Combined Isothermal Titration and Differential Scanning Calorimetry Define. Three-State Thermodynamics of fals-associated Mutant Apo SOD1 Dimers

Transcription

1 Supporting Information for: Combined Isothermal Titration and Differential Scanning Calorimetry Define Three-State Thermodynamics of fals-associated Mutant Apo SOD1 Dimers and an Increased Population of Folded Monomer Helen R. Broom, 1 Kenrick A. Vassall, 1,2 Jessica A.O. Rumfeldt, 1 Colleen M. Doyle, 1 Ming Sze Tong, 1 Julia M. Bonner, 1,3 and Elizabeth M. Meiering 1* 1 Department of Chemistry, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada 2 Present address: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada 3 Present Address: Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA Corresponding Author * Department of Chemistry, University of Waterloo, Waterloo, ON N2L 3G1, Canada. Tel.: , Ext ; Fax: ; meiering@uwaterloo.ca 1

2 Supplementary Methods Models Used for Thermodynamic Analysis of Homodimeric Protein Folding. The simplest model for the unfolding mechanism of a homodimeric protein is a reversible two-state transition with concerted dissociation and unfolding of the native dimer (N 2 ) to unfolded monomers (U), which can be expressed as: N 2 K(T) 2U (S1) [N 2 ] = P dimer (1 α) [U] = 2P dimer (α) (S2) (S3) where K(T) is the equilibrium constant for unfolding at any temperature T, P dimer is the total protein concentration in M dimer and α is the extent of the unfolding reaction. K(T) is defined as: K(T) = [U]2 = 4P dimerα 2 [N 2 ] (1 α) (S4) To determine the extent of the unfolding reaction at any temperature, eq S4 can be rearranged as: α = K(T)+ K(T)(K(T)+16P dimer) 8P dimer (S5) The heat capacities of the native ( C p N 2 ) and unfolded (C p U ) protein are usually taken to vary linearly with temperature 1-4 (see Material and Methods): C p N 2 = A + Bt (S6a) C p U = E + Ft (S6b) where A (E) and B (F) are the intercept and slope of the folded (unfolded) baseline and t is the temperature in C. The change in heat capacity of unfolding ( C p ) can be determined at any temperature: C p (t) = (E A) + (F B)t (S7) 2

3 The specific calorimetric enthalpy of the unfolding transition in cal (g protein) -1 as a function of temperature can be written as: h cal (t) = t t ref C p dt = h cal (t ref ) + (E A)(t t ref ) + 1 (F B)(t 2 2 t 2 ref ) (S8) where t ref is a reference temperature typically set at the temperature of half completion and h cal (t ref ) is the h cal at t ref. Note that when the pre and post transition baselines have the same slope, the C p is constant with temperature and eq S8 simplifies to h cal (t) = h cal (t ref ) + (E A)(t t ref ) which has been shown to be a reasonable approximation over 1, 3, 5, 6 modest temperature intervals. For fitting, h cal (t) is extrapolated to 0 C to give: h 0 = h cal (t ref ) (E A)(t ref ) 1 (F B)(t 2 2 ref) (S9) Now h cal can be calculated at any temperature t using: h cal (t) = h 0 + (E A)t (F B)t (S10) 2 The temperature-dependence of K is given by the van t Hoff equation: dlnk(t) dt = H vh(t) RT 2 = β h cal(t) RT 2 (S11) where β = H vh (T) h cal (T) and R is the gas constant, T is the temperature in units of Kelvin, and ΔH vh is the van t Hoff enthalpy for the change in enthalpy for unfolding. Note that ΔH vh has units of cal (mol cooperative unit) -1 which, for this model, is cal (mol dimer) -1 and is distinct from the calorimetrically determined change in enthalpy H cal = h cal. ΔH vh reflects the steepness of the transition and H cal is proportional to the area under the endothermic peak. Here, β is used as a fitting parameter to allow these two terms to differ. In a strictly two-state transition the ratio of 3

4 ΔH vh to H cal is unity and β is equal to the molecular weight of the cooperative unit, in this case the dimer. In non-2 state transitions, for example involving intermediate formation, the ratio < 1; if samples are not normalized correctly for protein concentration the ratio can be higher or lower than 1. To obtain K as a function of temperature, eq S10 can be substituted into eq S11, integrated and rearranged giving: T d ln K(T) = ln K = β T ref K ref R T T ref h cal (T) T 2 dt (S12) R β ln (K(T) K ref ) = AA ( 1 T 1 T ref ) + BBln ( T T ref ) + CC(T T ref ) (S13) where: AA h (E A) 1 2 (273.15)2 (F B) BB (E A) (273.15)(F B) CC 1 (F B) 2 T = t Solving for K gives, K(T) = K ref exp {[AA ( 1 T 1 T ref ) + BBln ( T T ref ) + CC(T T ref )] R β } (S14) The total measured heat capacity (C p,total ) corresponds to a baseline heat capacity (C p, baseline ) plus the transition excess heat capacity resulting from the absorption of heat which drives the unfolding reaction (C p,excess ): C p,total = C p,baseline + C p,excess (S15) C p, baseline is given by: C p,baseline = (1 α(t))c p N 2 + α(t)c p U (S16) 4

5 The C p,excess is given by: C p,excess = ( α T ) h cal(t) (S17) The partial derivative α can be solved analytically at any T (substituting K with eq S4): T dlnk(t) dt = H vh(t) RT 2 = β h cal (T) RT 2 = 2 dα + 1 dα α dt 1 α dt (S18) which can be rewritten as: dα = β h cal(t) α(1 α) dt RT 2 2 α (S19) Note that parameters such as h cal are defined at a specific temperature regardless of the temperature units so that h cal (T) is the same value as h cal (t). Combining S19 and S17, the excess specific heat can be written as: C p,excess = β h 2 cal (t) α(1 α) RT 2 2 α (S20) Dimer Three-State with Monomer Intermediate Unfolding Model. For homodimeric proteins, more complex unfolding transitions may be observed. While many different unfolding mechanisms are possible, thermal unfolding is often described using a three-state transition model with a folded monomeric intermediate (M). This three-state transition model involves two steps: (1) dimer dissociation, followed by (2) monomeric intermediate unfolding: K 1 (T) N 2 2M K 2(T) 2U (S21) [N 2 ] = P dimer (1 α 1 ) [M] = 2P dimer α 1 (1 α 2 ) [U] = 2P dimer α 1 α 2 (S22) (S23) (S24) 5

6 where K 1 (T) and α 1, and K 2 (T) and α 2 are the equilibrium constant and extent of the unfolding reaction at any temperature for the first (N 2 2M) and second (M U) unfolding transitions, respectively. K 1 (T) = [M]2 = 4P α 2 1 (1 α 2 ) 2 [N 2 ] dimer 1 α 1 K 2 (T) = [U]2 α 1 = = α 2 2 [M] 2 (1 α 2 ) P dimer K 1 (T)(1+ K 2 (T)) 2 (S25a) (S25b) (S26a) α 2 = K 2(T) 1+ K 2 (T) (S26b) The specific heat capacity for M is assumed to be linear with temperature as for N 2 and U in eqs S6a and S6b. C p M = C + Dt (S27) where C and D are the intercept and slope of the folded (unfolded) baseline. The change in heat capacity of dimer dissociation (ΔC p,n2 2M) and monomer unfolding (ΔC p,m U ) can be determined at any temperature: ΔC p,n2 2M (t) = (C A) + (D B)t ΔC p,m U (t) = (E C) + (F D)t (S28a) (S28b) Using the same procedure outlined in eqs S8-S14, the equations for K 1 and K 2 as a function of temperature are: K 1 (T) = K ref 1 exp {[AA1 ( 1 1 ) + BB1ln ( T ) + CC1(T T T T ref 1 T ref 1 )] R } (S29a) ref 1 β K 2 (T) = K ref 2 exp {[AA2 ( 1 1 ) + BB2ln ( T ) + CC2(T T T T ref 2 T ref 2 )] R } (S29b) ref 2 β where: 6

7 AA1 h (C A) 1 2 (273.15)2 (D B) BB1 (C A) (273.15)(D B) CC1 1 (D B) 2 AA2 h (E C) 1 2 (273.15)2 (F D) BB2 (E C) (273.15)(F D) CC2 1 (F D) 2 and K ref-1 and K ref-2 are the equilibrium constants at the reference temperatures for dimer dissociation and monomer unfolding, respectively (which in the present study we take to be the experimental temperature for measurement of K ref-1 i.e. 37 C, and the temperature of the midpoint for monomer unfolding where K ref-2 =1, respectively, see below and Table S1). Using eqs S18, S25a and S25b: dα 1 dt = (β 1 h cal1 (T) RT 2 + α 2β 2 h cal2 (T) RT 2 ) α 1(1 α 1 ) 2 α 1 (S30a) dα 2 dt = β 2 h cal2 (T) 2RT 2 α 2 (1 α 2 ) (S30b) For simplicity the subscripts 1 and 2 were used for the parameters and h cal as well as, to refer to dimer dissociation and monomer unfolding, respectively, so that h cal1 h caln2 2M and h cal2 h calm U. Also: C p,excess = dα 1 dt h cal1 (t) + (dα 1 dt α 2 + dα 2 dt α 1) h cal2 (t) C p,baseline = f N2 C p N 2 + f M C p M + f U C p U (S31) (S32) where f N2, f M and f U are the fractions of native dimer, monomer intermediate and unfolded monomer, respectively, and can be calculated using: 7

8 f N2 =(1 α 1 ) f M=α1 (1 α 2 ) f U=α1 α 2 (S33a) (S33b) (S33c) where f N2 + f M + f U = 1 (S33d) Methods for Simplifying the Dimer Three-State with Monomer Intermediate Unfolding Model. Due to the additional parameters in the three-state model compared to the two-state model, fitting individual thermograms to eq 3 resulted in high uncertainties in the fitted values. Accordingly, multiple datasets were fit globally (Matlab R2013b, The MathWorks Inc.) using shared parameters. The slopes of the monomer intermediate and unfolded monomer baselines were set equal to that of the native baseline (i.e., B=D=F), making the common assumption that ΔC p of unfolding is temperature independent (Figure S3), 1, 3, 5 which has been shown to be reasonable over limited temperature ranges as used here. 7 The intercepts of the intermediate (C) and unfolded (E) baselines were defined relative to the intercept of the native baseline using temperature-independent values for the change in heat capacity upon dimer dissociation to monomer intermediate (ΔC p,n2 2M) and the change in heat capacity upon monomer intermediate unfolding (ΔC p,m U ): C= A + ΔC p,n2 2M (S34a) E= A + ΔC p,n2 2M + ΔC p,m U (S34b) ΔC p,n2 2M was set to 1.7 kcal (mol dimer) -1 C -1 (and the corresponding value in units of kcal g -1 C -1 obtained by dividing by the molecular weight of the dimer), the average value measured 8

9 using Kirchoff analysis of ITC data for SOD1 variants where the enthalpy of dimer dissociation was measured as a function of temperature. 4, 8, 9 For the three-state model, ΔC p,n2 2M + ΔC p,m U = ΔC p.n2 2U (total ΔC p of apo SOD1 unfolding from folded dimer to unfolded monomers); the value of ΔC p,m U was obtained by subtracting ΔC p,n2 2M from the experimentally determined value for ΔC p,n2 2U of 3.3 kcal (mol dimer) -1 C -1, 1 which gives a value of 1.6 kcal (mol dimer) -1 C -1 or 0.8 kcal (mol monomer) -1 C -1. This approach is consistent with the observations that 4, 10 mutations typically cause little change in ΔC p and that the average directly fitted values of ΔC p,n2 2U for apo SOD1 mutants (Table 1) are close to the value of 3.3 kcal (mol dimer) -1 C -1 determined for pwt and G93 mutants. 11, 12 Of note, lower values of ΔC p,n2 2U for A4V and V148G (Table 1) are likely related to increased monomer formation, which is most pronounced for these dimer interface mutants (Figure 1). To further simplify the fitting, the parameters of the first transition (dimer dissociation) were fixed to values determined by ITC (Figure S1). 10 Specifically, the K ref-1 was set to the value measured by ITC, K d,n2 2M, and the associated t ref-1 (T ref-1 ) was fixed to 37 C ( K), the temperature where ITC was performed. The h cal1 (t ref 1 ) was fixed to the value determined by ITC at 37 C, h caln2 2M. Thus, in fitting data to eqs S31 and S32, the globally shared fitted parameters were: t ref 2, h cal2 (t ref 2 ) (same as h calm U (t ref 2 )), β1 = β2, and parameters defining the slope and intercept of the native baselines. Two-State Monomer Unfolding Model. DSC data for apo mutants showing evidence for significant monomer formation (A4V, H46R, and V148G) were also analyzed using a monomer two-state unfolding model describing a reversible transition from folded monomer (M) to 9

10 unfolded monomers (U), M U. 4 Individual thermograms were fit (using Origin 5.0, Microcal Inc) to eq S35: C p = (C + Dt)(1 α) + (E + Ft)α + β h 2 cal (t0.5 )α(1 α) (S35) RT 2 where C p is the total specific heat absorption at temperature t (in C); C and E are the intercepts of the folded and unfolded baselines, respectively; D and F are the slopes of the folded and unfolded baselines, respectively; R is the universal gas constant; β is the ratio of van t Hoff to calorimetric enthalpy multiplied by the molecular weight of the SOD dimer; Δh cal is the specific calorimetric enthalpy of unfolding at t; α is the extent of the unfolding reaction; and t 0.5 is the temperature at which unfolding is half complete (i.e α =0.5). Predicting ΔC p,n2 2M Based on Changes in Solvent Accessible Surface Area (ΔASA). The polar and non-polar contributions to ΔASA (ΔASA p andδasa np, respectively) between dimer and dissociated monomers were determined using the crystallographic structures for apo SOD1 wildtype (1HL4) using: ΔASA p = ASA mona-p + ASA monb-p ASA dimer-p (S36a) ΔASA np = ASA mona-np + ASA monb-np ASA dimer-np (S36b) where ASA mona-p and ASA monb-p are the polar, ASA mona-np and ASA monb-np are the non-polar solvent accessible surface areas of the folded monomers A and B, respectively, which together make up the dimer in the crystal structure, and ASA dimer-p and ASA dimer-np are the polar and non-polar solvent accessible surface areas, respectively, of the folded dimer. These values were calculated using InterProSurf. 1 10

11 equations: The ΔC p,n2 2M can be predicted using ΔASA p and ΔASA np and the empirically derived ΔC p,n2 2M = x ΔASA np x ΔASA p 13 (S37a) ΔC p,n2 2M = x ΔASA np x ΔASA p 14 (S37b) ΔC p,n2 2M = x ΔASA np x ΔASA p 15 (S37c) ΔC p,n2 2M = x ΔASA np x ΔASA p 5 (S37d) An average ΔC p,n2 2M value of 0.45 ± 0.12 kcal (mol dimer) -1 C -1 was determined based on eqs S37a-d and using predicted ΔASA np and ΔASA p of 1262 Å 2 and 258 Å 2, respectively. 16 Calculation of Thermodynamic Parameters. Assuming a temperature-independent ΔC p, which has been shown to be a reasonable approximation over modest temperature intervals 17 the ΔH as a function of temperature is: H(T) = H(T ref ) + C p (T T ref ) (SA1) where T ref is a reference temperature in degrees Kelvin, and ΔH(T ref ) and ΔH(T) are the change in enthalpy of unfolding at T ref and T, respectively, noting again that T t and ΔH(T ref ) is the same value as ΔH(t ref ). The temperature-dependence for the change in entropy for unfolding, S, is given by: S(T) = S ref + C p ln ( T T ref ) (SA2) The above equations can be combined to give the Gibbs-Helmholtz equation: G(T) = RT ln K(T) = H(T ref ) (1 T ) + C T p [T (T ref ) T ln ( T )] ref T ref (SA5) 11

12 The calculations for figures and tables were performed as follows using information in Table S1. 1. H cal at t ref in cal (mol dimer) -1 was calculated by multiplying h cal (t ref ) by the corresponding. 2. G as a function of temperature was calculated using eq SA5 and the associated H cal and C p.(refer to Table S1). 3. Fractions (f) were calculated by solving the quadratic equations using equilibrium constants for the associated transitions (K 1, K 2 ) calculated as a function of temperature using the corresponding G values. For each temperature, f U (two-state) or f M (three-state) were determined by solving the quadratic equation using the equilibrium constants K (two -state) or K 1 and K 2 (three-state) which were calculated from G at each temperature using equation SA5 and a given protein concentration. For the three-state model, f U was then calculated knowing f U = f M K 2. The fraction of the final species f U (two -state) and f N2 (three-state) were determined knowing they add to As a check, fractions were also calculated for the dimer three-state model using eqs S33a-c, S26a,b and S29a,b using the appropriate fitted or fixed parameters given in Table S1. For the dimer two-state model, fractions can also be calculated by using the values and eqs S5 and S14 where f N2 = (1 α) and f U = α. 12

13 Supplementary Results We used several approaches to assess the uncertainties in monomer stability as well as total protein stability for the three-state fits. Because the ΔC p,n2 2M could not be measured for all SOD1 variants due to low heats of dissociation at low temperature, the data were also fit with the maximum ΔC p,n2 2M determined experimentally (2.2 kcal (mol dimer) -1 C -1 ) 1, 3, 5 corresponding to an estimated upper limit of mutational effects, to evaluate how changes in ΔC p may impact monomer stability. In general, mutations have been found to have little effect on ΔC p for global protein unfolding (ΔC p,n2 2U), 10 and ITC experiments show that ΔC p,n2 2M also varies little upon 4, 7, 11, 12 mutation of SOD1. Highly non-conservative substitutions at buried positions of hydrophobic residues by hydrophilic residues or vice versa have, however, been reported to change ΔC p by up to ~40%. 10 We found that comparable increase in ΔC p,n2 2M (by ~0.6 kcal (mol dimer) -1 C -1 with simultaneous decrease of ΔC p,m U by 0.3 kcal (mol monomer) -1 C -1 to keep ΔC p,n2 2U constant as 3.3 kcal (mol dimer) -1 C -1 ) has relatively small effects on ΔG M U calculated at the t avg of 51.2 C (on average ±0.1 kcal (mol monomer) -1 ) and at 37 C (on average ±0.2 kcal (mol monomer) -1 ). Because dimer dissociation was measured at 37 C, changes in ΔC p,n2 2M have no effect on ΔG N2 2M(37 C), whereas ΔG N2 2M(t avg ) is decreased by ~0.2 kcal (mol dimer) -1. Thus, for three-state fitting of DSC data, treating ΔC p as a constant is further substantiated as reasonable, and changes in ΔC p have little impact on monomer stability. We also confirmed that potential aggregation at high temperature, which we also examined previously, 18 has little effect on fitted values, by varying the amounts of fitted data beyond the peak of the unfolding endotherm from a maximum of the apparent end of the endotherm peak to a minimum of ~25% of the high temperature side of the endotherm. Fitting 13

14 various amounts of the endotherm to the three-state model has little impact on total stability: ± kcal (mol dimer) -1 at t avg, and ± kcal (mol dimer) -1 at 37 C. Also, similar stability values were obtained when ΔH vh and ΔH cal were set to equal to each other (Table S1). Based on these analyses, effects of aggregation can be effectively minimized by excluding high temperature data from the fit, with little effect on the measured stability. 14

15 Table S1. Treatment of parameters for DSC data fitting to dimer unfolding models a parameters two-state three-state N 2 2U N 2 2M (1) M U (2) t ref C t 0.5, fit a 37 C t 0.5, fit h cal (t ref ) cal g -1 fit fit fit g mol -1 fit MW dimer MW dimer or fit with 1 = 2 or fit with 1 = 2 A cal g -1 C -1 fit fit na B cal g -1 C -1 fit fit na C cal g -1 C -1 fit A + ΔC p,n2 2M A + ΔC p,n2 2M D cal g -1 C -1 B B B E cal g -1 C -1 na b na C + ΔC p,m U F cal g -1 C -1 na na B c P dimer M dimer fixed fixed fixed For calculations: K ref d C p 2P dimer K ITC 1 kcal (mol dimer) -1 C = 4P dimer (f U ) 2 + K(f U ) - K 4P dimer (f M ) 2 + K 1 (1+K 2 )f M K 1 f U = f M K 2 1 = f N2 + f U f N2 + f M + f U a The parameters for global fits using the dimer two-state or the dimer three-state with monomeric intermediate models were set to defined values or allowed to float and so fit as specified above. b na indicates not applicable. c Protein concentration fixed to values determined by UV absorbance. Although endotherms were normalized by g protein, the fitting equation uses units of M dimer. The molecular weight of SOD1 used for pwt and mutants (MW dimer ) was g/mol. d The K ref is not a fit parameter but is defined for each model. 4,6 15

16 Table S2. Thermodynamic parameters for apo SOD1 determined from global three-state fits using different shared parameters. H N2 2M G N2 2M H t M U G M U G M U G M U SOD1 0.5, M U (kcal (mol (kcal (mol variant a ( C) c (kcal (mol (kcal (mol (kcal (mol (kcal (mol dimer) -1 ) dimer) -1 ) monomer) -1 ) monomer) -1 ) monomer) -1 ) monomer) -1 ) 37 C b 37 C b c,d t avg 37 C e,f d, f d,g t avg t avg pwt c,h na 10.2 ± 0.7 na na 3.4 ± 0.5 na na pwt (30.8 ± 8.8) (10.3 ± 0.5) 59.5 ± ± (3.0, 1.9) 1.2 (1.3, 0.7) na pwt i 8.8 ± ± ± ± na V148I (11.4 ± 2.2) (8.9 ± 0.2) 60.1 ± ± (5.7, 4.2) 2.3 (2.4, 1.6) -1.2 V148I i 7.5 ± ± ± ± nd G93S (17.6 ± 4.6) (8.4 ± 0.3) 49.2 ± ± (2.0, 1.5) -0.4 (-0.3, -0.6) 1.5 H46R (16.2 ± 4.4) (8.4 ± 0.4) 62.5 ± ± (5.4, 4.7) 2.6 (2.6, 2.3) -1.4 H46R i 25.0 ± ± ± ± nd E100G (16.0 ± 4.8) (8.0 ± 0.4) 48.0 ± ± (1.6, 2.1) -0.5 (-0.4, -0.3) 1.7 G37R (7.8 ± 1.8) (7.6 ± 0.2) 50.3 ± ± (1.8, 3.0) -0.2 (-0.3, -0.3) 1.4 G37R i 42.7 ± ± ± ± nd H43R (23.0 ± 1.4) (7.5 ± 0.0) 47.6 ± ± (1.7, 1.9) -0.6 (-0.6, -0.6) 1.8 G93A (14.0 ± 2.0) (7.2 ± 0.3) 47.4 ± ± (1.7, 1.7) -0.7 (-0.6, -0.7) 1.9 G93A i 22.0 ± ± ± ± nd I113T (30.2 ± 2.5) (7.1 ± 0.2) 46.7 ± ± (1.5, 1.7) -0.7 (-0.6, -0.7) 1.9 I113T i 27.2 ± ± ± ± A4T (39.2 ± 3.8) (7.1 ± 0.2) 43.6 ± ± (0.9, 1.3) -1.1 (-1.0, -1.1) 2.3 A4T i 13.7 ± ± ± ± nd A4S (9.0 ± 2.6) (7.0 ± 0.0) 46.3 ± ± (1.1, 1.0) -0.9 (-1.2, -1.3) 2.1 A4S i 46.5 ± ± ± ± nd 16

17 G93R (45.6 ± 1.8) (6.7 ± 0.1) 49.1 ± ± (3.1, 2.9) -0.6 (-0.5, -0.5) 1.7 A4V (37.2 ± 3.8) (6.4 ± 0.3) 50.9 ± ± (2.2, 2.4) -0.1 (-0.1, -0.1) 1.2 A4V i 35.9 ± ± ± ± nd V148G (50.6 ± 1.4) (5.9 ± 0.3) 48.6 ± ± (2.3, 2.1) -0.5 (-0.5, -0.5) 1.7 V148G i 54.8 ± ± ± ± nd na, not applicable; nd, not determined. a For each mutant, the scans at different protein concentrations used in the global fitting are those listed in Table 1, with the exception of pwt, where concentrations 0.20, 0.21, 0.40, 0.44, 0.85, 1.50, and 3.0 were fit. b Numbers in the brackets were determined by ITC and fixed in the DSC three-state fits. c Errors are the uncertainty in fitted values. d t avg is 51.2 C, the average of all t 0.5 values obtained from the two-state fits (Table 1). e ΔG M U values calculated at physiological temperature. f Values are determined from fits allowing ΔH vh /ΔH cal to vary. Monomer stability was also determined using a higher ΔC p,n2 2M (2.2 kcal (mol dimer) -1 C -1 ), and these values are the first values shown in brackets. Data were also fit with ΔH vh and ΔH cal set equal, and these are the second values shown in brackets. Uncertainties in monomer stability were approximated from the range of values obtained from these 3 different fitting procedures (Table 2). g ΔΔG = ΔG pwt - ΔG mutant, a positive value indicates lower stability of the mutant relative to pwt; values are calculated at t avg, where monomer stability is best defined. h ΔG N2 2M and ΔG M U were also determined by globally fitting urea denaturation curves at 37 C to a three-state model with monomer intermediate. i ΔG M U values obtained by fitting additional parameters ΔH N2 2M and ΔG N2 2M (ie. not fixing these parameters to the values obtained by ITC) and setting ΔH vh and ΔH cal equal, for mutants with more than 3 datasets. This fitting method returns values with high uncertainty; therefore, the values from the fixed fits give more reliable comparison of relative stabilities. 17

18 Table S3. Thermodynamic parameters for monomer two-state unfolding of apo SOD1. SOD1 variant [SOD1] (mg ml -1 ) t 0.5 ( C) a C p,m U (kcal (mol monomer) -1 ) H vh (t 0.5 ) (kcal (mol monomer) -1 ) a A4V ± ± 10.6 A4V ± ± 7.9 A4V ± ± 5.1 A4V ± ± 2.5 A4V ± ± 4.4 A4V ± ± 2.6 H46R ± ± 18.8 H46R ± ± 6.6 H46R ± ± 2.2 H46R ± ± 4.7 H46R ± ± 2.5 V148G ± ± 4.3 V148G ± ± 0.6 V148G ± ± 0.3 V148G ± ± 0.2 V148G ± ± 0.1 V148G ± ± 0.8 a Uncertainty estimates in fitted parameters are from the fitting program. 18

19 Figure S1. Representative raw ITC data obtained at 37 C for SOD1 mutants. (A) G93R, (B) H43R and (C) I113T. Each peak represents the measured heat for a small volume injection of protein solution into the ITC sample cell. The heat associated with each injection (q i ) was determined by integrating the power versus time trace. Data were fit to a dimer dissociation model 10, 19, 20 according to q i = V H d ([M i ] [M i 1 ] (1 v V ) f m [M o ] v V ) + q dil, where ΔH d is the enthalpy change of dissociation from native dimer to two monomers, calculated per mol monomer. [M] o is the total concentration of apo SOD1 (monomer units) in the syringe, [M i ] and [M i-1 ] are the concentrations of apo SOD1 monomer in the ITC cell after injection i and i-1, respectively, v is the volume of each injection, V is the ITC reaction cell volume, q dil is a correction factor for the heat associated with sample dilution unrelated to dissociation, and f m is the fraction of protein in the syringe that exists as free monomer, which can be expressed as 19

20 f m = 1 4[M 0 ] ( K d + K d 2 + 8K d [M o ]). The data were fit using Microcal Origin 7.0 (Microcal Inc) with ΔH d, K d and q dil as floating parameters. 20

21 Figure S2. Plots of lnp dimer versus 1/T 0.5 used to determine molecularity, n, for apo SOD1 variants. The lnp dimer values are plotted versus 1/T 0.5 values from the dimer two-state fits for a representative set of apo variants (Table 1), and fit to a straight line using linear regression. Note that the midpoint of the thermal unfolding transition is a relatively well defined experimental value that is affected little by fitting to different unfolding models. The values of slope from these linear fits were used to determine n (summarized in Table 1) using eq 2, as described in the Material and Methods. Values of n are related to the inverse of the slope values. In this plot, the data for H46R and A4V have the steepest slopes, and hence the lowest average n values of ~1.3 and ~1.6, respectively, consistent with these mutants having higher populations of monomer (Figure 7). The lower slopes for the other SOD1 variants correspond to higher n values approaching ~2, consistent with predominantly dimer unfolding. When molecularities were calculated using H cal similar trends were observed but there was more scatter in the data, likely relating to the typically higher experimental error in H cal. 21 Taken together, the molecularity analyses are consistent with dimer unfolding with varying levels of monomer, in agreement with trends in t 0.5 values with protein concentration (Figure 3, Table 1). 21

22 Figure S3. Three-state thermal denaturation of apo SOD1. The parameters for each transition in the total heat of unfolding (black curve) can be used to simulate endotherms for two separate protein transitions, dimer dissociation (red curve) and monomer unfolding (blue curve). In the three-state global fitting approach used here, K 1 (same as K d,n2 2M) and h cal1 (same as h caln2 2M ) which characterize dimer dissociation, were set to the values determined by ITC at 37 C. The slopes of the monomer intermediate and unfolded monomer baselines were set equal to that of the native baseline, making the common assumption that ΔC p of unfolding is 4, 22 temperature independent. The intercepts of the intermediate and unfolded baselines were defined relative to the intercept of the native baseline (solid grey line) according to temperatureindependent values for ΔC p,n2 2M and ΔC p,m U (see Materials and Methods). Thus, the unfolded monomer and dimer baselines, grey dashed and dotted lines respectively, were defined based on C p,n2 2M 1.7 kcal (mol dimer) -1 C -1 and C p,m U 1.6 kcal (mol dimer) -1 C -1 ; The only floating parameters were t ref-2 and h cal2 (t ref 2 ) (same as h calm U (t ref 2 )), β1 = β2, and parameters defining the intercept (A) and slope (B) of the native baselines (solid grey line). Note, because of the way the baselines are defined, the blue and red traces do not add to make black as they do in Figure 7 with baselines subtracted. 22

23 SUPPORTING INFORMATION REFERENCES (1) Sturtevant, J. M. (1987) Biochemical Applications of Differential Scanning Calorimetry, Annu. Rev. Phys. Chem, (2) Shriver, J. W., and Edmondson, S. P. (2009) Defining the stability of multimeric proteins, Methods Mol Biol 490, (3) Becktel, W. J., and Schellman, J. A. (1987) Protein stability curves, Biopolymers 26, (4) Stathopulos, P. B., Rumfeldt, J. A., Karbassi, F., Siddall, C. A., Lepock, J. R., and Meiering, E. M. (2006) Calorimetric analysis of thermodynamic stability and aggregation for apo and holo amyotrophic lateral sclerosis-associated Gly-93 mutants of superoxide dismutase, J Biol Chem 281, (5) Makhatadze, G. I., and Privalov, P. L. (1995) Energetics of protein structure, Adv Protein Chem 47, (6) Tamura, A., Kojima, S., Miura, K., and Sturtevant, J. M. (1995) A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. II. Replacements at the interface of dimer formation, Val13, J Mol Biol 249, (7) Prabhu, N. V., and Sharp, K. A. (2005) Heat capacity in proteins, Annu Rev Phys Chem 56, (8) Gomez, J., Hilser, V. J., Xie, D., and Freire, E. (1995) The heat capacity of proteins, Proteins 22, (9) Privalov, P. L., and Makhatadze, G. I. (1990) Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects, J Mol Biol 213,

24 (10) Broom, H. R., Rumfeldt, J. A., Vassall, K. A., and Meiering, E. M. (2015) Destabilization of the dimer interface is a common consequence of diverse ALS-associated mutations in metal free SOD1, Protein Sci. (11) Robic, S., Guzman-Casado, M., Sanchez-Ruiz, J. M., and Marqusee, S. (2003) Role of residual structure in the unfolded state of a thermophilic protein, Proc Natl Acad Sci U S A 100, (12) Gribenko, A. V., Keiffer, T. R., and Makhatadze, G. I. (2006) Amino acid substitutions affecting protein dynamics in eglin C do not affect heat capacity change upon unfolding, Proteins 64, (13) Spolar, R. S., Livingstone, J. R., and Record, M. T., Jr. (1992) Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water, Biochemistry 31, (14) Murphy, K. P., and Freire, E. (1992) Thermodynamics of structural stability and cooperative folding behavior in proteins, Adv Protein Chem 43, (15) Myers, J. K., Pace, C. N., and Scholtz, J. M. (1995) Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding, Protein Sci 4, (16) Negi, S. S., Kolokoltsov, A. A., Schein, C. H., Davey, R. A., and Braun, W. (2006) Determining functionally important amino acid residues of the E1 protein of Venezuelan equine encephalitis virus, J Mol Model 12, (17) Rumfeldt, J. A., Galvagnion, C., Vassall, K. A., and Meiering, E. M. (2008) Conformational stability and folding mechanisms of dimeric proteins, Prog Biophys Mol Biol 98,

25 (18) Loladze, V. V., Ermolenko, D. N., and Makhatadze, G. I. (2001) Heat capacity changes upon burial of polar and nonpolar groups in proteins, Protein Sci 10, (19) Burrows, S. D., Doyle, M. L., Murphy, K. P., Franklin, S. G., White, J. R., Brooks, I., McNulty, D. E., Scott, M. O., Knutson, J. R., Porter, D., and et al. (1994) Determination of the monomer-dimer equilibrium of interleukin-8 reveals it is a monomer at physiological concentrations, Biochemistry 33, (20) Velazquez-Campoy, A., Leavitt, S. A., and Freire, E. (2004) Characterization of proteinprotein interactions by isothermal titration calorimetry, Methods Mol Biol 261, (21) Tamura, A., and Sturtevant, J. M. (1995) A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. I. Hydrophobic replacements at the position of Met103, J Mol Biol 249, (22) Privalov, P. L., and Potekhin, S. A. (1986) Scanning microcalorimetry in studying temperature-induced changes in proteins, Methods Enzymol 131,