity exhibited a
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[2]
where F0 is the fluorescence intensity in the absence of quencher, DF is the change in fluorescence when the quencher is introduced, [Q] is the molar concentration of quencher, fa is the fractional accessibility of tryptophan to quencher, and KQ is the effective quenching constant, the product of the collisional rate constant and the fluorescence decay time. Modified Stern– Volmer plots for AK in urea are shown in Fig. 4. The linear relationship between F0/DF and 1/[Q] indicates suitability of analysis by the modified Stern–Volmer relationship. A substantial increase in KQ for AK begins after 2 M urea (Fig. 5), which is consistent with unfolding of the protein. Arginine kinase in the absence of denaturant exhibits nearly complete accessibility to acrylamide ( fa Å 0.99 { 0.03), while in 0.5 M urea this decreases to fa Å 0.89 { 0.02. Urea gel electrophoresis. The linearity of the urea concentration gradient in the polyacrylamide gel was confirmed by the urea gel calibration assay procedure. Initially, native homogeneous AK exhibits a faster migrating band in the region of 0.5 to 3 M urea after 100 min of UG–PAGE (Fig. 6). At 150 and 180 min, this transition is more evident with formation of three distinct regions. By 260 min, the gel exhibits an almost complete separation of a distinctly more mobile band in the 0 to 2 M urea region, followed by a faint, diffuse band from 2 to 3.5 M urea and then a slower, homogeneous band from 3.5 to 8 M urea. The time course of UG–PAGE, starting with denatured AK, exhibits essentially the same pattern of migration (gels not shown). DISCUSSION
Previous equilibrium and kinetic studies of creatine kinase (7, 24), a dimeric phosphagen kinase, revealed
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FIG. 2. Intrinsic fluorescence intensity and emission wavelength maximum of AK and anisotropy of AEDANS-AK as a function of urea concentration. Samples of AK (0.37 mg/ml) in buffer (0.05 potassium Hepes, pH 8.0, with 2 mM DTT) were incubated for 1 h in urea solutions at 247C. Samples were then diluted 1 to 5 in buffer containing the same urea concentrations. For the intrinsic fluorescence, excitation was at 295 nm and emission maxima (l) were determined from spectral scans between 300 and 400 nm. Relative fluorescence was measured at 356 nm and corrected for the presence of urea in the solvent and the direct effect of urea on the emission of tryptophan (s) (see text). For anisotropy measurements, samples contained 0.30 mg/ml AEDANS-AK (h) in 0.05 M potassium Hepes, pH 8.0, containing 2 mM DTT. After treatment with urea for 1 h, anisotropy was measured with excitation at 325 nm and emission was collected through two T-formatted Schott-type 470 cuton filters.
the occurrence a folding intermediate. Owing to the complexities of analyzing protein folding in the presence of subunit association in a multimeric protein, we examined AK, a simpler, albeit functionally analogous and structurally homologous (4), monomeric protein to search for and describe the structural features of a potentially analogous folding intermediate. During the course of this work, it was observed that transfer of native AK into dilute urea produced an apparently more compact protein structure which retained catalytic activity. Examination of a summary of the degree of parameter changes investigated as a function of urea concentration (Fig. 7) is consistent with a multiphasic model for denaturation. Focusing on the changes occurring in dilute denaturant reveals features characteristic of a structure more compact than the native state. The increase in activity in dilute denaturant has been observed for several other enzymes (25–27). This is generally attributed to a change in polypeptide flexibility in the domain of the active site. For AK, other results suggest a decrease in polypeptide flexibility in the active site (an increase in anisotropy of AEDANSderivatized AK), but as noted below, the steady-state anisotropy may be reflecting both localized and/or overall macromolecular rotation. Above 0.5 M urea, the cat-
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alytic activity of AK is the parameter most sensitive to denaturation. For purposes of comparison with other parameters only, assumption of a two-state model reveals a DGH2O of inactivation of 3.5 kcal/mol (28). Native AK possesses 16% a-helix, determined from the molar ellipticity at 222 nm by the method of Chen et al. (19). The 35% increase in molar ellipticity at 222 nm of AK between 0.25 and 2.5 M urea indicates not only resistance to denaturation of secondary structure but, apparently, the formation of more a-helix. The increase in secondary structure correlates with the urea gradient gel demonstrating a more compact protein state of AK in this range of denaturant concentration (see below). The stability of the secondary structure is revealed by a value of DGH2O Å 10.7 kcal/mol for the transition in circular dichroism and indicates that the structural feature most resistant to denaturation is the a-helix. The decrease in intrinsic fluorescence of AK in dilute urea may be due to more quenching within the protein or to exposure of the tryptophans to the aqueous media. That the decrease in intensity is not accompanied by a red shift in the emission maximum is consistent with internal quenching and the occurrence of a more compact conformation in dilute denaturant. The transition
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FIG. 3. Perrin plots for AK-AEDANS in urea. Samples of AK-AEDANS (2.0 ml of 0.25 mg/ml in 0.05 M potassium Hepes buffer, pH 8.0, and 2 mM DTT) were treated with increasing concentrations of sucrose at 257C. Steady-state anisotropy was measured as described for Fig. 4. Urea concentrations: (l), 0; (j), 0.5 M; (.) 3.5 M; (m) 4.5 M; (l) 6 M.
FIG. 4. Modified Stern–Volmer plots for quenching of the intrinsic fluorescence of AK. Samples (0.3 mg/ml) in 0.05 M potassium Hepes, pH 8.0, containing 2 mM DTT were titrated with aliquots from a 6 M stock solution of acrylamide. Fluorescence excitation wavelength was 295 nm and emission wavelength was 348 nm. Inner filter corrections were generally negligible. (s) 0 M urea; (l) 1 M urea; (,) 2 M urea; (.) 3 M urea; (h) 4 M urea; (j) 6 M urea; (n) 8 M urea.
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FIG. 5. Quenching constants (KQ) and fractional accessibility for AK with acrylamide as a function of the urea concentration. Values for KQ were determined from the modified Stern–Volmer plots as illustrated in Fig. 6. Fractional accessibility (l) and KQ (M01) (s).
FIG. 6. Time course of AK electrophoresis in urea gradient polyacrylamide gels. Native AK samples (0.50 mg/ml) in 0.01 Tris/HCl, pH 8.0, and 10 mM mercaptoethanol were subjected to electrophoresis (87C) for the indicated times and then gels were removed and stained with 0.1% Coomassie blue. Additional details are given in the text.
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FIG. 7. Summary of parameter changes in properties of AK as a function of urea concentration. fapp Å (pi 0 pD)/(pN 0 pD) 1 100, where pN , pD , and pi are the parameter values for the native state (zero urea), the value at the maximum change in parameter, and the intermediate parameter values, respectively. (,), Fluorescence emission maximum; (.), circular dichroism; (s), activity; (l) anisotropy.
to a higher intensity between 2 and 4 M urea signifies release of this quenching upon denaturation (5, 24) and possibly increased exposure of ionized tyrosines (29). When acrylamide is used as an extrinsic quencher, the quench constant for AK doubles between 2 and 4 M urea, implying a significant degree of unfolding, in agreement with the circular dichroism results. However, AK exhibits an anomalous decrease in fractional accessibility in dilute denaturant which is consistent with formation of a more compact conformation. Treatment of AK with urea greater than 1.5 M urea is clearly accompanied by a significant decrease in anisotropy, as expected for a protein undergoing denaturation. The small but experimentally significant increase in AEDANS-AK anisotropy (r) in low concentrations of urea indicates restriction of some depolarizing rotation, either locally or globally, although changes in fluorescence lifetime (t) or the limiting anisotropy (r0) could also account for the change in steady-state anisotropy, as described by the Perrin equation rss Å r0/ (1 / t/u), where u is the rotational correlation time. The biphasic dependence of the reciprocal of the anisotropy on viscosity in different concentrations of urea suggests the presence of multiple conformational states, which complicates the evaluation of the limiting anisotropy. Data not shown also suggest considerable heterogeneity in the fluorescence decay time of AE-
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DANS-AK in intermediate concentrations of urea. Consequently, time-resolved decay of the anisotropy could not be used to determine the structural origins of the increase in anisotropy observed in dilute urea. Urea gradient gel electrophoresis directly displays protein folding intermediates (2) and is important for confirming that intermediates detected by parameter changes under equilibrium conditions are not summation effects due to the simultaneous presence of native and denatured states. For AK, the distinct descending band observed early in electrophoresis may indicate a more compact conformation for the protein between 0.25 and 1.5 M urea, although this does not appear to be precisely the same range of denaturant in which other parameters point to a compact structure. The broad, diffuse band between 1.5 and 3 M urea corresponds to a folding/unfolding transition at a slower rate than the rate of electrophoresis (2). This is also the range for the midpoints in the unfolding transitions of AK, revealed by the changes in anisotropy (2.0 M urea), intrinsic fluorescence (3.0 M urea), and molar ellipticity at 222 nm (2.5 M urea). The slowest migrating band between 3 and 8 M urea represents the unfolded protein. Since the identical gel pattern is obtained when beginning with denatured AK, the refolding pathway may be the rapid reverse of the unfolding pathway (30). Compared with native protein, the structure of AK which exists in 0.5 M urea has (a) more circular dichroic absorbance in the far ultraviolet indicative of more helical content, (b) less fractional accessibility to tryptophans by acrylamide, (c) a significant decrease in intrinsic fluorescence, suggesting greater quenching between tryptophans, and (d) an increase in the anisotropy. From these data, it seems reasonable to suggest that the overall shape of AK in dilute urea is more compact than in the native solvent. Without meaningful time-resolved anisotropy data, it is not possible to evaluate the degree of tertiary flexibility, which may, in any event, be too slow for detection by timeresolved fluorescence analysis. However, the retention of catalytic activity suggests the absence of a largescale disruption of overall structure in dilute denaturant. The structure described does not appear to correspond to any of the four classes of protein folding intermediates found in acid solution (31). The persistence of catalytic activity, still accompanied by clear, if not dramatic, changes in conformation, suggests that this structure may be a thermodynamically accessible state even in the absence of urea. We have previously demonstrated that conformational isoforms of lobster muscle AK occur in the native solvent environment (15). The presence of dilute urea may shift the equilibrium to highly populate an isoform that is
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still active but with the apparently collapsed conformation described. Whether this would still be considered a folding intermediate on the path to the more populated native conformation in benign solvent is uncertain. ACKNOWLEDGMENTS Supported in part by grants (S.H.G.) from the American Heart Association, Florida Affiliate, and the National Science Foundation (9120260).
REFERENCES 1. Kim, P. S., and Baldwin, R. L. (1990) Annu. Rev. Biochem. 59, 631–660. 2. Creighton, T. E. (1979) J. Mol. Biol. 129, 235–264. 3. Ptitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., and Razgulyaev, O. I. (1990) FEBS Lett. 262, 20–24. 4. Anosike, E. O., Moreland, B. H., and Watts, D. C. (1975) Biochem. J. 145, 535–543. 5. Grossman, S. H., Pyle, J., and Steiner, R. J. (1981) Biochemistry 21, 6122–6128. 6. Grossman, S. H., Gray, K. A., and Lense, J. J. (1986) Biochim. Biophys. Acta 248, 234–242. 7. Grossman, S. H. (1994) Biochim. Biophys. Acta 1209, 19–23. 8. Creighton, T. E. (1984) Adv. Biophys. 18, 1–20. 9. Kuby, S. A., Nody, L., and Lardy, H. A. (1954) J. Biol. Chem. 210, 65–82. 10. Virden, R., and Watts, D. C. (1964) Comp. Biochem. Physiol. 13, 161–177. 11. Fiske, C. H., and Subbarow, Y. (1925) J. Biol. Chem. 56, 375– 400.
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12. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. 13. Virden, R., Watts, D. C., and Baldwin, E. (1965) Biochem. J. 94, 536–544. 14. Laemmli, U. K. (1970) Nature 227, 680–685. 15. Grossman, S. H. (1991) Biophys. J. 59, 590–597. 16. Lakowicz, J. R., Cherek, H., and Balter, A. (1981) J. Biochem. Biophys. Methods 5, 131–146. 17. Grossman, S. H. (1989) Biochemistry 28, 4894–4902. 18. Dumas, C., and Camonis, J. (1993) J. Biol. Chem. 268, 21599– 21605. 19. Chen, Y. H., Yang, J. T., and Martinez, H. M. (1972) Biochemistry 11, 4120–4131. 20. Talke, H., and Schubert, G. E. (1965) Klin. Wochenschr. 43, 174– 175. 21. Lakowicz, J. R. (1983) Principles of Fluorescence Spectroscopy, Plenum, New York. 22. Eftink, M. R., and Ghiron, C. A. (1981) Anal. Biochem. 114, 199– 227. 23. Lehrer, S. S. (1971) Biochemistry 10, 3254–3263. 24. Grossman, S. H., and Mixon, D. (1985) Arch. Biochem. Biophys. 236, 797–806. 25. Johnson, C. M., and Price, N. C. (1987) Biochem. J. 245, 525– 530. 26. Ma, Y. Z., and Tsou, C. L. (1991) Biochem. J. 277, 207–211. 27. Liang, S. J., Lin, Y. X., Zhou, J. M., Tsou, C. L., Wu, P., and Zhou, Z. (1990) Biochim. Biophys. Acta 1038, 240–246. 28. Pace, C. N. (1975) Crit. Rev. Biochem. 3, 1–43. 29. Yao, Q. Z., Tian, M., and Tsou, C. L. (1984) Biochemistry 23, 2740–2744. 30. Creighton, T. E. (1980) J. Mol. Biol. 137, 62–80. 31. Fink, A. L., Calciano, L. J., Goto, Y., Kurotsu, T., and Palleros, D. R. (1994) Biochemistry 33, 12504–12511.
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