Mechanism and Thermodynamics of Guanidinium Chloride-induced Denaturation of ALS-associated Mutant Cu,Zn Superoxide Dismutases

doi:10.1016/j.jmb.2005.10.042

J. Mol. Biol. (2006) 355, 106–123

Mechanism and Thermodynamics of Guanidinium Chloride-induced Denaturation of ALS-associated Mutant Cu,Zn Superoxide Dismutases Jessica A. O. Rumfeldt1, Peter B. Stathopulos1, Avijit Chakrabarrty2 James R. Lepock3 and Elizabeth M. Meiering1* 1

Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry and Department of Chemistry University of Waterloo Waterloo, Ont. Canada N2L 3G1 2

Departments of Medical Biophysics and Biochemistry Ontario Cancer Institute University of Toronto, 610 University Avenue, Toronto Ont. Canada M5G 2M9 3

Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto Toronto, Ont. Canada M5G 2M9

Mutations in human copper zinc superoxide dismutase (hSOD) that are associated with amyotrophic lateral sclerosis (ALS) have been proposed to destabilize the protein and thereby enhance toxic protein aggregation. In previous studies, denaturation of metallated (holo) hSODs was found to be irreversible, and complicated by the formation of intermolecular disulfide bonds. Here, ALS-associated mutations (E100G, G93A, G85R and A4V) are introduced into a pseudo wild-type background containing no free cysteine residues. The guanidinium chloride-induced denaturation of the holo proteins is generally found to be highly reversible (except for A4V, which tended to aggregate), enabling quantitative analysis of the effects of the mutations on protein stability. Denaturation and renaturation curves were monitored by tryptophan fluorescence, circular dichroism, enzyme activity, chemical cross-linking and analytical sedimentation, as a function of equilibration time and protein concentration. There is strong kinetic hysteresis, with curves requiring exceptionally long times (many days for pseudo wild-type) to reach equilibrium, and evidence for the formation of kinetic and equilibrium intermediate(s), which are more highly populated at lower protein concentrations. The effects of metal dissociation were included in the data fitting. The full protein concentration dependence is best described using a three-state model involving metallated native dimer, metallated monomeric intermediate and unfolded monomers with no bound metals; however, at high protein concentrations the unfolding approaches a two-state transition with metal binding to both the native dimers and unfolded monomers. We show that the E100G, G93A and G85R mutations decrease overall protein stability, largely by decreasing monomer stability with little effect on dimer dissociation. Comparison of the chemical denaturation data with ALS disease characteristics suggests that aggregation of some mutant hSOD may occur through increased population of partially folded states that are less stable than the monomeric intermediate and accessed from the destabilized holo protein. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: superoxide dismutase; amyotrophic lateral sclerosis; protein stability and folding; dimeric protein; guanidine hydrochloride denaturation

Abbreviations used: hSOD, human superoxide dismutase; DG, change in Gibbs free energy; m, dependence of DG unfolding on denaturant concentration; GdmCl, guanidinium chloride; ALS, amyotrophic lateral sclerosis; DSC, differential scanning calorimetry; ICP-AES, inductively coupled plasma atomic emission spectroscopy. E-mail address of the corresponding author: [email protected]

Introduction Mutations in human copper zinc superoxide dismutase (hSOD) are associated with the familial form of the neurodegenerative disease amyotrophic lateral sclerosis (fALS).1 hSOD is a dimeric enzyme that catalyzes the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen. Each subunit consists of a flattened

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

107

Chemical Denaturation of ALS-associated hSOD

Figure 1. Ribbon representation of the crystal structure of holo WT hSOD showing ALS-associated mutations. The protein is a dimer, with each subunit consisting of 153 amino acid residues, which form a flattened Greek-key b-barrel made up of eight antiparallel b-strands. Each monomer contains in the active site one copper (dark grey spheres) and one zinc (black spheres). Positions for mutations characterized in this study (A4V, G93A, G85R and E100G) are shown in black. This Figure was prepared using MOLMOL72 and PDB coordinates 1HL5.73

Greek-key b-barrel made up of eight antiparallel b-strands, and binds one copper ion and one zinc ion in the active site region (Figure 1). The copper undergoes successive reduction and oxidation during enzyme catalysis, while the zinc performs a structural role. To date, more than 110, primarily missense, mutations in hSOD have been linked with ALS†.2 fALS represents about 5–10% of all ALS cases, with mutations in hSOD accounting for w20% of fALS cases and being the major known cause of ALS; fALS is clinically indistinguishable from sporadic ALS. Currently, there is very little in the way of treatment for this rapidly progressive and invariably fatal disease. The ALS-associated mutations in hSOD are chemically diverse and are distributed throughout the structure of the protein. Owing to the autosomal dominant pattern of fALS disease inheritance, mutant SODs are thought to have a gain of toxic function. There are two main types of hypotheses, which may not be mutually exclusive, for the toxic function: new oxidative function and increased protein aggregation, perhaps owing to decreased protein stability.3,4 Despite extensive in vivo and in vitro studies on SOD, to date no general correlations between properties of mutant SODs and disease characteristics have been identified, and the molecular mechanisms for the gain of function remain unclear. We have undertaken in-depth studies of the folding and aggregation of hSOD in vitro in order to identify possible mechanisms by which mutations may alter folding and enhance aggregation. Fully metallated (holo) hSOD is remarkably stable against thermal unfolding5 and chemical denaturation by detergents and guanidinium † http://www.alsod.org

chloride (GdmCl).6 Partially metallated and unmetallated (apo) hSOD have considerably lower stability than holo protein7,8 and tend to have an increased propensity to aggregate.8–10 Chemical denaturation of apo hSODs has been reported to be reversible;11 however, previous studies of holo or partially metallated hSOD have found that chemical and thermal denaturation were irreversible5,11 or the extent of reversibility was not reported. Systematic studies of reversibility and a thermodynamic analysis of chemical denaturation for holo hSOD, the major form of protein in cells,12 have not been reported. We find here that chemical denaturation of holo hSOD involves formation of kinetic and equilibrium intermediates, shows pronounced kinetic hysteresis, and is highly reversible. We have employed a pseudo wild-type (pseudoWT) form of hSOD, in which free cysteine residues at positions 6 and 111 have been mutated to alanine and serine, respectively, greatly increasing the reversibility of unfolding and preventing the formation of aberrant intermolecular disulfide bonds.5 The pseudoWT has essentially the same activity, stability and structure as the wild-type protein,5,8,13 making it a useful background in which to study the effects of mutations. Several ALS-associated mutant holo hSODs (Figure 1) are found to unfold with a similar mechanism as pseudoWT, although the rates and equilibria for structural transitions are altered. The reversible unfolding allows the effects of mutations on thermodynamic stability to be analyzed. Interesting relationships are observed between the chemical denaturation data and the properties of the mutants in vivo that have implications for understanding the molecular bases for the disease.

Results GdmCl-induced denaturation of holo hSOD is highly reversible The refolding of GdmCl-denatured holo pseudoWT hSOD after dilution of the denaturant was found to be fully reversible, based on fluorescence, pyrogallol activity and circular dichroism (CD) measurements. Various mutant proteins also refold with high reversibility (see later sections). When pseudoWT is refolded from 6 M to 0.6 M GdmCl, at 25 8C, pH 7.8, the percentage of native activity is approximately 50%, 75% and 100% after 25 min, one day and two days, respectively; nearly 100% of native fluorescence is regained after 25 min. Since correct metallation is required for full activity12 and activity is proportional to the amount of copper bound in the active site,14 it appears therefore that the folded structure as measured by fluorescence is formed within 25 min of refolding but the metals are not yet in their correct positions. Activity is fully regained at all protein concentrations upon renaturation to low GdmCl concentration (below 2 M). The reversibility of unfolding is further

108

Chemical Denaturation of ALS-associated hSOD

demonstrated in the denaturation and renaturation curve experiments described below. Decrease in fluorescence between 0 and 1 M GdmCl is not due to a major structural change For pseudoWT hSOD, there is a marked nonlinear decrease in fluorescence (Figure 2(a)), but no significant change in CD or activity, between 0 M and w1 M GdmCl. A similar decrease in fluorescence but not CD is also observed for mutants (see below).

Normalized Fluorescence

(a) 10 9

GdmCl-induced denaturation and renaturation curves reach equilibrium at long incubation times

8 7 6 5 0

1

2

3 4 [GdmCl] (M)

5

6

(b) 5.0 Midpoint ([GdmCl] (M))

The fluorescence change does not appear to be caused by an ionic strength effect, since a similar change in fluorescence is observed over 0 to 2.6 M GdmCl when ionic strength is kept constant using NaCl, and fluorescence does not change for 0 to 1.8 M NaCl (data not shown). There does not seem to be a major structural transition over this range of GdmCl concentration since no kinetic unfolding transition is detected by stopped-flow fluorescence (data not shown) and 1H–15N heteronuclear single quantum correlation (HSQC) spectra acquired as a function of GdmCl concentration show only very small shifts in peak positions (Supplementary Data). Accordingly, in order to simplify data analysis, points at low GdmCl concentrations were not included during the data fitting to the various denaturation models described below.

4.5

4.0

3.5

0

20

40 60 Time (Days)

80

Figure 2. Denaturation and renaturation data as a function of time for holo pseudoWT hSOD. (a) GdmClinduced denaturation (open symbols) and renaturation (filled symbols) curves of holo pseudoWT hSOD at 25 8C, pH 7.8. Denaturation curves monitored by fluorescence were measured at 4 h (,), nine days ($), and 47 days (B), and renaturation curves monitored by fluorescence were measured at four days (%) and 28 days (C). The decrease in fluorescence between 0 M and 1 M GdmCl does not appear to involve a major structural transition (see the text) and so data over this range were sometimes omitted for clarity in subsequent Figures. Unfolding of pseudoWT is highly reversible at 1 mM protein as curves become coincident at long incubation times. (b) Apparent midpoints of the GdmCl curves where half of the maximal signal change has occurred are plotted for denaturation (open symbols) and renaturation (filled symbols) curves of 10 mM pseudoWT hSOD with time. Midpoints were obtained by fitting curves monitored by fluorescence (7,:) and activity (B,C) (see also Figure 4(c)) to a two-state unimolecular unfolding model.74

GdmCl-induced denaturation and renaturation curves for pseudoWT hSOD were monitored by fluorescence (Figure 3), CD and activity (Figure 4) for a range of protein concentrations. Mutant curves were also monitored by fluorescence (Figure 5) and CD (Supplementary Data). There is pronounced kinetic hysteresis in the structural transition, with both unfolding and refolding being very slow. Of the order of w40–80 days is required for curves of pseudoWT to approach equilibrium, as assessed by coincidence of renaturation and denaturation curves. Curves for pseudoWT are fully coincident for 1 mM protein (Figure 2(a)), though there is a small discrepancy at higher protein concentrations (Figures 2(b) and 3). While there is some loss of signal at high GdmCl concentrations at the higher protein concentrations and very long incubation times, generally the extended incubation time does not substantially change the overall features of the curves. Also, mass spectrometry analysis of 0 and 6 M GdmCl samples incubated for w60 days showed no change in mass of the hSOD, indicating no significant covalent changes at long incubation times, and the protein remains fully active in the region of the native baseline long after (e.g. 25 days, Figures 2(b) and 4(c)) equilibrium is reached. Comparison of the apparent transition midpoints as a function of incubation time suggests that equilibrium is reached sooner for renaturation than for denaturation (Figure 2(b)). The lack of coincidence for renaturation and denaturation curves at higher protein concentrations may be related to slower unfolding at higher protein concentrations (data not shown). For data fitting (see below), renaturation and denaturation curves were analyzed separately for pseudoWT at 5 mM and 10 mM to assess possible differences due to hysteresis. Mutants tend to reach equilibrium more rapidly, and so coincident renaturation and denaturation curves were averaged together for data fitting.

109

Chemical Denaturation of ALS-associated hSOD

(a)

(b)

Normalized Fluorescence

7.5

7.5

7.0

7.0

6.5

6.5

6.0

6.0

5.5

5.5 5.0

5.0 0

1

2

3

4

5

6

Normalized Fluorescence

(c)

1

2

3

4

5

6

5

6

(d) 7.5

7.5

7.0

7.0

6.5

6.5

6.0

6.0

5.5

5.5 5.0

5.0

1

2

3 4 [GdmCl] (M)

5

6

1

2

3 4 [GdmCl] (M)

Figure 3. Denaturation and renaturation data as a function of protein concentration for holo pseudoWT hSOD. Unfolding transitions equilibrated at 25 8C for between 40 and 75 days before fluorescence measurements are shown for (a) 0.2 mM, (b) 1 mM, (c) 5 mM and (d) 10 mM protein concentration. Each plot is an average of at least two curves. Renaturation (C) and denaturation ($) curves are shown for 5 mM and 10 mM protein. At 0.2 mM renaturation curves are omitted due to the large amount of scatter. At 1 mM renaturation and denaturation curves are coincident and are therefore shown averaged together (,). The bold lines represent fits to the three-state monomeric intermediate model with ( ) and without (- - -) metal dissociation (parameters given in Table 2). The thin lines were generated using the two-state dimer model with (—) and without (/) metal dissociation and the parameters given in Table 2. For 0.2 mM, lines for the two-state model were generated using DGtot calculated from the three-state fits.

Intermediate observed during unfolding kinetics The increasing biphasic character of the GdmCl curves with decreasing protein concentration is suggestive of the formation of intermediates. In order to investigate this further, the kinetics of unfolding in GdmCl were monitored by fluorescence for pseudoWT (Figure 6) and mutants (data not shown). The unfolding kinetics are generally double-exponential, with a smaller amplitude fast phase and a larger amplitude slow phase. The amplitudes of the kinetic data match the fluorescence change in equilibrium curves (i.e. there is no missing amplitude). The two kinetic phases may be indicative of two sequential steps, with the first phase corresponding to the transition from native dimer to intermediate, and the second phase being the transition from intermediate to unfolded protein. Consistent with this, the smaller amplitude for the fast phase appears to correspond to the smaller amplitude for the first transition of the denaturation curve, and the larger amplitude of

the slow phase matches the larger amplitude second transition in GdmCl curves (Figure 6(b)). The kinetic data were used to define the fluorescence of intermediates during data fitting (see Materials and Methods); detailed analysis of the kinetic data will be reported elsewhere. Evidence for monomeric intermediate by sedimentation analysis Analytical ultracentrifugation was conducted in different concentrations of GdmCl to further investigate the oligomeric nature of the intermediate. Sedimentation velocity measurements were obtained using ultraviolet (UV) absorbance optics for pseudoWT hSOD freshly dissolved in 4 M GdmCl at 2 mM or 80 mM protein (Figure 7). Data acquisition was completed within 2 h. Over this time interval the faster, smaller amplitude phase should be largely “complete”, whereas the slower kinetic phase should have proceeded very little. Thus the kinetic unfolding intermediate should be relatively

110

Chemical Denaturation of ALS-associated hSOD

are consistent with a monomeric intermediate being populated at low protein concentrations.

[q ]231 x 10–3 (deg cm2 dmol–1)

(a) 0.0

Evidence for monomeric intermediate from chemical crosslinking –0.5

–1.0 1

2

3

4

5

6

1

2

3

4

5

6

[q ]216 x 10–3 (deg cm2 dmol–1)

(b)

(c)

–1.5 –2.0 –2.5 –3.0 –3.5

1.2

Fraction unfolded

1.0 0.8 0.6 0.4 0.2 0.0 –0.2 0

2

4 [GdmCl] (M)

6

Figure 4. GdmCl-induced renaturation curves of pseudoWT hSOD monitored by (a) CD at 231 nm, (b) CD at 216 nm, and (c) activity (C) and fluorescence (B) at pH 7.8, 25 8C after w70 days incubation. The lines represent fits to the two-state model with ( ) and without (- - -) metal dissociation. At equilibrium, the activity of the native state is as expected for freshly thawed protein and fraction unfolded plots of activity and fluorescence are superimposable indicating that very little aggregation or inactivation of the protein has occurred during sample incubation.

highly populated under these conditions. Note that at increased protein concentration, the relative amplitude of the fast phase decreases, hence there may be decreased accumulation of intermediate. The sedimentation coefficient distribution at 2 mM SOD is significantly shifted to lower values compared to the distribution at 80 mM. These concentrationdependent changes in sedimentation experiments

The association state of the protein during denaturation was also investigated using covalent crosslinking with glutaraldehyde for samples incubated in GdmCl. Crosslinking data for GdmCl curves at equilibrium, for 1 mM and 10 mM protein, were consistent with data obtained by optical probes, but did not allow the association state of the equilibrium intermediate to be unambiguously identified, most likely due to the relatively low population of the intermediate at equilibrium. Crosslinking experiments were also conducted for GdmCl unfolding curves incubated for 6 h, when there should be a higher population of intermediate. The amount of dimer measured by crosslinking at 6 h is plotted versus GdmCl concentration for 1 mM and 8 mM pseudoWT in Figure 8(a), with corresponding fluorescence data shown for comparison in Figure 8(b); similar results were obtained for E100G (data not shown). As with fluorescence denaturation, the midpoint of the crosslinking transition shifts to higher GdmCl concentration at increased protein concentration. By 4 M GdmCl, crosslinking indicates that hSOD is completely monomeric; however, by fluorescence this GdmCl concentration is only partway through the transition. This is an indication that at low protein concentration a monomeric form of hSOD with a significant amount of tertiary structure is present in the transition region. At 8 mM protein, the intensity of the dimer band by crosslinking does not decrease until the fluorescence transition begins at approximately 3.4 M GdmCl. Both crosslinking and fluorescence transitions are mostly complete by 5 M GdmCl, with the fluorescence transition appearing somewhat steeper. It therefore appears as though at 8 mM protein a dimeric form of hSOD persists throughout the whole transition. Thus, as for optically monitored denaturation and sedimentation data, the cross-linking data are consistent with increased population of monomeric intermediate at low protein concentration. Fitting to two-state and three-state models: population of a monomeric equilibrium intermediate at low protein concentration Given that native hSOD is a homodimer that binds a total of four metals ions, there are many hypothetical mechanisms, involving various intermediates differing in oligomeric nature and metallation, that could, in principle, describe the denaturation mechanism. Information about metal binding may be obtained by studying denaturation as a function of excess free metal; this was not possible for hSOD, however, due to complications of protein precipitation. Consequently, we used an

111

Chemical Denaturation of ALS-associated hSOD

Normalized Fluorescence

(a) 11

(b) 11

10

10

9

9

8

8

7

7

0

1

2

3

4

5

6

0

Normalized Fluorescence

(c) 13

(d) 13

12

12

11

11

10

10

9

9

8

8

0

Normalized Fluorescence

(e)

1

2

3

4

5

1

0

2

1

3

2

4

3

5

6

4

5

(f)

13

10.0

12 9.5

11

9.0

10

8.5

9

8.0

0

1

2 3 4 [GdmCl] (M)

5

6

0

1

2

3

4

5

6

7

[GdmCl] (M)

Figure 5. Protein concentration dependence of curves for (a) and (b) E100G, (c) and (d) G85R and (e) G93A and timedependence for (f) A4V. For (a)–(d), curves at equilibrium are shown for protein concentrations of 0.2 mM ($), 1 mM (:), 5 mM (B) and 10 mM (,). Data below 1 M GdmCl were not included in the fitting (see the text) and are shown grayed out. Curves for G93A at 5 and 10 mM are not shown due to the large amount of scatter. Each curve is an average of at least two experiments. For E100G, lines represent the fit of each individual curve to the three-state monomeric intermediate model (a) without and (b) with metal dissociation. For G85R, the continuous line represents the fit of the curve at 1 mM protein using the three-state monomeric intermediate model (c) without and (d) with metal dissociation. Broken lines are simulated for 0.2, 5 and 10 mM protein using the corresponding model and the DG1 and DG2 determined from the fit at 1 mM protein. (e) For G93A, lines represent fits to the three-state monomeric intermediate model with (—) and without (- - -) metal dissociation. (f) The GdmCl-induced denaturation curve of 1 mM A4V after 40 days at 25 8C (*) is very scattered, most likely due to aggregation of the protein. Consequently, the data cannot be fit to any unfolding model. As a comparison, denaturation curves incubated for 6 h at 25 8C are shown for pseudoWT (C) and A4V (B).

alternative approach whereby information on metal binding is determined by measuring holo protein denaturation as a function of protein concentration, taking into account that the dissociated metals will have effects analogous to additional protein subunits according to the stoichiometry of binding.

The holo hSOD samples contained no significant excess metal beyond that bound to the protein, since samples were dialyzed exhaustively against water with metal levels below the limit of inductively coupled plasma atomic emission spectroscopy (ICP-AES) detection (!1 ppb) and chemical

112

Chemical Denaturation of ALS-associated hSOD

(a)

1 c(s) Distribution

16

Fluorescence (a.u)

15 14 13 12

0.8

2 µM 80 µM

0.6 0.4 0.2 0

11

0

0.5

1

1.5

2

Sedimentation Coefficient (S)

10 0

50

100

150

200

250 residuals [OD]

1.0

Residuals

0.0 –1.0 0.5

6.1

–0.5 0

50

100 150 Time (minutes)

200

2

3

6

250

(b) 6

5

4

3 1

4

5

6.2

6.3

6.4

6.5

6.6

0.02 0 –0.02 6.2

0.0

Normalized Fluorescence

0.02 0 –0.02

6.3

6.4

6.5

6.6

6.7

6.8

6.9

7

Figure 7. Analytical ultracentrifugation of pseudoWT hSOD in 4 M GdmCl, pH 7.8, 25 8C. The upper panel shows sedimentation coefficient distributions from numerical solutions to the Lamm equation. The middle and lower panels show the residuals from the fit for the 2 mM and 80 mM samples, respectively. Sedimentation velocity measurements were obtained using UV absorbance optics for protein freshly dissolved at 2 mM (/) or 80 mM (—) in 4 M GdmCl. Data acquisition was completed in 2 h. Based on fluorescence-monitored kinetics the smaller amplitude faster kinetic phase, that is tentatively assigned to formation of intermediate, should be largely completed over this time interval, whereas very little of the larger amplitude slower kinetic phase will have occurred. The sedimentation coefficient distribution at 2 mM SOD is significantly shifted to lower values compared to the distribution at 80 mM, consistent with formation of monomeric intermediate at 2 mM protein.

7

[GdmCl] (M)

Figure 6. Fluorescence-monitored unfolding kinetics of pseudoWT hSOD in 5 M GdmCl, pH 7.8, 25 8C are double-exponential. (a) Kinetic data are shown fit to a single (- - -) and double (—) exponential equation with a linear downward drift. The drift appears to be caused by photobleaching, since the fluorescence signal recovers when the shutter is closed and then reopened after w30 s. Residuals of the single-exponential with tZ1980 s (upper panel) and double-exponential fit with t1Z208 s and t2Z 2745 s (lower panel) indicate that the kinetics are best described by a double-exponential. (b) Comparison of equilibrium and kinetic fluorescence values for pseudoWT hSOD. Curves are for 1 mM pseudoWT hSOD (B) at equilibrium (averaged data), and ($) after 6 h, pH 7.8, 25 8C. The equilibrium native baseline is shown (—). Filled symbols represent the fluorescence corresponding to (C) the native baseline plus the amplitude for the fast phase, and (&) the native baseline plus the amplitudes of the fast and the slow phases (total amplitude). Since unfolding is extremely slow at 4, 4.5 and 4.75 M GdmCl, the final fluorescence for kinetic fits was fixed (indicated by open squares) to values based on the equilibrium denaturation curve measurements.

denaturation and DSC data obtained for multiple preparations of the proteins gave consistent results. For fitting of data to various models, metal dissociation from the native state was considered to be negligible; this is reasonable, given that native hSOD has extremely high affinity for Cu (Kdw10K18 M) and Zn (Kdw10K14 M)15 and that the protein remains stable and active in high denaturant. In the following, we consider models without and with metal dissociation in order of increasing complexity. Details of the data fitting are given in Materials and Methods, and equations for different models are summarized in Table 1. At high protein concentration, the GdmCl transition for holo hSODs appears monophasic, suggestive of a two-state transition between the native metallated dimeric state and the unfolded state. Two limiting scenarios involve metals being fully bound or fully dissociated from the unfolded state. Metal binding in the unfolded state has been observed for other proteins that bind cofactors

113

Chemical Denaturation of ALS-associated hSOD

dimer intensity

0.4

monomer + dimerintensity

(a)

0.3

0.2

0.1

0.0 9 Normalized Fluorescence

(b)

8

7

6

5 1

2

3

4

5

6

[GdmCl] (M)

Figure 8. Chemical cross-linking of hSOD. (a) Crosslinking and (b) fluorescence of (;) 1 mM and (6) 8 mM pseudoWT hSOD after 6 h incubation in GdmCl. Based on cross-linking at 1 mM protein, a dimeric species of hSOD is not detected above w4 M GdmCl, whereas the fluorescence changes are observed up to w5 M GdmCl, consistent with formation of a monomeric intermediate. In contrast, at 8 mM protein, cross-linking indicates that a dimeric species is present to the end of the fluorescence transition at w5.5 M GdmCl, consistent with decreased population of a monomeric intermediate at increased protein concentration.

with very high affinity in the native state.16 If the protein concentration is above the Kd for metal dissociation from the unfolded state, the equilibrium unfolding can be treated as a two-state transition with no metal dissociation. When the

hSOD curves are fit to a two-state transition between folded dimer and unfolded monomers, with or without metal dissociation, values of DG, the Gibbs free energy of unfolding, and m, the denaturant dependence of the free energy of unfolding, increase with increasing protein concentration (data not shown). When m is fixed at 8 kcal (mol dimer)K1 MK1 for the two-state model without metal dissociation (a value based on the expected increase in solvent-accessible surface area upon unfolding, see Materials and Methods), and 15.8 kcal (mol dimer) K1 M K1 for the two-state model with metal dissociation (a best fit empirical value) there is a deviation from the line of best fit for data points at low GdmCl concentrations, which becomes very pronounced at low protein concentrations (Figure 3). Thus, a dimer two-state model clearly does not account for the full protein concentration dependence of the curves. However, at the highest protein concentration (10 mM), the data points are close to the fitted two-state line (Figure 3(d)), and fitted values are very similar for curves monitored by different structural probes (Table 2). Thus, the curves approach two-state behaviour at high protein concentrations. Sedimentation (Figure 7) and chemical crosslinking (Figure 8) provide evidence for formation of a monomeric intermediate, which may also be apparent in GdmCl curves at lower protein concentrations (Figures 3 and 5). Curves were therefore also fit to three-state models involving native dimer, monomeric intermediate and unfolded monomers, with and without metal dissociation in the intermediate and unfolded states. The model with no metal binding to the intermediate or unfolded states gave poor fits, as values of DG1 decreased and DG2 increased with protein concentration; furthermore, this model predicts no shifts in the second half of the GdmCl transition with protein concentration, and such shifts are clearly observed (Figures 3 and 5). Thus, the intermediate appears to have bound metals. This conclusion is further supported by several lines of evidence: improved data fits for metallated intermediate model (see below), a higher midpoint

Table 1. Equations used for the bimolecular unfolding models with and without metal dissociation No metal dissociation

Metal dissociation in the unfolded state

Two-state dimer

Three-state monomeric int.

Two-state dimer

Three-state monomeric int.

N242UH KZ [UH]2/[N2]

N242ID2UH K1 Z [I]2/[N2]

N242UC2CuC2Zn KZ[U]2[Cu]2[Zn]2/ [N2]Z[U]6/[N2]a

N242ID2UC2CuC2Zn K1Z[IH]2/[N2]

PZ ½N2 C ½UH=2 FnCFuH Z1 FuHZ[UH]/(2P) 0Z 4PðFuH Þ2 C KFuH KK Yo Z Yn C Fu ðYuH KYn Þ

K2Z[UH]/[I] PZ ½N2 C ½I=2C ½UH=2 Fn C Fi C FuH Z 1 FiZ[I]/(2P) 0Z 4PF2i C K1 ð1C K2 ÞFi KK1 Yo Z Yn C Fi ðYi KYn C K2 ðYu KYn ÞÞ

PZ ½N2 C ½U=2 FnCFuZ1 FuZ[U]/(2P) 0Z 64P5 ðFu Þ6 C KFu KK Yo Z Yn C Fu ðYu KYn Þ

K2Z[U][Cu][Zn]/[I]Z[U]3/[I]a PZ ½N2 C ½IH=2C ½U=2 Fn C Fi C Fu Z 1 FuZ[U]/(2P) 0Z 64P5 F6u =ðK1 K2 ÞC 4P2 F3u C K2 Fu KK2 Yo Z Yn C ð4P2 F3u =K2 ÞðYi KYn ÞC Fu ðYu KYn Þ

Abbreviations used are: N2, fully metallated native dimer; I, fully metallated monomeric intermediate; UH, unfolded state with one copper atom and one zinc atom bound; U, unfolded state with no metals bound; P, total protein concentration expressed as dimer equivalent; Yn, Yi, Yj, YuH, Yu, are the measured signals of each species. a In the models with metal dissociation, free [Cu] and [Zn] are assumed to be equal to the concentration of unfolded protein.

114

Chemical Denaturation of ALS-associated hSOD

(by O1 M GdmCl) for the first part of the curve for holo hSOD than is observed for apo hSOD;17 no significant changes in the geometry of metalbinding ligands in an engineered monomeric variant of hSOD18 and binding of cofactors to GdmCl unfolded proteins.16 Fitting of data to the three-state models involving native metallated dimer, metallated monomeric intermediate, and unfolded monomers either with or without bound metals is illustrated in Figure 5, with fitted values summarized in Table 2. Note that the analysis (with different models) indicates the conditions under which metals are bound, but it does not directly define the mode or quantify the Kd for binding. For the model with no metal dissociation in the unfolded state, the DG1 (dimer dissociation) remains relatively constant between

1 mM and 10 mM for both mutant and pseudoWT; however, the DG2 (monomer unfolding) increases systematically with protein concentration whereby the midpoint of the 10 mM curve is at a higher denaturant concentration than predicted by the fit at 1 mM (Figure 5(c)). The model with metal dissociation accounts for the observed protein concentration dependence more fully, since both DG1 and DG2 are relatively constant between 1 mM and 10 mM protein (Table 2, Figure 5(d)). Note that relative errors increase at higher protein concentration where the curves become more monophasic, and neither model can fit the data at 0.2 mM. Comparing the parameters obtained from fitting to each three-state monomeric model, DG1 is approximately the same for both, whereas DG2 is significantly higher using the model involving

Table 2. Thermodynamic parameters for GdmCl curves determined for holo pseudoWT and ALS-associated mutant hSOD Two-state dimer no metal dissociation a

Model

Protein

Probe

WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT WT G85R G85R G85R G85R G85R G85R G85R G85R E100G E100G E100G E100G G93A G93A

Fl Fl Fl CD216 CD231 Fl CD216 CD231 Activity Fl CD216 CD231 Fl CD216 CD231 Activity Fl Fl Fl CD216 CD231 Fl CD216 CD231 Fl Fl Fl Fl Fl Fl

Protein conc. (mM) 0.2e 1f 5g 5g 5g 10g 10g 10g 10g 5e 5e 5e 10e 10e 10e 10e 0.14f 1f 5f 5f 5f 10f 10f 10f 0.2f 1f 5f 10f 0.2f 1f

Three-state monomeric intermediate no metal dissociation b

Two-state dimer with metal dissociation c

Three-state monomeric intermediate with metal dissociation d

DGtot N242UH (kcal (mol dimer)K1)

DG1, N242I (kcal (mol dimer)K1)

DG2, I4UH (kcal (mol monomerK1)

DGtot, N242UC 2CuC2Zn (kcal (mol dimer)K1)

DG1, N242I (kcal (mol dimer)K1)

DG2, I4UC CuCZn (kcal (mol monomerK1)

nd 32.7G0.5 33.0G0.4 34.4G0.4 35.6G0.3 35.0G0.2 34.2G0.3 34.8G0.2 34.7G0.3 35.0G0.4 35.7G0.3 36.2G0.2 36.2G0.2 36.1G0.3 36.4G0.2 36.1G0.3 nd 25.2G0.2 26.5G0.3 26.1G1.0 26.7G0.2 26.7G0.2 26.6G1.3 26.8G0.3 nd 30.0G0.4 31.9G0.2 33.0G0.2 nd 28.5G0.4

11.3G0.3 12.7G0.2 12.0G0.3 12.1G0.7 12.7G0.9 12.6G0.2 12.2G0.7 12.7G0.6 na 12.7G0.4 12.3G0.8 14.9G2.0 nd 13.3G1.0 13.2G0.5 na 10.4G0.5 12.2G0.5 12.1G0.6 12.1G1.0 14.4G4.0 12.7G0.7 12.7G2.6 nd 10.6G0.6 12.3G0.6 12.8G0.5 14.3G1.5 10.0G0.2 12.1G0.4

9.6G0.1 9.9G0.1 10.4G0.1 10.7G0.2 11.1G0.2 11.0G0.1 10.7G0.2 10.8G0.2 na 11.0G0.2 11.2G0.2 10.6G1.0 nd 11.2G0.4 11.5G0.2 na 5.7G0.2 6.3G0.2 7.1G0.3 6.9G0.5 6.1G2.0 7.0G0.3 7.0G1.2 nd 8.1G0.2 8.6G0.2 9.4G0.2 9.3G0.7 7.4G0.1 8.2G0.2

nd 89.9G0.9 87.7G0.6 90.6G0.6 92.9G0.6 90.3G0.4 89.1G0.5 90.0G0.4 89.7G0.6 91.6G0.8 92.8G0.6 93.9G0.4 92.6G0.4 92.5G0.5 93.3G0.3 92.6G0.5 nd 75.1G0.4 74.8G0.7 73.9G0.8 75.3G0.2 73.9G0.4 73.8G0.7 74.1G0.4 nd 84.9G0.8 85.4G0.4 86.4G0.3 nd 82.0G0.7

11.3G0.3 12.7G0.2 12.0G0.2 12.0G0.7 12.8G1.1 12.5G0.5 12.1G0.7 12.7G0.6 na 12.9G0.4 12.4G1.0 nd nd 13.2G1.1 13.4G0.5 na 10.4G0.3 12.5G0.3 12.8G0.1 12.9G1.8 nd 13.2G1.4 nd nd 10.7G0.5 12.3G0.6 12.8G0.3 16.1G7.0 10.0G0.2 12.2G0.3

40.2G0.3 38.9G0.1 38.1G0.2 38.9G0.3 39.8G0.4 38.9G0.3 38.2G0.3 38.5G0.3 na 39.5G0.4 40.0G0.9 nd nd 39.7G0.6 40.0G0.3 na 31.8G0.2 31.3G0.2 31.0G0.6 30.6G1.1 nd 30.3G0.7 nd nd 36.8G0.5 36.2G0.3 36.3G0.2 35.2G3.6 35.1G0.2 35.1G0.3

nd, Data could not be reasonably fit to the specified equilibrium model; na, activity data were not fit to the three-state model as activity of the intermediate is unknown. a m value fixed to 8 kcal (mol dimer)K1 MK1. b m1 value fixed to 1.8 kcal (mol dimer)K1 MK1 and m2 fixed to 3.1 kcal (mol monomer)K1 MK1. c m value fixed to 15.8 kcal (mol dimer)K1 MK1. d m1 value fixed to 1.8 kcal (mol dimer)K1 MK1 and m2 fixed to 7 kcal (mol monomer)K1 MK1. e Two or more denaturation curves were averaged together for fitting. f Multiple renaturation and denaturation curves were averaged together for fitting. g Two or more renaturation curves were averaged together for fitting.

115

Chemical Denaturation of ALS-associated hSOD

metal dissociation (Table 2). This is because the equilibrium constant describing the first transition is the same for both models, whereas for the second transition, K2 has a higher molecularity in the model involving metal dissociation (see Table 1). GdmCl denaturation and renaturation curves for G85R, G93A, E100G and A4V In order to investigate the effects of ALSassociated mutations on hSOD folding and stability, GdmCl denaturation and renaturation curves were also obtained for the mutants G85R, G93A, E100G and A4V (Figure 5). As for pseudoWT, denaturation of G85R, G93A and E100G is highly reversible, based on coincidence of denaturation and renaturation curves and the complete regain of activity upon refolding. However, increased scatter in GdmCl curves suggests that aggregation occurs before equilibrium is reached for A4V (Figure 5(f)) and for G93A at high protein concentration (data not shown). Denaturation and renaturation curves for the mutant proteins tend to reach equilibrium faster than for pseudoWT: within about two days for G85R, about 25 days for G93A and about 40 days for E100G. The protein concentration dependences of the mutant curves monitored by fluorescence and CD and the curve fitting trends are very similar to those for pseudoWT. For all the mutants, the threestate model with metallated monomeric intermediate and unmetallated unfolded monomers best describes the protein concentration dependence and the data at high protein concentration can be fit reasonably well by the two-state model with no metal dissociation (Figure 5, Table 2). This further confirms that the curves are not significantly distorted at long incubation times, and suggests that all the proteins unfold by a similar mechanism. All of the mutants are destabilized relative to pseudoWT, since GdmCl-induced transitions are shifted to lower denaturant concentrations. The changes in thermodynamic stability of the mutant

proteins relative to pseudoWT (DDGZDGmutK DGwt) were estimated for both limiting cases using the two-state fitted values at 10 mM protein (DGs from renaturation experiments were used for pseudoWT) and the three-state values, DG1 and DG2 averaged for 1 mM and 5 mM protein where these values are best defined by the data (Table 3). The DDGs for the models involving metal dissociation are larger than the corresponding values with no metal dissociation; this is related to the dependence of the metal dissociation model DG values on the free energy of the metals. For G93A and A4V, DDGtotal cannot be estimated from the two-state fitting owing to aggregation at high protein concentration but could be estimated from the three-state fits using DDGtotal Z DDG1 C 2 !DDG2 .

Discussion Fully metallated hSOD undergoes reversible unfolding with strong kinetic hysteresis The pseudoWT hSOD as well as the ALSassociated mutants A4V, E100G, G93A and G85R used in this study were fully metallated. Previous preparations of pseudoWT, E100G and G93A using this expression system were also found to be fully metallated and active.5,19 Evidence for full metallation of the proteins studied here is: Cu and Zn are in the correct 1:1 ratio based on ICP-AES, full specific activity of the proteins, and a single endotherm by DSC. These results are in contrast to other studies that have reported heterogeneous metallation of mutant hSOD prepared using various bacterial, yeast and insect cell expression systems and purification protocols.7,17,20–22 The major form of hSOD in cells is the metallated protein,12 and so it is pertinent to characterize the folding behaviour of holo hSODs in order to gain insights into possible aggregation mechanisms of the protein. Our studies represent the first clear demonstration

Table 3. Difference in Gibbs free energy of unfolding between hSOD pseudoWT and ALS mutants

Model

Two-state dimer no metal dissociation

Mutant

DDGtotal (kcal (mol dimer)K1)

G85R E100G G93A

K8.0G0.5a K7.5G1.8b K1.7G0.3a K2.5G0.7b K4.0G0.6b

Three-state monomeric intermediate no metal dissociation DDG1 (kcal (mol dimerlK1)

DDG2 (kcal (mol monomer)K1)

0.1G1.5b

K3.8G0.5b

0.1G0.6b K0.6G0.3b

K1.3G0.2b K1.7G0.1b

Two-state dimer with metal dissociation DDGtotal (kcal (mol dimer)K1) K16.2G0.6a K15.6G1.2b K4.0G0.5a K5.3G0.8b K8.1G0.5b

Three-state monomeric intermediate with metal dissociation DDG1 (kcal (mol dimerlK1)

DDG2 (kcal (mol monomer)K1)

0.2G0.7b

K7.9G0.5b

0.1G0.6b K0.5G0.3b

K2.7G0.3b K3.8G0.2b

DDGZDGmutKDGwt. a DDG values were determined for 10 mM protein where the transition appears close to two-state. Data were averaged for all three probes when available. b DDG values were averaged using data from all available probes for 1 mM and 5 mM protein. Fitted values were less well defined by the data for 10 mM and 0.2 mM protein and so these were not included in the average. For the three-state monomeric intermediate model DDGtotal Z DDG1 C 2 !DDG2 .

116 and systematic analysis of highly reversible GdmCl-induced denaturation of fully metallated SODs, based on fluorescence, CD and activity measurements. There is, however, very strong kinetic hysteresis, so that pseudoWT SOD in the transition region must unfold or refold for longer than a month in order for denaturation and renaturation curves to reach equilibrium. During denaturation and renaturation there is evidence for formation of at least one intermediate, based on biphasic equilibrium GdmCl curves (Figures 2, 3 and 5) and kinetics (Figure 6). It should be noted that the biphasic curves obtained here at short incubation times are comparable to holo hSOD curves reported previously, which were generally incubated for between 20 min and 20 h.11,17,23,24 Renaturation curves and reversibility of unfolding were not characterized in detail in the previous studies; our results suggest that these curves were not at equilibrium. Also, generally, the metallation state of the protein was variable or quantitative values were not reported. The reasons for the strong kinetic hysteresis reported here are likely related to the unusually high stability of the protein, in particular, the strong stabilization imparted by metal binding.8,25 In vitro unfolding rates for the holo protein are much slower than for the apo protein;8,26,27 hence, metal binding slows unfolding. However, in vitro folding of apo hSOD is relatively rapid,26 suggesting that metal binding may also slow folding, perhaps by creating kinetic traps due to non-native-like metal binding. It is not clear whether slow refolding involving metallation is relevant in vivo, where other proteins, such as the copper chaperone for SOD,28 may facilitate metal incorporation. Nature of intermediates and fitting of GdmCl curves There is substantial evidence that hSOD forms a monomeric intermediate, which is increasingly populated as protein concentration is decreased. Formation of a monomeric intermediate is indicated by the protein concentration dependence of sedimentation (Figure 7), chemical cross-linking (Figure 8), and GdmCl curves (Figures 3 and 5), since as protein concentration increases there appears to be less monomer present at low GdmCl concentrations (wfirst half of curve), as expected for a transition from native dimer to a monomeric intermediate. Previous measurements of sedimentation velocity as well as fluorescence anisotropy and decay of holo hSOD (exact metal content was not reported) after relatively short (hours) incubation times in GdmCl were also interpreted in terms of formation of a monomeric intermediate.23 It is not possible to determine from available data whether the kinetic and equilibrium intermediate(s) are the same species; however, the equivalence of kinetic and equilibrium fluorescence

Chemical Denaturation of ALS-associated hSOD

changes (Figure 6(b)), and the evidence for monomeric intermediate in both kinetic unfolding (Figure 6(a)) ultracentrifugation (Figure 7) and crosslinking experiments (Figure 8), and at equilibrium (Figures 3 and 5), suggests that they are similar. A monomeric intermediate may be expected to be reasonably stable, given the following considerations. Firstly, it has been possible to successfully engineer a monomeric form of hSOD by substituting charged residues into the dimer interface.29 Secondly, the Cu,Zn SOD from Escherichia coli is naturally monomeric,30 and completely unrelated dimer interfaces are found in other Cu, Zn SODs.31,32 Thirdly, an analysis based on per residue surface area/interface area for distinguishing whether protein components are ordered (stable) or disordered (unstable) when separated from their complexes33 predicts that hSOD will form a stable monomer. The model that best accounts for the observed protein concentration dependence of the GdmCl curves is a three-state transition involving metallated dimer, metallated monomer intermediate and unfolded monomers with no bound metals. Several lines of evidence, including denaturation midpoints, data fitting to various models and the very strong binding of metals by native dimer and engineered monomer, support that the intermediate is metallated (see Results). The three-state metallated monomeric intermediate model with metal dissociation from the unfolded state describes the protein concentration dependence of hSOD quite well (the DGs are constant between 1 mM and 10 mM, Table 2) and the fits more closely resemble experimental curves, particularly at the end of the transition (w3.5 M GdmCl) than the three-state fits with no metal dissociation from the unfolded state, particularly at lower protein concentrations (Figure 5(a) and (b)). Further support for the applicability of the three-state fitting models is that the change in free energy upon dissociation of native dimer to monomeric intermediate obtained from fitting to a three-state model, DG1 value of w13 kcal (mol dimer)K1 (Table 2), agrees well with measurements of dissociation by size-exclusion chromatography (0.16 nM for wild-type hSOD at pH 7.8, corresponding to 13.4 kcal (mol dimer)K1)34 and sedimentation equilibrium experiments (w10K8 M or lower).35 Although the data for 1–10 mM protein are fit reasonably well by the three-state model, there is a clear discrepancy at 0.2 mM protein, where the fluorescence of the intermediate appears to be increased (Figures 3 and 5) and values for a threestate fit diverge from values obtained at higher protein concentrations (Table 2). The change in intermediate fluorescence and apparent stability may be due to increased population of partially metallated intermediate species, which may be increasingly populated as the protein concentration nears the Kd for metal dissociation. Note, however,

Chemical Denaturation of ALS-associated hSOD

that a three-state model with unmetallated monomeric intermediate does not give good fits for the GdmCl curves, again indicating that metals are bound to the intermediate at higher protein concentrations. Another possible explanation for the changes at 0.2 mM protein is aggregation of the intermediate; renaturation curves at 0.2 mM consistently showed scatter. Also, the native baseline is not well defined at 0.2 mM and this could interfere with obtaining a proper fit. Although there is additional complexity in the denaturation mechanism at low protein concentration than is accounted for by the models considered here, the experiments and data analysis presented herein provide a more detailed explanation and understanding of the nature of intermediates and unfolded species populated, and the role of bound metals in the GdmCl denaturation of holo hSOD. Unfolding mechanism approaches two-state at high protein concentration At high protein concentration, the unfolding transition for holo hSOD becomes close to twostate, since inflection due to intermediate formation in the GdmCl curves is not as apparent and fits to a two-state model are good. The DGtotal from the model without metal dissociation (obtained from the two-state fit or by adding the DGs from the three-state fit) increase with protein concentration but level off at 10 mM. This is evidence that metal dissociation from the unfolded state is decreasing over this range, as would be expected when the protein concentration increases into a range where it becomes similar to the Kd. This type of behaviour has been observed for other proteins that bind cofactors, such as the Cu-binding protein azurin, cytochrome b562 and flavodoxin; chemical denaturation of these proteins has been conducted at protein concentrations higher than the Kd for the unfolded protein, in order to simplify the data analysis.16 The very high affinity of native hSOD for Cu and Zn, and the relatively close proximity of the metalbinding ligands in the primary sequence of the protein, make it physically reasonable that metals can associate with the unfolded protein. In addition to the evidence from analysis of the GdmCl curves, NMR measurements of hydrodynamic radius have found evidence for binding of metals to unfolded pseudoWT.36 We obtained further evidence for metal association with the unfolded protein from further NMR titration and dialysis experiments. Also, DSC experiments of holo hSODs provide evidence for metal association in the thermally unfolded state, and DSC measures of DGtotal are in agreement with values obtained by chemical denaturation (P.B.S., J.A.O.R., Clare A. Siddall, J.R.L. & E.M.M., unpublished data). Both the twostate analysis with bound metals and the three-state analysis appear to have a reasonable physical basis, and so can be used to analyze the effects of mutations on holo hSOD stability, as discussed below.

117 Effects of ALS-associated mutations on holo hSOD stability and aggregation Protein destabilization due to mutations has been linked to increased aggregation for many misfolding diseases, including familial amyloid polyneuropathy, light chain amyloidosis, prion diseases and serpinopathies37 and has also been proposed to be involved in enhanced hSOD aggregation in ALS.4,38 The holo mutant hSODs studied have decreased stability relative to pseudoWT, as measured by GdmCl (Table 3) and thermal denaturation.8 Owing to irreversible denaturation of wild-type protein, changes in thermodynamic stability upon mutation have not been determined in previous DSC studies of hSOD5,7 but may be estimated from the equilibrium GdmCl curves reported here. Data analysis using both the twostate and three-state models reveals that the E100G, G93A and G85R mutations are slightly to moderately destabilizing, owing primarily to a decrease in monomer stability with little change in the energetics of dimerization. Since the mutations are far from the dimer interface (Figure 1), these results are structurally reasonable. We have also conducted DSC analysis of reversible unfolding of these mutants in the pseudoWT background, and the results are very similar to those obtained by GdmCl denaturation (P.B.S., J.A.O.R., C. Siddall, J.R.L. & E.M.M., unpublished data). The magnitudes of destabilizing effects of the mutations are similar to those typically seen for similar types of mutations at solvent-exposed sites.39,40 It is of interest to consider the specific effects of the individual mutations. G93 is a highly conserved residue at position iC3 in a type I turn.41 Glycine is the only residue that is statistically preferred at this position, since only glycine can readily adopt the required left-handed helical phi, psi values that facilitate the return of the chain to run antiparallel to the original direction.42 Mutating glycine residues in turns to other residues (such as Ala, Asn or Asp) has been found to destabilize other proteins by w1.3–2.3 kcal molK1.43,44 This is comparable to the DDG2 of K2.0 kcal (mol monomer)K1 for G93A. Note that this mutation causes changes in structure and dynamics not only in the turn, but also in an adjacent loop (residues 35–42),45 which may also affect stability. E100 makes a surface ion pair with K30; the DDG 2 value for E100G of K0.85 kcal (mol monomer)K1 is within the range typically seen for a mutation of a single residue involved in this type of surface electrostatic interaction (w0.1–1.3 kcal molK1).46,47 The DDG2 of K4.0 kcal (mol monomer)K1 for G85R is relatively larger. G85 is a conserved residue located near the edge of the active site, in the centre of the fifth b-strand.41 There is evidence that mutating G85 to R may significantly alter copper binding. 48 The structural consequences of the mutation have been determined for yeast Cu,Zn SOD: the larger arginine side-chain appears to be readily accommodated at this fairly exposed site,

118 with only small structural changes occurring very close to the site of mutation (PDB codes 1f18 for G85R and 2jcw for wild-type).49 This suggests that the destabilization may be caused largely by weakened metal binding in the monomer. It should be noted that although binding may be weakened, in vitro metallated protein prepared here and by others27 has wild-type-like metal content and activity. Also, the protein concentration dependence of the curves for G85R is the same as for the other hSODs, suggesting that the unfolding mechanism applies. In A4V, a larger hydrophobic residue is inserted into the dimer interface. Such a mutation may be expected to weaken dimer association. However, thermodynamic effects could not be determined by chemical denaturation due to aggregation. Aggregation in misfolding diseases is generally thought to occur via partially folded states.37,50 In this study there is generally little aggregation in the denaturation and renaturation curves for pseudoWT, E100G, G93A and G85R, despite the formation of prominent kinetic and equilibrium intermediate(s). Since GdmCl may interfere with aggregation, it is not clear whether the intermediate(s) are prone to aggregate. Note that the mutations will decrease the population of the monomeric intermediate by decreasing its stability, and will increase the population of species less stable than the intermediate. Since toxicity is proposed to be proportional to the amount of aggregating species, this suggests that it may be more destabilized forms of hSOD (e.g. due to loss of metals, monomerization or disulfide bond reduction) that are prone to aggregate and cause disease, as has been suggested in other studies.8,10,17 To date, the identification of general correlations between properties of fALS-associated mutant hSOD and disease characteristics has proven elusive. For a given mutation, there are large variations in the age of disease onset, and disease duration, while also variable, may be more predictable.51,52 The clinical data for individual fALS hSOD mutations are generally limited to a small number of individuals, which may interfere with identifying clear correlations between disease characteristics and properties of mutant proteins.2 Bearing these limitations in mind, it is nevertheless interesting to consider relationships between results of denaturation studies and disease characteristics. One notable observation is the pronounced tendency of A4V to aggregate in these and other studies;45,53 this may be related to the short disease duration for this mutation.51 In contrast, E100G has been suggested to be associated with a relatively long disease duration;51,52 this mutant tends not to aggregate in GdmCl curves and has the highest stability of the mutants studied here. G93A has intermediate stability; based on the relatively limited patient data available the disease duration is not particularly short or long.52 For G85R, the disease duration is not unusual considering the small number of reported cases;51 however, this mutation in mice models has been reported to be

Chemical Denaturation of ALS-associated hSOD

especially toxic, since it causes disease characteristics at very low levels of mutant protein and the disease duration in mice is particularly short.54 Notably, this mutant is the most destabilized of the ones studied here. Thus, there is a tendency for disease duration to be shorter as the extent of destabilization of the holo mutants increases. Previous studies have proposed a correlation between shorter disease duration and increased destabilization of apo hSOD.17 Comparing thermal melting temperatures (Tm),8 apo E100G, G93A, and A4V have similar Tms (w10 deg. C lower than pseudoWT) but apo G85R has significantly higher Tm (w5 deg. C lower than pseudoWT), which may not fit the trend. In contrast, for the holo proteins, G85R has significantly lower Tm than the other mutants, as well as significantly lower chemical denaturation midpoint (Figure 5). This suggests that the effects of G85R may not be exerted through destabilization of the apo protein, but rather through destabilization of the holo protein, i.e. aggregation may occur via forms of hSOD that are accessed more readily from the destabilized holo mutant than from holo wild-type. Given the limited data, it is not yet possible to draw general conclusions regarding effects of mutations and disease characteristics; however, it seems likely that the many different hSOD mutations associated with fALS may exert their effects in many different ways: through alterations in protein thermodynamic stability by various mechanisms including monomer destabilization, weakened metal binding or dimer destabilization, or by increased protein fragmentation or covalent modification,55 as well as perhaps through kinetic effects, such as increased rates of unfolding or slowed rates of folding.8 These different mechanisms may unmask an intrinsic tendency of hSOD to aggregate through increased population of partially folded states upon protein destabilization, which may also occur in sporadic ALS56 and Huntington’s disease.57 The systematic and quantitative analysis of the mechanism and thermodynamics of fALS mutant hSOD unfolding established herein has provided new insights regarding possible mechanisms of protein aggregation in ALS, and provides the basis for further kinetic and thermodynamic studies to characterize the molecular basis for these mechanisms in further detail.

Materials and Methods Preparation of recombinant holo hSOD Expression vectors and purification protocols for holo pseudoWT and E100G, A4V, G93A and G85R hSOD have been described.8 Recombinant pseudoWT and mutant hSOD were expressed and purified using a modification of the procedure of Getzoff et al.58 in which a Poros PE or HP2 column replaced the DEAE column. The final yield was typically w20–40 mg of protein per liter of culture. Proteins were fully metallated within 5–10% (1 mol each

119

Chemical Denaturation of ALS-associated hSOD

of Cu and Zn per mol per monomer of SOD) as measured by ICP-AES (Solutions Analytical Laboratory, University of Waterloo, Waterloo, Ontario, Canada)). DSC analysis revealed a single endotherm at high temperature,8 confirming full metallation, since incorrect metallation gives rise to additional endotherms at lower temperatures due to unfolding of incorrectly metallated species7 (data not shown). Protein concentration was determined using the Lowry assay. Activity measurements Activity of purified recombinant hSOD was checked routinely by measuring the amount of hSOD required for 50% inhibition of auto-oxidation of pyrogallol.59 Briefly, pyrogallol (8 mM in 10 mM HCl) was added to 3 ml of buffer (50 mM Tris (pH 8.2), 1 mM diethylene triamine penta acetic acid (DTPA), 25 8C) to a final concentration of 25 mM just prior to addition of hSOD. The concentration of hSOD was adjusted so that the volume of hSOD added (typically 10 ml) would decrease the rate of pyrogallol auto-oxidation by between 40% and 60%. The rate of pyrogallol auto-oxidation was determined as above without the addition of hSOD. Activity was reproducible within experimental error from one preparation to another, and was within 1500–1700 units/mg. GdmCl denaturation and renaturation curves Denaturation and renaturation curve experiments were conducted at 25 8C in 20 mM Hepes buffer at pH 7.8. Stock SOD protein solutions in 20 mM Hepes (pH 7.8) were diluted tenfold into different concentrations of GdmCl in 20 mM Hepes and equilibrated at 25 8C for 6 h to O80 days in a thermally equilibrated water-bath or incubator. Fluorescence was measured using a Fluorolog3-22 (Instruments SA, Edison, NJ) equipped with a thermostatted cuvette holder with excitation and emission wavelengths of 282 nm and 360 nm, respectively. CD was measured using a J715 CD spectropolarimeter (Jasco), at 25 8C, with a 0.01 cm path-length cell and a 2 nm bandwidth. Activity was measured as described above. The effect of GdmCl on the activity was corrected at each concentration. GdmCl increases the rate of pyrogallol auto-oxidation and, due to the high ionic strength, decreases the activity of hSOD. GdmCl curve analysis Thermodynamic parameters were obtained by analyzing the GdmCl-induced denaturation and renaturation curves using two-state and three-state dimeric transition models described in Table 1. The dependence of activity, fluorescence or CD signal (Yo) with GdmCl is related to the mol fraction of the different species present at each GdmCl concentration using the following equations:60 Yo Z SXi Yi

(1)

where Xi is the mol fraction of species i and Yi is the signal (measured using activity, fluorescence or CD) of species i. The signal of each species was allowed to have a dependence on the GdmCl concentration: Yi Z YH2 O;i C si ½GdmCl

(2)

where YH2 O;i is the signal of the species i in 0 M GdmCl and si is the dependence of Yi on GdmCl concentration. The mol fraction of each species can be used to determine equilibrium constants (K) and the difference in Gibbs free

energy between two states, DG, at each GdmCl concentration, which can then be used to determine the free energy difference at 0 M GdmCl, DGH2 O , assuming a linear dependence of free energy with GdmCl concentration, m, according to the equation: DG ZKRT ln K Z DGH2 O C m½GdmCl

(3)

where R is the gas constant and T is the absolute temperature. For each model in Table 1, the fraction of a particular reference species was defined in terms of the equilibrium constant(s) and total protein concentration using the equations for molar fraction. The total signal (equation (1)) was then defined in terms of the fraction of this reference species and equilibrium constants where needed. Data fitting Equilibrium curves were fit to the above bimolecular unfolding models using Matlab software, version 7.0.4. In all cases, the many parameters for the fitting models (i.e. six for two-state model, ten for models with intermediates) could not simultaneously all be accurately fit using a single curve, and so the following procedures were used to determine the values as accurately as possible. The native and unfolded baselines in CD and fluorescence curves were determined using linear regression and fixed for all fitting models. The slope for the native baseline was determined at 10 mM protein, where the population of native dimer is highest (see Results), scaled according to protein concentration as necessary and fixed for subsequent fitting. The native baseline for CD at 216 nm was set to have a slope of zero, which was confirmed by measuring 10 mM pseudoWT after a short (2 h) incubation. For the three-state models, the intermediate fluorescence or CD signal was fixed at a value one-quarter of the way through the transition and s was set to zero (for equation (2)), based on the inflection of the 1 mM fluorescence curves and on the amplitudes of the two unfolding kinetic phases (see Results, Figure 6). At 0.2 mM the point of inflection occurred at a higher fluorescence value, therefore the intermediate fluorescence was set at a value half-way through the transition. For curves monitored by activity, the native and unfolded baseline slope and the unfolded activity were fixed at zero. For the two-state dimer models, the m value was either allowed to float or was fixed. Without metal dissociation, the m value was fixed at 8 kcal (mol dimer)K1 MK1, which is an average value obtained by calculating m based on the change in solvent-accessible surface area for unfolding, DASA, as determined using empirical equations (7.3 kcal (mol dimer)K1 MK1)61 or using the surface areas for the holo dimer (PDB code 1SOS)13 calculated using GetArea 1.162 and for the unfolded state using the Rose lab website† (with standard option)63,64 (9.4 kcal (mol dimer)K1 MK1). The m value for the model involving metal dissociation is expected to be higher due to increased molecularity of the transition and was fixed to 15.8 kcal (mol dimer)K1 MK1, which is an average value obtained from fitting the 10 mM curves. The m values for three-state models were defined as follows. Using the above procedures for calculating surface areas and assuming the monomer intermediate structure was the same as the structure of the monomer in the holo dimer crystal gave low m1 values that predicted significant dimer dissociation at low GdmCl, which does not occur † http://roselab.jhu.edu/

120 based on crosslinking data (Figure 8), and equilibrium analytical centrifugation.35 The m1 value was therefore set to the lowest value (1.8 kcal (mol dimer)K1 MK1) that did not predict dissociation at 0 M GdmCl. The m2 value was then fixed at either 3.1 kcal (mol monomer)K1 MK1 or 7 kcal (mol monomer)K1 MK1 corresponding to a total m of 8 kcal (mol dimer)K1 MK1 or of 15.8 kcal (mol dimer)K1 MK1as was used for two-state dimer fit without or with metal dissociation, respectively (see above). The higher m1 value suggests that the monomeric intermediate is significantly expanded compared to the structure of the monomer in the dimer crystal structure. Consistent with this, the solution structures of the holo, Zn, and apo forms of an engineered monomeric form of pseudoWT hSOD have increased disorder compared to dimeric holo hSOD.29,65,66 The m2 value of 3.1 kcal (mol monomer)K1 MK1 is comparable but somewhat small compared to values obtained for other monomeric proteins of this size.61 Again, for the metal dissociation model the m2 value of 7 kcal (mol monomer)K1 MK1 is higher owing to the higher molecularity of the transition. It should be noted that the m values can be varied considerably without a substantial reduction in the quality of fits, i.e. m values are difficult to determine with great accuracy from the data, hence they have simply been defined within a physically reasonable range. NMR spectroscopy 1

H–15N HSQC spectra were recorded at 298.2 K on a Bruker Avance DMX 600 MHz spectrometer equipped with a triple resonance xyz-gradient probe, and data were processed using Felix97.0 (MSI, Inc.). NMR samples contained w0.4 mM protein reduced with 5 mM ascorbate in 20 mM Hepes (pH 7.8), and 0–2 M GdmCl in 93% H2O/7% 2H2O. Assignments obtained at pH 5.0 in phosphate buffer67 were transferred to pH 7.8 by obtaining 1H–15N HSQC spectra as a function of pH over this range as well as obtaining a 15N-edited NOESY HSQC at pH 7.8. Only small changes were observed upon changing the pH from 5.0 to 7.8. GdmCl-induced unfolding kinetics Kinetic experiments were performed under the same solution conditions as equilibrium experiments: 10 mM stock hSOD protein in 20 mM Hepes (pH 7.8), was diluted tenfold into various GdmCl solutions in 20 mM Hepes (pH 7.8), that had been pre-equilibrated at 25 8C. Fluorescence was immediately monitored using a Fluorolog3-22 (Instruments SA, Edison, NJ) at 25 8C, with excitation and emission wavelengths of 282 nm and 360 nm. Equilibrium ultracentrifugation Sedimentation experiments were performed at 20 8C on a Beckman XLI Analytical Ultracentrifuge using an AN50-Ti rotor. Sedimentation velocity measurements using double-sector charcoal-Epon cells equipped with sapphire windows were performed on samples (2 or 80 mM) centrifuged at 50,000 rpm and concentration distributions were determined using absorbance optics. A total of 200 scans were obtained. The partial specific volume and density of the sample were calculated using the program SEDNTERP68 from the amino acid sequence and buffer composition, respectively. Data analysis was performed using the program sedfit.69

Chemical Denaturation of ALS-associated hSOD

Covalent crosslinking Dimeric hSOD was intramolecularly crosslinked using glutaraldehyde.70 Reaction conditions were optimized for specific intramolecular dimeric crosslinking by performing experiments as a function of glutaraldehyde concentration, protein concentration and time. Glutaraldehyde (5% (w/v)) was added to hSOD solution (1–8 mM protein in 0–8 M GdmCl, 20 mM Hepes, pH 7.8) and incubated at ambient temperature for 2 min. The reaction was stopped by the addition of NaBH4, then the GdmCl was diluted by adding water, and the protein was precipitated by adding deoxycholic acid and trichloroacetic acid.71 Precipitated protein was pelleted by centrifugation, washed with ice-cold acetone and then resolubilized in gel loading buffer, boiled, and separated by SDS-PAGE. Coomassie stained gels were digitized (BioRad Gel Doc, BioRad Laboratories, Inc., Hercules, CA) and band intensities were quantified (Quantity One, version 4.3.1, BioRad Laboratories, Inc.).

Acknowledgements We thank Joe Gaspar, Mike Ditty and Jan Venne for assistance with NMR experiments and analysis, Colin Campbell and Matthew Scorah for assistance with Matlab, and a reviewer for helpful comments. This research was funded by the Neuromuscular Research Partnership, an initiative of the ALS Society of Canada, Muscular Dystrophy Society and Canadian Institutes of Health Research, grants to E.M.M. and J.R.L. and to A.C.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2005.10.042

References 1. Rosen, D. R., Siddique, T., Patterson, D., Figlewicz, D. A., Sapp, P., Hentati, A. et al. (1993). Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 362, 59–62. 2. Radunovic, A. & Leigh, P. N. (1999). ALSODatabase: database of SOD1 (and other) gene mutations in ALS on the Internet. European FALS group and ALSOD consortium. Amyotroph. Lateral Scler. Other Motor Neuron Disord. 1, 45–49. 3. Cleveland, D. W. & Liu, J. (2000). Oxidation versus aggregation–how do SOD1 mutants cause ALS? Nature Med. 6, 1320–1321. 4. Bendotti, C. & Carri, M. T. (2004). Lessons from models of SOD1-linked familial ALS. Trends Mol. Med. 10, 393–400. 5. Lepock, J. R., Frey, H. E. & Hallewell, R. A. (1990). Contribution of conformational stability and reversibility of unfolding to the increased thermostability of human and bovine superoxide dismutase mutated at free cysteines. J. Biol. Chem. 265, 21612–21618.

Chemical Denaturation of ALS-associated hSOD

6. Malinowski, D. P. & Fridovich, I. (1979). Subunit association and side-chain reactivities of bovine erythrocyte superoxide dismutase in denaturing solvents. Biochemistry, 18, 5055–5060. 7. Rodriguez, J. A., Valentine, J. S., Eggers, D. K., Roe, J. A., Tiwari, A., Brown, R. H., Jr & Hayward, L. J. (2002). Familial amyotrophic lateral sclerosis-associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide dismutase. J. Biol. Chem. 277, 15932–15937. 8. Stathopulos, P. B., Rumfeldt, J. A., Scholz, G. A., Irani, R. A., Frey, H. E., Hallewell, R. A. et al. (2003). Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro. Proc. Natl Acad. Sci. USA, 100, 7021–7026. 9. Rakhit, R., Cunningham, P., Furtos-Matei, A., Dahan, S., Qi, X. F., Crow, J. P. et al. (2002). Oxidation-induced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis. J. Biol. Chem. 277, 47551–47556. 10. Furukawa, Y. & O’Halloran, T. V. (2005). ALS mutations have the greatest destabilizing effect on the apo, reduced form of SOD1, leading to unfolding and oxidative aggregation. J. Biol. Chem. 17, 17266–17274. 11. Cardoso, R. M., Thayer, M. M., DiDonato, M., Lo, T. P., Bruns, C. K., Getzoff, E. D. & Tainer, J. A. (2002). Insights into Lou Gehrig’s disease from the structure and instability of the A4V mutant of human Cu,Zn superoxide dismutase. J. Mol. Biol. 324, 247–256. 12. McCord, J. M. & Fridovich, I. (1969). Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055. 13. Parge, H. E., Hallewell, R. A. & Tainer, J. A. (1992). Atomic structures of wild-type and thermostable mutant recombinant human Cu,Zn superoxide dismutase. Proc. Natl Acad. Sci. USA, 89, 6109–6113. 14. Rigo, A., Terenzi, M., Viglino, P., Calabrese, L., Cocco, D. & Rotilio, G. (1977). The binding of copper ions to copper-free bovine superoxide dismutase. Properties of the protein recombined with increasing amounts of copper ions. Biochem. J. 161, 31–35. 15. Crow, J. P., Sampson, J. B., Zhuang, Y., Thompson, J. A. & Beckman, J. S. (1997). Decreased zinc affinity of amyotrophic lateral sclerosis-associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitrite. J. Neurochem. 69, 1936–1944. 16. Wittung-Stafshede, P. (2002). Role of cofactors in protein folding. Accts. Chem. Res. 35, 201–208. 17. Lindberg, M. J., Tibell, L. & Oliveberg, M. (2002). Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: decreased stability of the apo state. Proc. Natl Acad. Sci. USA, 99, 16607–16612. 18. Ferraroni, M., Rypniewski, W., Wilson, K. S., Viezzoli, M. S., Banci, L., Bertini, I. & Mangani, S. (1999). The crystal structure of the monomeric human SOD mutant F50E/G51E/E133Q at atomic resolution. The enzyme mechanism revisited. J. Mol. Biol. 288, 413–426. 19. Liochev, S. I., Chen, L. L., Hallewell, R. A. & Fridovich, I. (1998). The familial amyotrophic lateral sclerosis-associated amino acid substitutions E100G, G93A, and G93R do not influence the rate of inactivation of copper- and zinc-containing superoxide dismutase by H2O2. Arch. Biochem. Biophys. 352, 237–239.

121 20. Lyons, T. J., Liu, H., Goto, J. J., Nersissian, A., Roe, J. A., Graden, J. A. et al. (1996). Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein. Proc. Natl Acad. Sci. USA, 93, 12240–12244. 21. Hayward, L. J., Rodriguez, J. A., Kim, J. W., Tiwari, A., Goto, J. J., Cabelli, D. E. et al. (2002). Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis. J. Biol. Chem. 277, 15923–15931. 22. Goto, J. J., Zhu, H., Sanchez, R. J., Nersissian, A., Gralla, E. B., Valentine, J. S. & Cabelli, D. E. (2000). Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis. J. Biol. Chem. 275, 1007–1014. 23. Mei, G., Rosato, N., Silva, N., Jr, Rusch, R., Gratton, E., Savini, I. & Finazzi-Agro, A. (1992). Denaturation of human Cu/Zn superoxide dismutase by guanidine hydrochloride: a dynamic fluorescence study. Biochemistry, 31, 7224–7230. 24. Stroppolo, M. E., Malvezzi-Campeggi, F., Mei, G., Rosato, N. & Desideri, A. (2000). Role of the tertiary and quaternary structures in the stability of dimeric copper, zinc superoxide dismutases. Arch. Biochem. Biophys. 377, 215–218. 25. Roe, J. A., Butler, A., Scholler, D. M., Valentine, J. S., Marky, L. & Breslauer, K. J. (1988). Differential scanning calorimetry of Cu,Zn-superoxide dismutase, the apoprotein, and its zinc-substituted derivatives. Biochemistry, 27, 950–958. 26. Lindberg, M. J., Normark, J., Holmgren, A. & Oliveberg, M. (2004). Folding of human superoxide dismutase: disulfide reduction prevents dimerization and produces marginally stable monomers. Proc. Natl Acad. Sci. USA, 101, 15893–15898. 27. Lynch, S. M., Boswell, S. A. & Colon, W. (2004). Kinetic stability of Cu/Zn superoxide dismutase is dependent on its metal ligands: implications for ALS. Biochemistry, 43, 16525–16531. 28. Culotta, V. C., Klomp, L. W., Strain, J., Casareno, R. L., Krems, B. & Gitlin, J. D. (1997). The copper chaperone for superoxide dismutase. J. Biol. Chem. 272, 23469–23472. 29. Banci, L., Benedetto, M., Bertini, I., Del Conte, R., Piccioli, M. & Viezzoli, M. S. (1998). Solution structure of reduced monomeric Q133M2 copper, zinc superoxide dismutase (SOD) Why is SOD a dimeric enzyme? Biochemistry, 37, 11780–11791. 30. Pesce, A., Capasso, C., Battistoni, A., Folcarelli, S., Rotilio, G., Desideri, A. & Bolognesi, M. (1997). Unique structural features of the monomeric Cu,Zn superoxide dismutase from Escherichia coli, revealed by X-ray crystallography. J. Mol. Biol. 274, 408–420. 31. Bourne, Y., Redford, S. M., Steinman, H. M., Lepock, J. R., Tainer, J. A. & Getzoff, E. D. (1996). Novel dimeric interface and electrostatic recognition in bacterial Cu,Zn superoxide dismutase. Proc. Natl Acad. Sci. USA, 93, 12774–12779. 32. Bordo, D., Matak, D., Djinovic-Carugo, K., Rosano, C., Pesce, A., Bolognesi, M. et al. (1999). Evolutionary constraints for dimer formation in prokaryotic Cu,Zn superoxide dismutase. J. Mol. Biol. 285, 283–296. 33. Gunasekaran, K., Tsai, C. J. & Nussinov, R. (2004). Analysis of ordered and disordered protein complexes reveals structural features discriminating

122

34.

35.

36.

37. 38. 39. 40.

41. 42. 43.

44. 45.

46. 47.

48.

49.

between stable and unstable monomers. J. Mol. Biol. 341, 1327–1341. Khare, S. D., Caplow, M. & Dokholyan, N. V. (2004). The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis. Proc. Natl Acad. Sci. USA, 101, 15094–15099. Doucette, P. A., Whitson, L. J., Cao, X., Schirf, V., Demeler, B., Valentine, J. S. et al. (2004). Dissociation of human copper–zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular basis for dimer stability. J. Biol. Chem. 279, 54558–54566. Assfalg, M., Banci, L., Bertini, I., Turano, P. & Vasos, P. R. (2003). Superoxide dismutase folding/unfolding pathway: role of the metal ions in modulating structural and dynamical features. J. Mol. Biol. 330, 145–158. Uversky, V. N. & Fink, A. L. (2004). Conformational constraints for amyloid fibrillation: the importance of being unfolded. Biochim. Biophys. Acta, 1698, 131–153. Julien, J. P. (2001). Amyotrophic lateral sclerosis. Unfolding the toxicity of the misfolded. Cell, 104, 581–591. Alber, T. (1989). Mutational effects on protein stability. Annu. Rev. Biochem. 58, 765–798. Gromiha, M. M., Oobatake, M., Kono, H., Uedaira, H. & Sarai, A. (2002). Importance of mutant position in Ramachandran plot for predicting protein stability of surface mutations. Biopolymers, 64, 210–220. Bordo, D., Djinovic, K. & Bolognesi, M. (1994). Conserved patterns in the Cu,Zn superoxide dismutase family. J. Mol. Biol. 238, 366–386. Hutchinson, E. G. & Thornton, J. M. (1994). A revised set of potentials for beta-turn formation in proteins. Protein Sci. 3, 2207–2216. Kim, J., Brych, S. R., Lee, J., Logan, T. M. & Blaber, M. (2003). Identification of a key structural element for protein folding within beta-hairpin turns. J. Mol. Biol. 328, 951–961. Predki, P. F., Agrawal, V., Brunger, A. T. & Regan, L. (1996). Amino-acid substitutions in a surface turn modulate protein stability. Nature Struct. Biol. 3, 54–58. Shipp, E. L., Cantini, F., Bertini, I., Valentine, J. S. & Banci, L. (2003). Dynamic properties of the G93A mutant of copper–zinc superoxide dismutase as detected by NMR spectroscopy: implications for the pathology of familial amyotrophic lateral sclerosis. Biochemistry, 42, 1890–1899. Sali, D., Bycroft, M. & Fersht, A. R. (1991). Surface electrostatic interactions contribute little of stability of barnase. J. Mol. Biol. 220, 779–788. Marti, D. N. & Bosshard, H. R. (2003). Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges. J. Mol. Biol. 330, 621–637. Ratovitski, T., Corson, L. B., Strain, J., Wong, P., Cleveland, D. W., Culotta, V. C. & Borchelt, D. R. (1999). Variation in the biochemical/biophysical properties of mutant superoxide dismutase 1 enzymes and the rate of disease progression in familial amyotrophic lateral sclerosis kindreds. Hum. Mol. Genet. 8, 1451–1460. Hart, P. J., Balbirnie, M. M., Ogihara, N. L., Nersissian, A. M., Weiss, M. S., Valentine, J. S. & Eisenberg, D. (1999). A structure-based mechanism for copper–zinc superoxide dismutase. Biochemistry, 38, 2167–2178.

Chemical Denaturation of ALS-associated hSOD

50. Kelly, J. W. (1996). Alternative conformations of amyloidogenic proteins govern their behavior. Curr. Opin. Struct. Biol. 6, 11–17. 51. Juneja, T., Pericak-Vance, M. A., Laing, N. G., Dave, S. & Siddique, T. (1997). Prognosis in familial amyotrophic lateral sclerosis: progression and survival in patients with glu100gly and ala4val mutations in Cu,Zn superoxide dismutase. Neurology, 48, 55–57. 52. Cudkowicz, M. E., McKenna-Yasek, D., Sapp, P. E., Chin, W., Geller, B., Hayden, D. L. et al. (1997). Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis. Ann. Neurol. 41, 210–221. 53. Leinweber, B., Barofsky, E., Barofsky, D. F., Ermilov, V., Nylin, K. & Beckman, J. S. (2004). Aggregation of ALS mutant superoxide dismutase expressed in Escherichia coli. Free Radic. Biol. Med. 36, 911–918. 54. Bruijn, L. I., Becher, M. W., Lee, M. K., Anderson, K. L., Jenkins, N. A., Copeland, N. G. et al. (1997). ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron, 18, 327–338. 55. Wang, J., Slunt, H., Gonzales, V., Fromholt, D., Coonfield, M., Copeland, N. G. et al. (2003). Copper-binding-site-null SOD1 causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a common feature. Hum. Mol. Genet. 12, 2753–2764. 56. Shibata, N., Hirano, A., Kobayashi, M., Sasaki, S., Kato, T., Matsumoto, S. et al. (1994). Cu/Zn superoxide dismutase-like immunoreactivity in Lewy body-like inclusions of sporadic amyotrophic lateral sclerosis. Neurosci. Letters, 179, 149–152. 57. Nishiyama, K., Murayama, S., Shimizu, J., Ohya, Y., Kwak, S., Asayama, K. & Kanazawa, I. (1995). Cu/Zn superoxide dismutase-like immunoreactivity is present in Lewy bodies from Parkinson disease: a light and electron microscopic immunocytochemical study. Acta Neuropathol. (Berl.), 89, 471–474. 58. Getzoff, E. D., Cabelli, D. E., Fisher, C. L., Parge, H. E., Viezzoli, M. S., Banci, L. & Hallewell, R. A. (1992). Faster superoxide dismutase mutants designed by enhancing electrostatic guidance. Nature, 358, 347–351. 59. Marklund, S. & Marklund, G. (1974). Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47, 469–474. 60. Park, Y. C. & Bedouelle, H. (1998). Dimeric tyrosyltRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions. J. Biol. Chem. 273, 18052–18059. 61. Myers, J. K., Pace, C. N. & Scholtz, J. M. (1995). Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4, 2138–2148. 62. Fraczkiewicz, R. & Braun, W. (1998). Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. J. Comput. Chem. 19, 319–333. 63. Creamer, T. P., Srinivasan, R. & Rose, G. D. (1995). Modeling unfolded states of peptides and proteins. Biochemistry, 34, 16245–16250. 64. Creamer, T. P., Srinivasan, R. & Rose, G. D. (1997). Modeling unfolded states of proteins and peptides. II. Backbone solvent accessibility. Biochemistry, 36, 2832–2835. 65. Banci, L., Bertini, I., Cantini, F., D’Onofrio, M. & Viezzoli, M. S. (2002). Structure and dynamics of

Chemical Denaturation of ALS-associated hSOD

66.

67.

68.

69.

copper-free SOD: the protein before binding copper. Protein Sci. 11, 2479–2492. Banci, L., Bertini, I., Cramaro, F., Del Conte, R. & Viezzoli, M. S. (2003). Solution structure of Apo Cu,Zn superoxide dismutase: role of metal ions in protein folding. Biochemistry, 42, 9543–9553. Banci, L., Bertini, I., Cramaro, F., Del Conte, R. & Viezzoli, M. S. (2002). The solution structure of reduced dimeric copper zinc superoxide dismutase. The structural effects of dimerization. Eur. J. Biochem. 269, 1905–1915. Laue, T. M., Shah, B. D., Ridgeway, T. M. & Pelletier, S. L. (1992). Computer-aided interpretation of analytical sedimentation data for proteins. In Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. & Rowe, A., eds), pp. 90–125, Royal Society of Chemistry, Cambridge, UK. Schuck, P. (2000). Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619.

123 70. Bond, J. M., Bannister, J. V. & Bannister, W. H. (1991). Polymers of Cu/Zn superoxide dismutase produced by cross-linking with glutaraldehyde. Free Radic. Res. Commun. 12, 545–551. 71. Lai, Z., McCulloch, J., Lashuel, H. A. & Kelly, J. W. (1997). Guanidine hydrochloride-induced denaturation and refolding of transthyretin exhibits a marked hysteresis: equilibria with high kinetic barriers. Biochemistry, 36, 10230–10239. 72. Koradi, R., Billeter, M. & Wuthrich, K. (1996). MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55. 73. Strange, R. W., Antonyuk, S., Hough, M. A., Doucette, P. A., Rodriguez, J. A., Hart, P. J. et al. (2003). The structure of holo and metal-deficient wild-type human Cu,Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis. J. Mol. Biol. 328, 877–891. 74. Pace, C. N. (1986). Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131, 266–280.

Edited by F. Schmid (Received 2 June 2005; received in revised form 16 September 2005; accepted 18 October 2005) Available online 8 November 2005