(Received for publication, April 1, 1995; and in revised form, June 15, 1995)
From the
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.
Human growth hormone (hGH) ()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.
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.
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.
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.
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
(). 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.
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
3 mg/ml. The time constants
and
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 and
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:
--,
; -
-,
, and -
-,
. 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.
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 -- and
-
- for
and
,
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,
, comprises less than 5% of the expected fluorescence
amplitude over the entire range of refolding final GdnHCl
concentrations. The amplitude of the
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
amplitude decreases to 10-20% of the expected relative
amplitude. The amplitude of the
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.
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 and
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,
, 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 folding phase is dominant for jumps to low denaturant
concentrations and decreases steadily through the equilibrium
transition region. The normalized amplitudes of the
and
folding phases are small in folding
conditions of low denaturant and increase steadily for jumps into the
transition region.
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.
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.
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) .