©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetic Analysis of the Folding of Human Growth Hormone
INFLUENCE OF DISULFIDE BONDS (*)

(Received for publication, April 1, 1995; and in revised form, June 15, 1995)

Karen M. Youngman Donald B. Spencer (§) David N. Brems (¶) Michael R. DeFelippis (**)

From the From Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We report the results of a stopped-flow kinetic evaluation of the folding of human growth hormone (hGH). The results are compared with those obtained for a disulfide-modified analog in which the four cysteine residues have been reduced and alkylated to form tetra-S-carbamidomethylated hGH in order to elucidate the role of disulfide bonds in the folding reaction. Multiple detection techniques were applied to monitor both refolding and unfolding processes initiated by guanidine hydrochloride concentration jumps. Using far-UV circular dichroism (CD) detection to monitor folding of hGH, we find that 70% of the secondary structure forms in a burst phase occurring within the stopped-flow dead time. Two slower phases were identified in the observable portion of the CD signal. Multiple kinetic phases were resolved when folding was monitored by intrinsic tryptophan fluorescence or near-UV absorbance as probes of tertiary structure, and the number of time constants required to fit the data depended on the hGH concentration and nature of the denaturant jump. The associated amplitudes also displayed strong dependence on the final denaturant concentration. Results obtained from the tetra-S-carbamidomethylated hGH studies demonstrate that the folding reactions of hGH are remarkably similar in the presence and absence of the disulfide bonds. Disulfide bond reduction in hGH is proposed to affect folding primarily by increasing the population of self-associated intermediate states in the folding pathway.


INTRODUCTION

Human growth hormone (hGH) (^1)is a single domain, globular protein containing 191 amino acids and having a molecular weight of approximately 22 kDa. There are two disulfide bridges present in the protein: one connecting distant parts of the molecule involving residues 53 and 165 (large loop) and another near the C terminus between residues 182 and 189 (small loop). hGH stimulates cell growth and affects other metabolic, physiologic, and anatomic processes(1, 2) . It has been demonstrated that the two disulfide bonds in hGH may be reduced and alkylated to form carbamidomethylated derivatives with full retention of growth-stimulating activity(3) . Therefore, hGH provides a unique system for comparing the folding properties of a protein in the presence and absence of its disulfide bonds. The difference in folding properties of proteins in the presence and absence of disulfide bonds has important implications for the comparison of in vitro and in vivo protein folding reactions.

Equilibrium denaturation studies have been previously reported for hGH and cysteine-modified forms of the protein(4) . The folding of hGH was shown to be a cooperative two-step process. Carbamidomethylation or carboxymethylation of the four cysteine residues decreased the stability of hGH by over 9 kcal/mol. Furthermore, carboxymethylation of hGH resulted in noncoincidence of equilibrium denaturation curves detected by different spectroscopic methods. From these results it was concluded that reduction of the disulfides in hGH decreases the stability of the native state relative to the intermediate folding states, leading to the population of stable intermediates under equilibrium conditions. A more recent evaluation (5) of the equilibrium folding properties of hGH indicates that both monomeric and self-associated intermediates can be populated at equilibrium depending on the solution conditions. These equilibrium data can be interpreted in terms of a folding model that is similar to the mechanism reported for bovine growth hormone (bGH)(6, 7, 8) . Equilibrium folding data reported on porcine growth hormone (9) is also consistent with such a model. The similarities in the equilibrium properties of these three proteins suggest that a general equilibrium folding mechanism might exist for growth hormones.

While the equilibrium folding properties of growth hormones from a number of species have been studied, the folding kinetics of only bGH have been reported to date(10) . This present report describes results obtained from a kinetic evaluation of the folding of hGH and a disulfide-modified form of the protein in which the four cysteine residues have been reduced and carbamidomethylated, tetra-S-carbamidomethylated human growth hormone (2-RCAM hGH). Stopped-flow rapid mixing techniques were used to initiate folding and unfolding reactions by GdnHCl concentration jumps, and multiple spectroscopic detection techniques were employed to probe the resultant changes in secondary and tertiary structure as a function of time. The kinetic results obtained for hGH and 2-RCAM hGH are compared, and the similarities to the folding kinetics of bGH are also discussed.


EXPERIMENTAL PROCEDURES

Materials

Human growth hormone was produced by recombinant DNA techniques at Eli Lilly and Company and had the naturally occurring amino terminus. GdnHCl was ultrapure from ICN Biochemicals (Cleveland, OH). Dithiothreitol and iodoacetamide were obtained from Sigma. D-Glucurono-6,3-lactone, used for stopped-flow CD calibration, was obtained from Aldrich. All other reagents were analytical grade or better and were obtained from standard sources. Water used to prepare solutions was purified by a Millipore Milli-Q Plus system.

Methods

Preparation of Tetra-S-carbamidomethylated Human Growth Hormone

The reduced 2-RCAM hGH was prepared as described previously (4) with the following modifications. The reaction was carried out with 1 g of hGH as starting material, and the quantities of dithiothreitol and iodoacetamide were increased proportionately. Instead of undergoing purification and desalting by column chromatography, the 200-ml reaction mixture was dialyzed extensively against 4 4 liters of deionized water that had been adjusted to pH 8.0 with NH(4)OH. The product was lyophilized and stored at -15 °C. The amino acid content of the product, including carbamidomethylation of all four cysteine residues, was verified by amino acid analysis.

Solution Preparation for Stopped-flow Measurements

Stock solutions of hGH and 2-RCAM hGH were prepared by dissolving protein in degassed 20 mM Hepes, pH 7.5, or degassed 20 mM Hepes containing 7 M GdnHCl, pH 7.5. The solutions were then filtered through Gelman Acrodisc 0.20 or 0.45-µm low protein binding filters. Protein concentrations were determined by UV absorption using the extinction coefficient of 18,890 M cm reported in (4) . The GdnHCl concentrations were determined by refractometry as described in (11) . Hepes buffer solutions were filtered and vacuum degassed, and GdnHCl solutions were filtered, sonicated, and vacuum degassed before each stopped-flow experiment.

For protein concentration dependence experiments, the initial hGH concentration ranges were 0.5-5.5 mg/ml for fluorescence and absorbance and 0.5-1.5 mg/ml for CD measurements. The stopped flow was programmed for concentration jumps from 7 to 3 M GdnHCl (a 2.3-fold dilution) for refolding and from 0 to 7 M GdnHCl (a 6-fold dilution) for unfolding experiments. To determine the GdnHCl-concentration dependence for refolding, a stock solution of 2.5 mg/ml hGH in 20 mM Hepes containing 8 M GdnHCl adjusted to pH 7.5 was prepared. This solution was diluted in the stopped flow to achieve the desired final GdnHCl concentration while maintaining a constant dilution of protein to 0.42 mg/ml. The GdnHCl concentration dependence for unfolding jumps was determined in a similar manner, except that the initial hGH was 2.5 mg/mL in 20 mM Hepes, pH 7.5. Similar conditions were used for the 2-RCAM hGH protein concentration- and GdnHCl-dependent refolding experiments except for minor modifications as noted in the figure legends.

Stopped-flow Instrumentation and Methodology

A Bio-Logic (Claix, France) stopped flow was used to initiate the folding reactions. The instrumentation consisted of an SFM-3 syringe module and MPS-51 controller/power supply. An IBM-compatible computer running the Bio-Logic, Bio-Kine acquisition software package was used to program the desired syringe drive sequences. The observation head/cuvette holder allowed for independent temperature regulation. All measurements were performed at 22 ± 1 °C (direct thermocouple measurement inside cuvette), and the observation head and syringes were differentially temperature-controlled using individual circulating water baths to minimize any optical artifacts caused by temperature changes upon mixing and dilution of GdnHCl. The observation head was outfitted with an HDS mixer and a hard stop electronic valve accessory supplied by Bio-Logic to prevent optical artifacts due to Schlieren effects.

Kinetic reactions were monitored by intrinsic tryptophan fluorescence, near-UV absorbance, or CD using an optical detection system supplied by Bio-Logic. The light source was a 150-W mercury-xenon lamp (Hamamatsu, Bridgewater, NJ) controlled by an ALX-210 (Bio-Logic) regulated DC power supply, and wavelength selection was accomplished using a variable wavelength grating monochromator (Jobin-Yvon, Longjumeau, France) with interchangeable fixed slit widths. The monochromatic light was delivered to the observation cuvette by a tapered, quartz fiber optic light guide (Bio-Logic). A shielded photomultiplier tube and PMS-400 photomultiplier tube controller/data processing unit (Bio-Logic) were used to detect the transient signals. Fluorescence or UV experiments were performed by changing the orientation of the photomultiplier tube relative to the light guide. For fluorescence measurements, the excitation wavelength was 290 nm, the detector was oriented at 90°, and appropriate cut-off filters (Corion, Holliston, MA) were placed in front of the photomultiplier tube to approximate the emission wavelength. Near-UV absorbance experiments were performed at 295 nm. To perform far-UV CD measurements, the light source was focused through a UV polarizer and a Hinds Instruments (Hillsboro, OR) 50 kHz photoelastic modulator powered by a PEM-90 controller set at quarter-wave retardation. When programmed for CD mode, the PMS-400 allowed direct readings of ellipticity in millidegrees. The wavelength for the CD experiments was 225 nm, and the CD optics were optimized and calibrated using solutions of D-glucurono-6,3-lactone in water and hGH dissolved in Hepes. Adjustments were made to the CD optical and electronic components so that signals for the calibration solutions exactly matched those obtained on an Aviv model 61DS circular dichroism spectrometer.

Different Bio-Logic stopped-flow quartz cuvettes were used, depending on the optical detection technique. Absorbance measurements were performed using either a TC-100/15 (10-mm pathlength, 40-µl volume) or TC-50/10 (5-mm pathlength, 15-µl volume) cuvette. The TC-100/15 cuvette was used for fluorescence measurements (excitation through the 1.5-mm pathlength and emission observed on the 10-mm pathlength). An FC-20 (2-mm pathlength, 54-µl volume) was used for CD experiments. The SFM-3 flow rate was generally programmed to 5 ml/s, translating into theoretical dead times of 8, 3, or 10.8 ms for the TC-100/15, TC-50/10, and FC-20 cuvettes, respectively.

Data Collection and Analysis

The stopped-flow instrumentation was set up for dual channel operation allowing simultaneous data collection of fast and slow time bases. Detector signals were electronically filtered using appropriate time constants. For fluorescence and absorbance experiments, 5 successive kinetic traces were generally averaged. A minimum of 25 kinetic curves was averaged for stopped-flow CD measurements. When appropriate, we recorded relevant base lines and used the calculation procedures described in (12) to estimate the amplitude change occurring within the dead time. The kinetic files were converted to ASCII format and downloaded to a Vax computer running the nonlinear least-squares program NLIN (SAS Institute, Cary, NC). The fast and slow channel kinetic data files were merged and fit to an equation (13) of the following form.

where A(t) is the total amplitude at time t, A is the amplitude at infinite time, A is the amplitude of the individual phases, i, at zero time and is the associated time constant. To determine the number of kinetic phases, the data were evaluated multiple times using containing 2-4 time constants. The function providing the best fit, based on the statistical output from the fitting routine, was used to obtain estimates for the time constants and amplitudes.

Normalization of Kinetic Amplitudes

The normalization procedure involves expressing the observed photomultiplier voltage signal for a particular kinetic phase as a fraction of the total expected voltage difference between the initial and final protein conformational states for a given denaturant concentration at equilibrium. The final fluorescence intensities were obtained from the A parameter in the kinetic amplitude response function. This parameter represents the photomultiplier voltage at infinite time, when the refolding or unfolding reactions have reached equilibrium. The amplitudes were plotted against the GdnHCl concentration and fit to a two-state model for an equilibrium unfolding transition(14) . The values of the initial fluorescence intensities expected at a particular denaturant concentration are obtained by linear extrapolation of the pre- and post-transition base lines to unfolding and refolding concentrations of GdnHCl, respectively. At a given final denaturant concentration, the expected amplitude change is calculated as the difference between the final voltage A and the extrapolated initial voltage.


RESULTS

Stopped-flow Circular Dichroism Detection of Native hGH Folding

The large helical content of folded hGH yields a strong far-UV CD signal at 225 nm of approximately -20,000 degreesbulletcm^2bulletdmol. As shown in Fig. 1A, the ellipticity in this wavelength region is virtually eliminated when the protein is dissolved in solutions containing >6 M GdnHCl. Therefore, far-UV CD provides an excellent detection method for observations of changes in secondary structure. Kinetic experiments were performed on solutions containing varying concentrations of hGH dissolved in 7 M GdnHCl that were diluted with buffer to a final GdnHCl concentration of 3 M. The 3 M final GdnHCl concentration was chosen because of problems encountered at lower denaturant concentration with aggregation resulting in light-scattering signals that overlapped with the folding kinetics. The higher level of GdnHCl eliminates this problem, and the denaturant concentration is dilute enough so that the protein is essentially folded. The aggregation problem was primarily observed when using fluorescence or near-UV absorbance detection where high hGH concentrations were required; however, to maintain experimental consistency, the GdnHCl concentration jumps were kept identical for these CD measurements.


Figure 1: Formation of secondary structure observed by refolding kinetics. A, far-UV CD spectra of hGH in 20 mM Hepes, pH 7.5 (--) and 20 mM Hepes containing 7 M GdnHCl, pH 7.5 (bulletbulletbulletbullet). B, kinetic trace of hGH refolding measured at 225 nm. Refolding was initiated by a GdnHCl concentration jump from 7 to 3 M. The solidline drawn through the data is a nonlinear fit using .



An example of a kinetic trace obtained from the dilution of 1.3 mg/ml hGH in 7 M GdnHCl is shown in Fig. 1B. It can be seen by comparison to the far-UV CD spectra in Fig. 1A that a significant portion (approximately 70%) of the reaction is unobservable and presumed to be lost in the stopped-flow dead time. Refolding reactions were studied in the hGH concentration range of 0.5-1.5 mg/ml. All of the kinetic traces could best be fit using two exponential terms, and the time constants are independent of hGH concentration over the range tested. The time constants for both phases are reported in Table 1as an average over the concentration range studied. The fractional amplitudes for these rate constants are also concentration-independent and contribute approximately equally to the total observed amplitude for the folding reaction. In some of the experimental trials, we utilized the calculation procedures described in (12) to obtain estimates for the fraction of expected reaction. From these analyses, we determined that the amplitudes for the two phases contribute almost equally (10-15% for each phase) to the total detectable reaction, and approximately 30% of the refolding process is actually observed.



Fluorescence- and Absorbance-detected Refolding: hGH and 2-RCAM hGH Concentration Dependence

The intrinsic tryptophan fluorescence spectrum of fully unfolded hGH at neutral pH is quenched and displays a 15-nm red shift relative to the native state(4) . These spectral changes provide a means to study folding of the tertiary structure by following differences in the tryptophan emission spectrum reflecting conformational changes around the tryptophan environment. To improve signal, higher initial hGH concentrations were required in these experiments. The hGH concentration dependence for refolding and unfolding reactions was studied in the range of 0.5-5.5 mg/ml (before dilution). For refolding experiments diluted to <1 M denaturant concentrations, precipitation resulted, and light-scattering signal changes overlapping with the folding reactions complicated analysis of the kinetic curves. This problem was overcome by performing refolding jumps from 7 M to 3 M GdnHCl.

The time constants obtained for fluorescence-detected refolding experiments are independent of the hGH concentration, and the results averaged over the range studied are reported in Table 1. Using nonlinear curve fitting, a total of four kinetic phases are resolved. One of the phases, designated with the subscript n.p. for new phase, is only resolved in experiments performed using initial hGH concentrations geq3 mg/ml. The time constants (3) and (1) are comparable with those obtained by stopped-flow CD measurements.

The near-UV absorbance spectrum of hGH is also sensitive to tertiary structure conformational changes. A spectral difference between native and GdnHCl-denatured recombinant derived hGH with a maximum change at 295 nm has been reported(4) . Kinetic reactions were also monitored by this detection technique. Refolding reactions were initiated with the same 7 to 3 M GdnHCl concentration jump, and the hGH concentration was varied in a similar manner to the fluorescence experiments. Results from these experiments are in good agreement with the fluorescence data. The four time constants obtained from the data analysis are listed in Table 1. Similar to the fluorescence data, the phase designated as could only be resolved above 3 mg/ml hGH. By extrapolation of the near-UV absorbance amplitude data, approximately 30% of the expected reaction is observed.

Similar concentration-dependent folding experiments were performed on 2-RCAM hGH. The effect of protein concentration on the 2-RCAM hGH folding reaction was explored by dilution of a GdnHCl-denatured protein sample from 7 to 2 M GdnHCl for final protein concentrations between 0.08 and 1.7 mg/ml. The results of the 2-RCAM hGH concentration-dependent folding experiment using fluorescence detection are presented in Fig. 2. Three kinetic phases were detected for the folding reaction at all protein concentrations, with time constants between 20 ms and 5 s. The (3) and (2) phases demonstrate a dependence on protein concentration, showing a 6-10-fold increase in time constant as the final protein concentration is increased from 0.08 to 1.8 mg/ml. The time constant of the slowest phase is less dependent on protein concentration over the range studied. The results of 2-RCAM hGH concentration-dependent folding jumps from 7 to 2 M GdnHCl monitored by UV absorbance spectroscopy (data not shown) are similar to those observed by fluorescence.


Figure 2: Effect of protein concentration on the fluorescence-detected kinetics of 2-RCAM hGH folding. Solutions containing varying amounts of 2-RCAM hGH in 7 M GdnHCl were diluted to a final denaturant concentration of 2.0 M GdnHCl. The time constants are defined by the following symbols: --, (1); --, (2), and --, (3). The errorbars represent the variation determined from three separate folding experiments at each protein concentration. A second order polynomial equation was used to obtain smoothlines through the data points in order to aid with visualization of the trends.



Fluorescence-detected Unfolding: hGH Concentration Dependence

The concentration dependence of the unfolding reaction was determined using similar conditions as described for refolding experiments, except that unfolding was initiated by a GdnHCl concentration jump from 0 to 7 M GdnHCl. A kinetic model comprising two time constants best described the data. The results listed in Table 1demonstrate that the two time constants are independent of hGH concentration. These values are in agreement with two of the time constants, (3) and (2), resolved in the refolding experiments. Most notably absent in these unfolding results, however, is the apparent concentration dependence at initial hGH concentrations above 3 mg/ml and the slow phase that was observed in all of the refolding experiments. Due to significant problems encountered with aggregation of 2-RCAM hGH under these experimental conditions, unfolding experiments could not be performed.

GdnHCl Concentration Dependence on Refolding and Unfolding of hGH

Three phases were observed in GdnHCl concentration-dependent stopped-flow fluorescence refolding experiments on hGH, with time constants ranging from 10 ms to about 10 s. These phases are designated (1), (2), and (3) in order of decreasing time constant. The fast phase, (3), is characterized by a strong dependence on final GdnHCl concentration, as shown in Fig. 3A. The two slow refolding phases have much less pronounced denaturant concentration dependences.


Figure 3: Fluorescence-detected GdnHCl concentration-dependent hGH refolding and unfolding kinetic data. A, the symbols representing the time constants for refolding experiments are the same as in Fig. 2. Unfolding time constants are represented by -black square- and -up triangle, filled- for (1) and (2), respectively. -▾- refers to unfolding jumps where only one phase was observed. B, amplitudes calculated relative to the total expected amplitude based on equilibrium fluorescence intensities as described under ``Experimental Procedures.'' The symbols are the same as in A.



Two phases were observed for GdnHCl concentration-dependent stopped-flow fluorescence unfolding of hGH (Fig. 3A). The time constants of these phases are 10 ms and 1 s under strongly denaturing conditions (6.7 M GdnHCl). The time constant of the faster of the two unfolding phases is strongly dependent on GdnHCl concentration, while the other phase has a less pronounced denaturant dependence. At final denaturant concentrations in the range of 4.5-4.7 M GdnHCl, only one unfolding phase could be resolved. Since it is difficult to determine if this single phase is related to either of the two unfolding phases (or both) observed under strongly denaturing conditions, the data points corresponding to these conditions are represented in Fig. 3A by a different symbol.

A direct comparison of the amplitudes for folding jumps with different final concentrations of denaturant is complicated by the strong dependence of the fluorescence intensity of hGH on GdnHCl concentration in both the pre- and post-transition base-line regions. Further complication arises from the lower fluorescence signals expected for folding jumps into the transition region where a mixed population of native and denatured protein is present. To facilitate meaningful comparisons of the amplitudes of kinetic phases for folding and unfolding jumps to different final GdnHCl concentrations, the observed amplitudes were normalized according to the procedure described under ``Experimental Procedures.'' A plot of relative fluorescence intensity as a function of final GdnHCl concentration is presented in Fig. 4. The values of the initial fluorescence intensities expected for folding jumps at a particular denaturant concentration are obtained by linear extrapolation of the equilibrium post-transition base lines to folding concentrations of GdnHCl. At a given final denaturant concentration, the expected amplitude change is calculated as the difference between the final voltage (A) and the extrapolated initial voltage.


Figure 4: Relative fluorescence intensities for hGH kinetic traces as a function of final GdnHCl concentration. Each data point represents the final intensity obtained following a particular GdnHCl folding jump. The data were analyzed assuming a two-state model as described in (14) .



The behavior of each of the three refolding amplitudes is quite distinct when the amplitudes are calculated relative to the total expected amplitude based on equilibrium fluorescence intensities (Fig. 3B). The amplitude of the slowest phase, (1), comprises less than 5% of the expected fluorescence amplitude over the entire range of refolding final GdnHCl concentrations. The amplitude of the (3) refolding phase is dominant at low final denaturant concentrations, increasing steadily from 25 to 40% of the expected amplitude over the range from 3.0 to 3.8 M GdnHCl. From 3.8 to 4.5 M GdnHCl, the (3) amplitude decreases to 10-20% of the expected relative amplitude. The amplitude of the (2) refolding phase increases sharply from less than 5% at 3 M GdnHCl to 60% at 4.5 M GdnHCl.

The amplitudes of the two fluorescence-detected, GdnHCl-dependent unfolding phases are very different (Fig. 3B). The relative amplitude of the slower phase is small, comprising 5-10% of the expected signal change for unfolding jumps. The relative amplitude of the faster unfolding phase is dominant over the entire unfolding range of 4.5-6.7 M GdnHCl and accounts for 80% of the expected fluorescence change under strong denaturing conditions and up to 100% of the expected fluorescence change for unfolding jumps into the transition region.

It should be noted that for unfolding jumps to final GdnHCl concentrations of 4.5-4.7 M, only one phase was observed with values of 100% or slightly greater for the fractional relative amplitudes. We could not assign a probable cause to amplitudes greater than 100% but conclude that they may reflect uncertainty in the values of the expected amplitudes in this region. Furthermore, these amplitudes cannot be compared relative to the others because of the difficulty in assigning the relatedness to either unfolding phase. For these reasons, the amplitudes corresponding to these time constants are not plotted in Fig. 3B.

GdnHCl Concentration Dependence on Refolding of 2-RCAM hGH

The folding kinetics of 2-RCAM hGH were investigated as a function of final GdnHCl concentration using similar procedures as described for hGH. The fluorescence-detected GdnHCl concentration dependence of the folding time constants and amplitudes is presented in Fig. 5, A and B, respectively. Three kinetic phases were detected for the folding reaction, with time constants between 20 ms and 5 s. The magnitudes of the time constants of these folding phases are very similar to those of the three folding phases observed for disulfide-intact hGH and are designated (1), (2), and (3) in order of decreasing time constant.


Figure 5: Effect of GdnHCl concentration on the fluorescence-detected kinetics of 2-RCAM hGH folding. A, folding was initiated by dilution of an unfolded protein sample in 7 M GdnHCl, 20 mM Hepes, pH 7.5 to the indicated denaturant concentration in 20 mM Hepes, pH 7.5. The initial protein concentration was 2.5 mg/ml, and the final protein concentration was 0.42 mg/ml. B, normalized amplitudes for the fluorescence-detected kinetics of 2-RCAM hGH folding obtained as described under ``Experimental Procedures.'' The conditions are the same as described for A. The symbols representing the time constants and associated amplitudes are the same as those described in the Fig. 2legend except for (--), which indicates the total amplitude determined from the sum of the amplitudes from each kinetic phase.



For the (1) and (2) folding phases, the time constant is maximal for folding jumps to a final GdnHCl concentration of about 2 M GdnHCl. For folding jumps to final GdnHCl concentrations greater than 2 M, the time constants of these phases decrease with increasing GdnHCl concentration. The fast folding phase, (3), is less dependent on the final denaturant conditions.

The behavior of GdnHCl concentration-dependent folding time constants detected by UV absorbance was similar to that observed by fluorescence (data not shown). Analysis of the UV absorbance-detected amplitudes, however, was hampered by poor signal-to-noise ratios.

The normalized amplitudes for 2-RCAM hGH folding jumps plotted as a function of final GdnHCl concentration are presented in Fig. 5B. Under folding conditions in low denaturant, only 40% of the expected amplitude is actually observed. As the final denaturant concentrations approach those of the equilibrium transition region, however, a progressively greater proportion of the reaction (up to 75% in the transition region) is detected. The undetected portion of the folding reaction must represent a transition(s) that occurs within the dead time of mixing of the stopped-flow instrument.

The amplitude of the (1) folding phase is dominant for jumps to low denaturant concentrations and decreases steadily through the equilibrium transition region. The normalized amplitudes of the (3) and (2) folding phases are small in folding conditions of low denaturant and increase steadily for jumps into the transition region.

Normalized Amplitudes as a Function of GdnHCl Concentration: Determination of Thermodynamic Parameters

The final fluorescence amplitude values determined for each refolding kinetic curve as a function of GdnHCl concentration were used to construct the curve for the hGH results shown in Fig. 4. Using the procedures described in (14) , these equilibrium results were analyzed assuming a two-state model to calculate estimates for the free energy of unfolding, DeltaG, in the absence of denaturant. The DeltaG and midpoint values are 13.8 kcal/mol and 4.5 M GdnHCl, respectively. Similar analysis of near-UV absorbance-detected data yielded a DeltaG of 12.2 kcal/mol and midpoint of 4.4 M GdnHCl (data not shown). These results are in good agreement with previously reported data on the equilibrium denaturation of hGH(4, 5) . A similar analysis was performed on the 2-RCAM hGH data, yielding a DeltaG of 4.4 kcal/mol and an equilibrium transition midpoint of 2.9 M GdnHCl (data not shown). These values also compare favorably with previously reported equilibrium results(4) .


DISCUSSION

Comparison of Folding in the Presence and Absence of Disulfide Bonds

The unfolded states of hGH and 2-RCAM hGH are likely different. However, evidence obtained from CD, fluorescence, and UV absorbance measurements indicates that the final folded states are identical(4) . 2-RCAM hGH has also been shown to be fully active in biological assays(3) , further supporting a folded state similar to hGH. These results strongly suggest that carbamidomethylation of the cysteine residues does not significantly perturb the structure of hGH. In consideration of these facts, the folding kinetics of both proteins may be compared.

For both forms of the protein and under folding conditions of low denaturant, a large fraction of the expected fluorescence and UV absorbance-detected signal change is lost in the dead time of mixing. At these same conditions, the time constants of the three folding phases observed for hGH were similar in magnitude to the three folding phases obtained for 2-RCAM hGH. The greatest difference observed between the folding kinetics of the two molecules at low denaturant is the larger amplitude of the slowest folding phase for 2-RCAM hGH compared with intact hGH.

Differences were also observed in the dependence of the time constants of the kinetic folding phases of hGH and 2-RCAM hGH on final protein concentration. The time constants of the two faster folding phases of 2-RCAM hGH demonstrated a marked dependence on final protein concentration, while the time constant for the slowest 2-RCAM hGH phase and those for each of the three corresponding hGH phases did not. However, at higher hGH concentrations an additional folding phase was observed. The observation of an increased dependence of the kinetic data on protein concentration for 2-RCAM hGH relative to disulfide-intact hGH could be evidence for a greater degree of intermolecular interaction in the folding of the reduced and alkylated form of the protein. Similarly, the additional phase encountered with disulfide-intact hGH at high concentration may be related to an aggregation phenomenon as argued for previously reported equilibrium results(5) .

It appears, in general, that the protein folding kinetics have not been drastically altered for the 2-RCAM hGH molecule relative to hGH. This is the case at least for the folding kinetics, where the same number of phases with similar relaxation times were detected for both proteins. These results indicate that the role played by the disulfide bonds in folding reactions is minimal, at least to the extent that the effects can be detected by the methods employed in the present study. This apparent lack of effect may result from the fast collapse of the molecule as evidenced by the loss of a significant amount of the CD, fluorescence, and UV signals within the dead time of stopped-flow mixing. A result of this burst phase may be to position the two cysteine residues of the large disulfide loop proximate to each other in a manner similar to the way in which they are constrained by the disulfide bond in intact hGH. If methods were available to detect denaturant-induced protein conformational changes on the time scale of the burst phase species, it might be observed that this species forms more readily in the case of intact hGH where the burst phase conformation is constrained by disulfide bonds.

Our kinetic folding data on hGH and its disulfide-reduced form may also be compared with the results of other investigations on the effects of natural disulfides, genetically engineered disulfides, and chemical cross-links on protein folding kinetics(15, 16, 17, 18, 19, 20, 21) . For the various proteins employed in these studies, the observed effects on the kinetics were specific to either changes in the rates of refolding or unfolding but not both. In each case cited, the effect of the cross-link was interpreted as resulting from the selective perturbation of particular states populated along the folding pathway. For hGH, where no dramatic effects are observed on the rates of folding phases common to the disulfide-intact and reduced forms, it is likely that any stabilizing or destabilizing effects of the disulfide bonds are on states populated early in the folding reaction that occur faster than the time resolution of the stopped-flow experiment, as was discussed above.

Comparison with bGH Folding

Folding kinetics have been reported for bGH (10) and can now be compared with our observations in this study. For bGH under refolding conditions, all of the expected absorbance change was observed, while only 16-30% of the expected amplitude was detected by far-UV CD. This data indicates that secondary structure forms earlier than tertiary structure. In contrast, for hGH refolding, no such estimation could be made since only a fraction of the reaction was observed by all detection techniques employed. In terms of refolding dependence on protein concentration, two of the three 2-RCAM hGH phases detected by fluorescence and the slower of the two bGH phases detected by absorbance were concentration-dependent, while the three hGH phases detected at lower concentrations by both fluorescence and absorbance were concentration-independent. Similarly, by far-UV CD the two folding phases detected for hGH were concentration-independent, while the single phase observed for bGH was concentration-dependent. In addition, two unfolding phases were resolved for hGH by fluorescence, whereas only one was observed for bGH by CD and near-UV absorbance. The differences between hGH and bGH folding may be related to relative stability, spectral properties, or kinetics of intermediates compared with the native state.

The stability of hGH toward GdnHCl-induced denaturation is known to be greater than that of growth hormones from bovine and porcine sources (4, 9, 22) . The observation of equilibrium intermediates and self-associated forms in 2-RCAM hGH and other nonhuman growth hormones was suggested to be related to the much lower stabilities of these latter species(4) . In the case of hGH, the intermediates were proposed to be destabilized relative to the native state and not significantly populated under equilibrium conditions. Reduction and alkylation of the disulfide bridges of hGH diminishes the stability differences between the native and intermediate states such that the denaturation behavior is similar to the nonhuman growth hormones with well populated equilibrium intermediates. We propose that the concentration dependence of the 2-RCAM hGH folding kinetics suggests an increased propensity for populating a self-associated intermediate compared with hGH and that this interpretation is consistent with the explanation offered for the equilibrium data.

Implications for Protein Folding in Vitro and in Vivo

Protein folding reactions of disulfide-containing proteins studied in vitro are typically conducted in the presence of the disulfide bonds. This situation is in contrast to the in vivo folding process, where disulfide cross-links may not be formed prior to the protein folding reaction. To more closely approximate in vivo folding conditions, protein folding investigations of disulfide-containing proteins should be conducted under conditions where the disulfides are reduced if possible. For hGH, the disulfides are not necessary for folding nor are they required for activity. The present study, therefore, has important implications for the comparison of in vitro and in vivo protein folding reactions, especially since few examples of kinetic folding investigations exist for naturally occurring disulfide-containing proteins in both intact and reduced forms. The results obtained for hGH show that the folding kinetics of the reduced and intact forms are remarkably similar in the portion of the folding reaction that can be observed by the stopped-flow technique. These results are interesting in light of the equilibrium data in which a significant destabilization of the reduced and alkylated form of hGH relative to the disulfide-intact protein was observed(4) . Finally, the results are important in terms of in vitroversusin vivo protein folding mechanisms, because they lend support to the application of folding models based on in vitro experiments to the more physiologically relevant issue of in vivo protein folding.

The increased propensity of 2-RCAM hGH to self-associate in vitro may also have relevance to the in vivo folding pathway. A similar effect of disulfide-reduced folding in vivo would clearly point out the necessity for molecular chaperones to assist in the folding of disulfide-reduced proteins in which misfolded intermediates might lead to aggregation and precipitation within the cell. Indeed, the formation of inclusion bodies in the heterologous expression of hGH in Escherichia coli could be inhibited by the co-expression of DnaK chaperonin (23) .

Relevance to the Protein Folding Problem

The framework model and the molten globule model are two theories on the mechanism of protein folding that have gained popularity in recent years(24, 25, 26, 27, 28, 29, 30) . In principle, both models are similar, since both propose early formation of secondary structure. The molten globule model seems to be a refinement of the framework model. The folding of bGH has been shown to be consistent with the framework model(10) , because formation of the secondary structure has been shown to clearly precede the tertiary structure. On the other hand, the majority of hGH secondary and tertiary structure forms within the stopped-flow dead time, so it is difficult to determine the actual sequence of early folding events. Due to this fact, it is unclear whether hGH follows the framework theory of protein folding. However, in a recent analysis of the ellipticities of early transient intermediates in refolding of 14 different proteins (31) it was found that a substantial amount of the secondary structure is generally formed in the earliest detectable intermediate. Our kinetic folding results on hGH are consistent with this observation.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: GDS Technology Inc., 25235 Leer Dr., Elkhart, IN 46514.

Present address: Pharmaceutics and Drug Delivery, Amgen Inc., Thousand Oaks, CA 91320.

**
To whom correspondence should be addressed. Tel.: 317-276-6027; Fax: 317-277-0833; DeFelippis\_Michael\_R{at}Lilly.com.

(^1)
The abbreviations used are: hGH, human growth hormone recombinant derived; bGH, bovine growth hormone; GdnHCl, guanidine hydrochloride; 2-RCAM hGH, tetra-S-carbamidomethylated human growth hormone; , time constant.


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