Protein Science (1998), 71352-1358. Cambridge University Press. Printed in the USA.
`Copyright 0 1998 The Protein Society
`
`Thermodynamic characterization of an intermediate
`state of human growth hormone
`
`ISABEL GOMEZ-ORELLANA, BRUCE VARIANO, JUDY MIUFL4-FRABONI,
`SAM MILSTEIN, AND DUNCAN R. PATON
`Emisphere Technologies, Inc., 15 Skyline Drive, Hawthorne, New York 10532
`(RECEIVED September 4, 1997; ACCEPTED March 13, 1998)
`
`Abstract
`The thermal denaturation of recombinant human growth hormone (rhGH) was studied by differential scanning calo-
`rimetry and circular dichroism spectroscopy (CD). The thermal unfolding is reversible only below pH 3.5, and under
`these conditions a single two-state transition was observed between 0 and 100°C. The magnitudes of the A H and ACp
`of this transition indicate that it corresponds to a partial unfolding of rhGH. This is also supported by CD data, which
`show that significant secondary structure remains after the unfolding. Above pH 3.5
`the thermal denaturation is
`irreversible due to the aggregation of rhGH upon unfolding. This aggregation is prevented in aqueous solutions of
`alcohols such as n-propanol, 2-propanol, or 1,2-propanediol (propylene glycol), which suggests that the self-association
`of rhGH is caused by hydrophobic interactions. In addition, it was found that the native state of rhGH is stable in
`relatively high concentrations of propylene glycol (up to 45% v/v at pH 7-8 or 30% at pH 3) and that under these
`conditions the thermal unfolding is cooperative and corresponds to a transition from the native state to a partially folded
`state, as observed at acidic pH in the absence of alcohols. In higher concentrations of propylene glycol, the tertiary
`structure of rhGH is disrupted and the cooperativity of the unfolding decreases. Moreover, the CD and DSC data indicate
`that a partially folded intermediate with essentially native secondary structure and disordered tertiary structure becomes
`significantly populated in 70-80% propylene glycol.
`Keywords: circular dichroism; differential scanning calorimetry; folding intermediates; human growth hormone;
`propylene glycol; protein folding
`
`a single-chain
`Recombinant human growth hormone (rhGH) is
`protein of 191 amino acids. Its tertiary structure is characterized by
`a four-helix bundle with two long loops (de Vos et al., 1992; Ultsch
`et al., 1994). The native state is stable and does not undergo sig-
`nificant conformational changes between pH 2-1 1 (Tumer et al.,
`1983; Kauffman et al., 1989; Abildgaard et al., 1992). Slight differ-
`ences observed in the near-UV CD spectra below pH 4 have been
`shown to stem from local adjustments in the tertiary structure after
`the protonation of carboxyl groups, and not from major tertiary
`structure alterations (Kauffman et al., 1989; DeFelippis et al., 1995).
`The guanidine hydrochloride (GuHCI) denaturation of rhGH has
`been extensively studied (Brems et al., 1990; DeFelippis et al.,
`al., 1996). rhGH unfolds at about 4.5 M
`1993, 1995; Bam et
`
`Reprint requests to: Isabel Gomez-Orellana, Emisphere Technologies,
`Inc., 15 Skyline Drive, Hawthorne, New York 10532; e-mail: igomez@
`emisphere.com.
`Abbreviations: CD, circular dichroism; AC,, heat capacity change; AG,
`Gihbs free energy change; AH, enthalpy change; AH,,,, enthalpy change at
`the unfolding temperature; DSC, differential scanning calorimetry; T,,,,
`temperature of the maximum in the heat capacity function; rhGH, recom-
`binant human growth hormone; AHUh/AH,,,, van't Hoff to calorimetric
`enthalpy ratio.
`
`GuHCl, and it is fully unfolded above 5 M GuHCl. The unfolding
`transition does not conform
`to a two-state process, and it was
`found that the Gibbs free energy of unfolding is protein concen-
`tration dependent due to the self-association of an intermediate that
`becomes populated during the unfolding. A similar behavior has
`been observed also for the GuHCl denaturation of porcine and
`bovine growth hormones (Have1 et al., 1986; Bastiras & Wallace,
`1992). Surfactants and protein fragments capable of forming am-
`phiphilic helices have been shown to reduce the self-association of
`this rhGH intermediate (DeFelippis et al., 1993; Bam et al., 1996);
`however, the thermodynamic characterization of this partially folded
`state has remained elusive.
`We have studied the thermal denaturation of rhGH using differ-
`ential scanning calorimetry (DSC) and circular dichroism spec-
`troscopy (CD). Our results show that the thermal denaturation is
`reversible only below pH 3.5, and that under these conditions the
`temperature-induced unfolding corresponds to a two-state transition
`from the native state to a partially folded state. No further unfold-
`ing of this intermediate was observed within the temperature range
`studied, 0-100 "C. Nevertheless, because of the absence of aggre-
`gation under the conditions of our experiments, we have been able
`to characterize the energetics of this partially folded state of rhGH.
`
`1352
`
`MYLAN INST. EXHIBIT 1094 PAGE 1
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`MYLAN INST. EXHIBIT 1094 PAGE 1
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`

`

`Intermediate states of human growth hormone
`
`1353
`
`(25°C) varies from 3.01 kcal/mol at pH 2 to 6.61 kcal/mol at
`pH 3.5.
`AG(T) = AH;(l - T/T,,,) + AC;(T - T, - T.lnT/Tm).
`
`(1)
`
`The thermal unfolding was also studied by CD. The unfolding
`profiles obtained by monitoring the changes in ellipticity in the far-
`and near-UV regions are shown in Figure 3. Both the far- and
`near-UV transitions were coincident at each pH studied between 2
`and 3, and the T,,, and AHm calculated from these CD measure-
`ments agreed with the values determined by DSC. Because the far-
`
`-6000 r
`
`I"
`-14000 I
`
`-1 0000
`
`$"
`i
`
`-22000 1
`20
`
`40 I
`
`E -20 t
`-30 '
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70
`
`80
`
`90
`
`100
`
`Temperature ("C)
`
`30
`
`40
`
`50
`
`60 70
`
`80
`
`90
`
`1
`
`0
`
`Temperature ("C)
`
`5000 L
`
`L
`0
`
`t
`2 0
`
`
`
`60
`80
`40
`Temperature ("C)
`
`100
`
`Fig. 1. Partial molar heat capacity of rhGH at different pH values.
`
`Results and discussion
`
`Thermal unfolding at different pH values
`Figure 1 shows the partial molar heat capacity of rhGH as a func-
`tion of temperature at different pH values. The reversibility of the
`unfolding was checked by performing repeated DSC scans of each
`sample, and it was found that the thermal denaturation of rhGH is
`reversible only below pH 3.5. Between pH 2-3.5, the thermal
`transition is independent of the rhGH concentration in the range
`0.2-2 mg/mL, which indicates the absence of aggregation upon
`unfolding. In addition, under these conditions the thermal unfold-
`
`ing is essentially a two-state process (AH,,/AHCaI - 1). Between
`
`pH 1-1.5, rhGH unfolds at about 42"C, but the data from these
`DSC scans were not analyzed because significant protein hydro-
`lysis was observed on samples heated to as low as 40°C.
`As shown in Figure 1, the thermal transition is highly sensitive
`to pH changes, which suggests that the unfolding is coupled to the
`protonation of carboxyl groups. Furthermore, the fact that the Tm
`and AH,,, vary with pH allowed the calculation of the heat capacity
`of unfolding from the plot of AH,,, versus T, (Fig. 2). The AC,, of
`1.13 kcal/K.mol determined from the linear fit of this plot com-
`pares well with the value of 0.92 kcal/K-mol obtained by sub-
`tracting the partial molar heat capacity of the native state from that
`of the unfolded state. The Gibbs free energy of unfolding was then
`calculated using Equation 1, with AC,, = 1.13 kcal/K.mol. AG
`
`40000
`55
`
`60
`
`65
`
`75
`8 5
`
`
`
`8 0
`
`70
`Tm ("C)
`
`Fig. 2. Unfolding enthalpies versus the unfolding temperatures determined
`at different pHs.
`
`Fig. 3. Thermal unfolding of rhGH at pH
`3 monitored by CD at (A)
`222 nm and (B) 295 nm. The measurements were performed on aliquots of
`the same sample because the unfolding is sensitive to slight pH changes
`(see Fig. 1). (C) Fraction of unfolded protein as a function of temperature,
`calculated from the unfolding measurements at 222 and 295 nm.
`
`80000 1
`90000 IT
`ACp = 1131 ?r 72 caVK.mol
`
`u
`2
`U
`
`1.5
`
`i
`
`0
`
`k
`
`r
`
`-0.5
`20
`
`30
`
`40
`
`60 70
`
`50
`Temperature ("C)
`
`l
`
`1
`
`
`
`0 295 nm
`
`80
`
`90 100
`
`MYLAN INST. EXHIBIT 1094 PAGE 2
`
`MYLAN INST. EXHIBIT 1094 PAGE 2
`
`

`

`1354
`
`Table 1. Experimental and calculated enthalpy
`and heat capacity of rhGH unfoldinga
`
`Experimental
`Calculatedb
`
`
`
`Calculated' 2.2
`
`
`
`A H (60 "C)
`(kcal/mol)
`
`52.5
`118.8
`
`149.5
`
`*CP
`(kcal/K.mol)
`
`1.1
`2.7 at 25 "C
`2.0 at 70°C
`
`aThe calculations were done using the coordinates from the Brookhaven
`Protein Data Bank file lhuw. Similar values were obtained using the co-
`ordinates from files lhgu or 3hhr.
`bValues calculated using the parameterization developed by Murphy and
`Freire (1992). Xie and Freire (1994), and Gomez et al. (1995).
`'Calculated using the model proposed by Spolar et al. (1992).
`
`12000
`
`10000
`
`8000
`
`6000
`
`4000
`
`2000
`
`0
`
`h 0"
`9
`
`I. Gomez-Orellana et al.
`
`50
`
`60
`
`ao
`70
`Temperature ("C)
`
`90
`
`loo
`
`and near-UV ellipticity report changes in the secondary and ter-
`tiary structure, respectively, the coincidence of the unfolding pro-
`files confirms that the thermal unfolding of rhGH is essentially a
`two-state process, as suggested by the AHuh/AHCur - 1 obtained
`from the DSC measurements.
`The different parameterizations proposed to estimate the AH
`and AC, of unfolding from the changes in solvent-accessible sur-
`face areas (Murphy & Freire, 1992; Spolar et al., 1992; Xie &
`Freire, 1994; Gomez et al., 1995) were used to calculate the thermo-
`dynamic parameters corresponding to the complete unfolding of
`rhGH. The calculated values are compared to our experimental
`data in Table 1. It can be seen that the calculated AH and ACp of
`unfolding are significantly higher than the experimental values,
`which suggests that the thermal transition observed by DSC and
`CD corresponds to a partial unfolding of rhGH. This interpretation
`is also supported by the CD spectra of rhGH at temperatures above
`the T,, which indicate that significant secondary structure remains
`after the unfolding (Fig. 4). Thus, both the CD and the thermo-
`dynamic data lead to the conclusion that the thermal unfolding of
`
`-30 1
`
`I
`
`200
`
`210
`
`220
`230
`Wavelength (nm)
`
`240
`
`250
`
`Fig. 5. Excess heat capacity of rhGH at pH 7.5
`
`rhGH corresponds to a transition from the native state (N) to a
`partially folded state (I),
`
`As shown in Figures 1 and 3, the unfolding temperature is
`relatively high, and no additional unfolding transitions were ob-
`served at temperatures up to 100°C. It is possible that further
`unfolding of this partially folded rhGH occurs in a noncooperative
`way as has been observed for other protein intermediates (Jeng &
`Englander, 1991; Griko et al., 1994). Some of these intermediates
`have shown increased unfolding cooperativity in high ionic strength
`solutions (Potekhin & Pfeil, 1989; Kuroda et al., 1992; Xie et al.,
`1995), but, unfortunately, the effect of the ionic strength on the
`unfolding of rhGH could not be investigated because the protein
`precipitates at acidic pH in NaCl solutions above 80 mM.
`Above pH 3.5, the thermal unfolding of rhGH is irreversible, as
`indicated by the lack of a transition in a repeated DSC scan of the
`same sample. The AHuh/AHcul is about 4, and the T,,, decreases
`with increasing concentrations of rhGH. These results suggest that
`the irreversibility is caused by aggregation upon unfolding. Al-
`though no precipitate or turbidity was observed in the samples
`after the temperature scans, the presence of aggregates in solution
`was confirmed by light scattering (data not shown).
`Between pH 7-8 the heat capacity function of rhGH shows two
`transitions: the first at about 80 "C, and the second at about 95 "C.
`No transition was observed in a repeated scan of the same sample.
`As the rhGH concentration was increased (0.2-1.5 mg/mL), the T,
`of the first transition decreased and the T,,, of the second transition
`increased (Fig. 5). This behavior suggests that the first transition
`involves the self-association of rhGH upon unfolding and that the
`second transition is coupled to oligomer dissociation. At protein
`concentrations above 1.5 mg/mL, the second transition was no
`longer observed, suggesting that the aggregation becomes more
`extensive and irreversible with increasing protein concentrations.
`
`Fig. 4. Far-UV CD spectra of rhGH at pH 3 below (15°C) and above
`(95 "C) the T , . For comparison, the CD spectra of rhGH unfolded in 6 M
`GuHCl are also plotted: pH 7.5 and 6 M GuHCl at 15 "C (dashed line);
`pH 3 and 6 M GuHCl at 90°C (open circles); pH 7.5 and 6 M GuHCl at
`90 "C (solid circles).
`
`Effect of alcohols on the thermal unfolding
`In aqueous solutions of either 10% v/v n-propanol, 10% 2-propanol,
`or 20% 1.2-propanediol (propylene glycol), the thermal denatur-
`ation of rhGH at pH 7-8 is characterized by a single, reversible
`
`MYLAN INST. EXHIBIT 1094 PAGE 3
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`MYLAN INST. EXHIBIT 1094 PAGE 3
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`

`

`Intermediate states of human growth hormone
`
`1355
`
`transition with a AHvh/AHcal - 1. Under these conditions, the
`thermal unfolding is independent of the protein concentration, in-
`dicating the absence of aggregation. At temperatures below the T',
`the far- and near-UV CD spectra of rhGH in these alcohol solu-
`tions are superimposable on the spectra obtained in the absence of
`alcohols, which suggests that the native conformation is not af-
`fected by these solvent conditions. Above the T , , the CD spectra
`of rhGH in these water/alcohol mixtures are similar to the CD
`spectrum of the intermediate obtained upon unfolding at acidic pH
`(Fig. 4). Therefore, the thermal unfolding in these mixtures may
`correspond to a partial unfolding as observed at acidic pH.
`The absence of aggregation in these alcohol solutions suggests
`that the self-association is caused by hydrophobic interactions be-
`tween apolar surfaces of rhGH that become exposed during the
`unfolding. These interactions are weaker in low polarity solvents
`such as water/alcohol mixtures, and, therefore, the aggregation is
`inhibited. Similarly, Bam et al. (1996) have shown that the self-
`association of rhGH in GuHCl decreases in
`the presence of sur-
`factants, an indication that this aggregation also stems from nonpolar
`interactions.
`Between pH 3.5 and 7, however, the aggregation upon unfolding
`could not be prevented completely, even in the presence of rela-
`tively high alcohol concentrations (40-50%). It is possible that the
`aggregation becomes more extensive as the pH approaches the
`isoelectric point of rhGH (pH - 5.3) (Pearlman & Bewley, 1993).
`Between pH 7-8, the CD spectra indicate that rhGH remains
`essentially native in propylene glycol concentrations up to 45%,
`and, surprisingly, rhGH unfolds in a cooperative, two-state fashion
`
`(AHUh/AHcar - 1) in 20-45% propylene glycol within this pH
`
`range. To be able to compare the thermal unfolding in the presence
`of propylene glycol with the unfolding in pure aqueous solution,
`the effect of propylene glycol on rhGH was also studied at pH 3,
`where the unfolding is reversible and uncoupled to aggregation in
`the absence of alcohols. At this pH, the CD spectra indicate that the
`native state is stable in propylene glycol concentrations up to 30%,
`and it was found that the thermal unfolding is reversible and co-
`
`operative (AHUh/AHcal - 1) in 0-30% propylene glycol. The mag-
`
`nitudes of the unfolding enthalpies and the CD spectra of rhGH
`above the T,,, indicate that under these conditions the thermal tran-
`sition corresponds to partial unfolding of rhGH. Thus, at pH 3 the
`
`unfolding process is essentially the same in the presence and in the
`absence of propylene glycol, and the cooperativity of the unfolding
`is not affected by propylene glycol concentrations below 30%.
`Figure 6 shows the variation of the T, and AH,,, with the pro-
`pylene glycol concentration at pH 3 and pH 7.5. AH,,, initially
`increases and then decreases with increasing propylene glycol con-
`centrations. A similar behavior has been reported for the AH,,, of
`hen egg white lysozyme and cytochrome c in different water/
`alcohol mixtures (Velicelebi & Sturtevant, 1979; Fu & Freire,
`1992). The 7'' varies linearly with the propylene glycol concen-
`tration. At pH 3, the y-intercept from the linear fit of the T,,, values
`is in agreement with the experimental T,,, in the absence of pro-
`pylene glycol. At pH 7.5, the y-intercept from the linear fit of the
`T,,, data is 91.3 "C, which presumably corresponds to the unfolding
`temperature without propylene glycol in the absence of aggrega-
`tion. The unfolding enthalpy corresponding to this T,,, was calcu-
`lated using the ACp and A H determined for the partial unfolding at
`acidic pH (Fig. 2). The value obtained, 86.8 kcal/mol, compares
`well with the AH, values determined from the DSC scans at
`different propylene glycol concentrations (Fig. 6B), which sug-
`gests that in the absence of aggregation the thermal unfolding at
`pH 7.5 would correspond to a partial unfolding as at acidic pH.
`In propylene glycol concentrations above 45% at pH 7-8 or
`30% at pH 3, the near-UV CD spectra of rhGH show differences
`from the spectra in the absence of propylene glycol. These differ-
`ences are more pronounced in 70-80% propylene glycol and are
`mainly observed in the region of the spectrum that is assigned to
`tryptophan residues (288-310 nm). The ellipticity in the 255-
`285 nm region, which is attributed to the optical activity of tyro-
`sines and phenylalanines
`(Bewley, 1979) appears less altered
`(Fig. 7A). These changes in the near-W ellipticity reflect alter-
`ations in the local environment and rotational freedom of the ar-
`omatic side chains. rhGH has a single tryptophan (Trp 86), which
`is mostly buried in the hydrophobic core of the protein (de Vos
`et al., 1992; Ultsch et al., 1994). The absence of ellipticity in the
`tryptophan region at 80% propylene glycol indicates that this res-
`idue has gained rotational freedom and that its local environment
`has changed, and, therefore, that the tertiary structure of rhGH is
`somehow disrupted. Most phenylalanines and tyrosines, on the
`other hand, are located on the surface of rhGH; thus, it is not
`
`90
`
`50 t
`
`4 0
`0
`
`I
`10
`
`I
`5 0
`
`/I+"
`f 60
`\
`t
`4 0 I
`0
`
`I
`I
`20
`30
`4 0
`[propylene glycol] (% v h )
`Fig. 6. A: Variation of the unfolding temperature with the propylene glycol concentration. The solid circle corresponds to the
`experimental T, at pH 3 in the absence of propylene glycol. This value was not included in the linear fitting. B: Variation of the
`unfolding enthalpy with the propylene glycol concentration. The solid diamond represents the calculated AH,,, at pH 7.5 in the absence
`of propylene glycol (see text).
`
`pH3
`
`1
`40
`
`A
`50
`
`10
`
`20
`
`I
`30
`
`[propylene glycol] (% v/v)
`
`MYLAN INST. EXHIBIT 1094 PAGE 4
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`MYLAN INST. EXHIBIT 1094 PAGE 4
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`

`

`1356
`
`I. Gomez-Orellana et al.
`
`10000
`
`0
`
`L-
`
`-10000
`
`I
`
`-
`-
`E
`n
`U -
`"E
`0
`0)
`v -20000
`-30000 i
`
`260
`
`280 300 320 340
`Wavelength (nm)
`
`360 380
`
`200
`
`,
`21 0
`
`,
`220 230 240
`Wavelength (nm)
`
`1
`
`B
`-
`250
`
`I
`
`Fig. 7. (A) Near- and (B) far-UV CD spectra of rhCH in 0, 50, and 80% v/v propylene glycol at pH 7.5 and 15 "C.
`
`surprising that tertiary structure alterations have a lower impact on
`the average ellipticity of these residues. No significant changes in
`the secondary structure were detected by CD, even in 80% pro-
`pylene glycol (Fig. 7B).
`The DSC scans performed at the propylene glycol concentra-
`tions in which the tertiary structure of rhGH appears to be altered
`(>45% at pH 7-8 or >30% at pH 3) show that the cooperativity
`of the unfolding decreases (AHvh/AHcal < 1) and the unfolding
`transition becomes broader and progressively less endothermic
`as the propylene glycol concentration is increased, until finally,
`at 80% propylene glycol, no
`transition was detected by DSC
`(Fig. SA). At this propylene glycol concentration, the thermal un-
`folding monitored by CD at 222 nm is characterized by a gradual
`change in the molar ellipticity with temperature (Fig. 8B), indi-
`cating that the absence of a detectable transition in the DSC scan
`is due to the low cooperativity of the unfolding under these con-
`ditions. This low unfolding cooperativity suggests that the tertiary
`structure of rhGH is largely disordered at this propylene glycol
`concentration, in agreement with the near-UV CD data (Fig. 7A).
`These observations, thus, lead to the conclusion that a partially
`
`folded form of rhGH, with native-like secondary structure and
`disordered tertiary structure, is significantly populated in 80% pro-
`pylene glycol.
`
`Intermediate states of rhGH
`Two partially folded states of rhGH have been characterized in this
`paper: (1) an intermediate with disordered tertiary structure but
`essentially native secondary structure that is stabilized in 80%
`propylene glycol at low temperatures (Fig. 7), and (2) an inter-
`mediate with a partially unfolded secondary structure that becomes
`populated at high temperatures and acidic pH (Fig. 4).
`Propylene glycol and other alcohols are known to induce the
`partial unfolding of proteins through the disruption of tertiary struc-
`ture interactions (Liu & Bolen, 1995; Shiraki et al., 1995; Dib
`et al., 1996; Kamatari et al., 1996; Schonbrunner et al., 1996;
`Hirota et al., 1997; Uversky et al., 1997). In general, alcohol-
`stabilized intermediates are characterized by having disordered
`tertiary structure and high alpha-helical content, and usually they
`contain non-native secondary structure elements because alcohols
`
`16000 r
`
`A
`
`12000
`
`t
`
`0
`
`_LC
`
`,
`20 30 40
`
`I
`
`B
`
`-5000
`
`L
`
`- -10000
`6
`'0 - -
`5?
`= -20000
`
`-15000
`
`1
`80
`
`I
`
`90
`
`-25000
`0
`
`20
`
`40
`60
`Temperature ("C)
`
`80
`
`1
`
`1
`
`1
`50 60
`70
`Temperature ("C)
`
`Fig. 8. A: Excess heat capacity of rhGH at pH 7.5 in different propylene glycol concentrations. B: Temperature-induced unfolding of
`rhCH at pH 7.5 in 40 and 80% propylene glycol monitored by CD at 222 nm.
`
`MYLAN INST. EXHIBIT 1094 PAGE 5
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`MYLAN INST. EXHIBIT 1094 PAGE 5
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`

`

`Intermediate states of human growth hormone
`
`induce the population of a-helical structure in regions of the pro-
`tein that are non-helical in the native state. The rhGH intermediate
`populated in 80% propylene glycol has the structural characteris-
`tics of alcohol-stabilized intermediates, but the secondary structure
`of this intermediate is predominantly native because rhGH is mostly
`helical. Similar rhGH intermediates have been previously observed
`in aqueous solutions of 1-propanol and acetonitrile (Wicar et al.,
`1994). Alcohol-stabilized intermediates of various proteins have
`been shown to be involved in early folding events (Buck et al.,
`1995; Hamada et al., 1996)
`and in the translocation of proteins
`across membranes (Bychkova et al., 1996). Thus, rhGH intermedi-
`ates analogous to the intermediate populated in propylene glycol
`could be biologically relevant or play a role in the folding mech-
`anisms of rhGH.
`The partially folded state stabilized at high temperatures and
`acidic pH, on the other hand, is an equilibrium folding intermedi-
`ate characterized by having the secondary structure partially un-
`folded. Equilibrium and kinetic folding intermediates are often
`alike (Jennings &Wright, 1993; Balbach et al., 1995; Roder, 1995;
`Chamberlain et al., 1996); thus, it is possible that this intermediate
`shares structural similarities with the intermediates detected in
`studies of the folding kinetics of rhGH (Youngman et al., 1995)
`and the partially folded forms of rhGH (equilibrium intermediates)
`populated during the unfolding in GuHCl (DeFelippis et al., 1993,
`1995; Bam et al., 1996). The structural and thermodynamic char-
`acterization of folding intermediates is of fundamental importance
`in understanding the mechanisms of protein folding and the inter-
`actions that determine the stability of native proteins. Until now
`the characterization of the folding intermediates of rhGH has been
`impeded by the aggregation of these intermediates under the con-
`ditions in which they become populated. We have identified con-
`ditions in which a partially folded form of rhGH is significantly
`populated without aggregation, and have characterized the ener-
`getics of this intermediate state. These conditions can now be used
`to characterize the structure of this partially folded state. A better
`understanding of the folding mechanisms of rhGH should be pos-
`sible from the thermodynamic information reported in this paper
`combined with future structural characterizations of this intermedi-
`ate state.
`
`Materials and methods
`
`Materials
`1,2-Propanediol (propylene glycol) was obtained from Sigma.
`2-Propanol and n-propanol were from Aldrich (Milwaukee, Wis-
`consin). Human growth hormone was obtained by recombinant
`DNA techniques. The primary structure of rhGH corresponds to
`that reported by Goeddel et al. (1979), and has Phe at the N-terminal.
`The purity of rhGH was confirmed by size exclusion and reversed-
`phase chromatography and mass spectrometry. The protein solu-
`tions were prepared by dialysis overnight, at 4"C, against 4 L of
`the appropriate buffer. The rhGH concentration was determined
`spectrophotometrically, using an extinction coefficient of 18,890
`M-' cm" at 278 nm (Brems et al., 1990). The buffers used were
`10 mM glycine/HCI (pH 1 to 3.5), 10 mM sodium acetate (pH 4
`to 5), and 10 mM sodium phosphate (pH 6 to 8).
`
`Dlflerential scanning calorimetry (DSC)
`A Nan0 Differential Scanning Calorimeter (Model 5100) (Calo-
`rimetry Sciences Corporation, Provo, Utah) was used for the ca-
`
`1357
`
`lorimetric experiments. The temperature scans were performed at
`1 "C/min on samples containing 0.2-2 mg/mL of rhGH. Decon-
`volution of the heat capacity function was performed with nonlin-
`ear least-squares fitting software developed by Emesto Freire (The
`Johns Hopkins University) (Freire & Biltonen, 1978; Freire, 1994;
`Xie et al., 1995).
`
`Circular dichroism (CD)
`CD spectra were recorded on a Jasco J-715 spectropolarimeter.
`The instrument was calibrated with an aqueous solution of 0.06%
`w/v ammonium d-camphor-10 sulfonate. Each spectrum was ob-
`tained as the average of four consecutive wavelength scans. Round
`thermostatic cells of 1 cm and 0.02 cm path length were used for
`the near- and f a r - W regions, respectively. The temperature was
`regulated with a Neslab RTE-111 temperature controller. Wave-
`length scans were performed on samples containing 0.5-1 mg/mL
`of rhGH.
`The unfolding measurements were performed on samples con-
`taining 1 mg/mL rhGH. The samples were heated at a constant rate
`(1 "C/min). The unfolding curves were analyzed assuming a two-
`state behavior, and the fraction of unfolded protein (P,) was cal-
`culated according to Equation 2,
`
`where Y represents the molar ellipticity at any temperature (T), and
`(Y, + b. T ) and (Y, + a . T ) are linear equations obtained from the
`least-squares fitting of the post-transition and pre-transition data,
`respectively.
`
`Sudace areas
`Solvent accessible surface areas were calculated with the program
`ACCESS, developed by Lee and Richards (1971) and imple-
`mented by T. Richmond and S. Presnell, using the coordinates
`from the files lhuw, lhgu, and 3hhr of the Brookhaven Protein
`Data Bank
`
`Acknowledgments
`
`We thank Emesto Freire for providing us with the program for the decon-
`volution of the excess heat capacity function. We also thank Suraj Kalbag,
`Carlos Salvador, and Elizabeth Harris for helpful discussions, and Amy
`Funkhouser and Dalton Guo for assistance in the experimental work.
`
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