A Single Outer Sphere Mutation Stabilizes apo- Mn Superoxide Dismutase by 35 C and. Disfavors Mn Binding.

Transcription

1 Supporting information for A Single Outer Sphere Mutation Stabilizes apo- Mn Superoxide Dismutase by 35 C and Disfavors Mn Binding. Anne-Frances Miller* and Ting Wang Department of Chemistry, University of Kentucky, Lexington Kentucky, 40506, United States S 1

2 Table S1. Melting temperatures and associated thermodynamic parameters. a WTholoMn 2+ SOD T m b C ΔH kj/mol ΔS J/mol K 67.4 ± ± ± ± ± ± ± ± ±700 WTapoMnSOD Q146EholoMnSOD Q146EapoMnSOD 87.9 ± ± ±100 a Thermodynamic parameters for each transition were extracted using the van't Hoff analysis provided in the CDpro software (JASCO). In brief: ΔG = ΔH -TΔS (eq. S1) and ΔG = -RT ln(k eq ) (eq. S2). Solving eq. 2 for ln(k eq ) we have ln(k eq ) = -ΔG /RT = -ΔH /RT +ΔS /R (eq. S3). This linear form of the van't Hoff equation can applied to DSC data that have been treated to account for the slope of the baseline before and after the transition being characterized. Once this has been done, each value of the ellipticity reveals the fraction of the sample that has undergone unfolding (f u ) and the fraction remaining in the folded form (f f, for the example of a transition that corresponds to unfolding). Thus each θ 222 vs. temperature point corresponds to a value of K eq = f u /f f and the temperature, T. A replot of ln(k eq ) vs. 1/T ideally conforms to a straight line for which the slope is -ΔH /R and the intercept is ΔS /R, yielding estimates of ΔH and ΔS which are valid only so long as they do not change significantly within the temperature range of the transition and applicable to that transition near its T m. S 2

3 For the WT, the enthalpies are comparable to published values of 430 kj/mol for interleukin-4, kj/mol for RNase A and 243 kj/mol for lysozyme, 2 although they are two-fold lower than values for WT-MnSOD determined calorimetrically. 3 The entropies of unfolding are also comparable to published values, of -790 J/mol K and -530 J/mol K for RNase A and lysozyme, respectively. For Q146E, our data indicate that the T m of 88 C describes apomn-protein whereas the event near 49 C pertains to Q146E-holoMnSOD. Q146E-apoMnSOD displays more than doubling of both the enthalpic stabilization of the folded state and the change in entropy associated with unfolding, compared to WT-apoMnSOD. Direct comparison with WT is complicated by the fact that these parameters apply at a much higher temperature, however the much larger ΔH in conjunction with the structurally conservative nature of the mutation 4 suggests that Glu at position 146 benefits from favourable electrostatic interactions with a nearby His in folded apomnsod, 5 given that charge is the major distinction between Glu and Gln in apomnsod. It is appealing to propose that a Glu - His + salt bridge forms in the mutant and that protonation of the participating His has the effect of diminishing the site's affinity for Mn 2+. Both the other two His could be protonated even in WT-apoMnSOD due to interactions with Asp167 and Glu170.B, respectively. Thus we speculate that mutation of Gln146 to Glu could cause protonation of the last neutral His ligand and thereby create a larger proton displacement barrier to metal ion acquisition. b Standard errors of individual fits averaged 0.6 C, with a maximum of 1.6 C. S 3

4 MnSOD Q146 FeSOD Q69 Figure S1. Ribbon diagram of one monomer of MnSOD showing that Gln146 comes from the C-terminal domain (yellow-red ribbon) whereas the analogous Gln69 of FeSOD derives from the N-terminal domain (violet blue - green). Ligand amino acids and the Tyr and Trp that hydrogen bond with the active site Gln are shown as sticks. The active site Mn ion is a purple sphere and two water molecules in the coordination sphere at cryogenic temperature are provided as red dots. Figure is based on 1D5N.pdb and generated using Chimera. 6 S 4

5 Figure S2. Comparison of circular dichroism signatures of the secondary structure of WT-holoMn-SOD, WT-apoMnSOD and Q146E-apoMnSOD. Neither the Q146E mutation nor Mn binding significantly affects the secondary structure content of MnSOD, based on far-uv CD. S 5

6 20$ 30$ 40$ 50$ 60$ 70$ 80$ 90$ 100$!6000$ Temperature#( C)#!8000$!10000$!12000$!14000$!16000$!18000$!20000$!22000$ θ 222 #(deg cm 2 dmol -1 )# Figure S3. Comparison of CD melts of samples dominated by WT-Mn 2+ SOD vs. WT- reconstituted_wtholomnsod as-isolated_wtholomnsod Mn 3+ SOD. Samples were WT-MnrecSOD (prepared by Mn removal followed by reconstitution with Mn 2+, 0.95 ±.05 Mn/site) or WT-holoMnSOD as-isolated (1.0 ± 0.1 Mn/site). 15 μm SOD (dimers) were equilibrated in 5 mm potassium phosphate buffer at ph 7.4 with 0.8 M GdmCl to prevent protein aggregation, and the temperature was increased from 20 to 100 at 1 /min. At each temperature point (5 C intervals) four spectra were collected and the average value of θ 222 was recorded. Although our apparatus did not permit control of the oxidation state of the Mn ion over the course of the experiments, as-isolated WT-holoMnSOD (substantially Mn 3+ ) displayed a sharp transition above 80 C near the reported calorimetric T m of 90 C whereas freshly-reconstituted WT-holoMnSOD (containing more Mn 2+ ) displayed greater change in θ 222 near the calorimetric T m of 69 C and samples resulting from other S 6

7 treatments or storage displayed results in between. Thus redox heterogeneity provides an explanation for the broad and somewhat variable shapes of the high-temperature portions of the WT-holoMnSOD melting curves, as well as the range of values produced for T m by simple two-state fits. S 7

8 Figure S4. High resolution thermal melts monitored via θ 222. Samples were WTapoMnSOD (prepared by Mn removal from WT-holoMnSOD as per the methods (0.01 ±.01 Mn/site), WT-MnrecSOD (prepared by Mn removal followed by reconstitution with Mn, 0.95 ±.05 Mn/site) or Q146E-apoMnSOD prepared by denaturation followed by reconstitution with Mn (0.13 ±.01 Mn/site). 15 μm SOD (dimers) were equilibrated in 5 mm potassium phosphate buffer at ph 7.4 with 0.8 M GdmCl to prevent protein aggregation, and the temperature was increased from 20 to 100 at 1 /min, θ 222 was measured with a time constant of 1 s at 1 C intervals. The thermodynamic parameters describing protein thermal denaturation assuming a two-state process N U were extracted using the van t Hoff analysis method provided by CDpro (Jasco Co) and are reported in Table S1, above. WT- apomnsod WT- holomnsod Q146E-.1Mn/site S 8

9 Temperature)( C))!7000% 20%!9000% 30% 40% 50% 60% 70% 80% 90% 100%!11000% Ellip%city)Θ 222 )(deg cm 2 dmo l31 ))!13000%!15000%!17000%!19000%!21000%!23000%!25000%!27000% 0.1 Mn/site 0.01Mn/site <0.005 Mn/site Figure S5. Comparison of melts of Q146E-MnrecSOD vs. Q146-apoMnSOD using absolute vertical scale. Temperature-induced unfolding was compared among samples of Q146E-MnSOD containing Mn in different fractions of the active sites. Samples were Q146E-apoMnSOD as-isolated ( 0.02 Mn/site, 0.03 Mn/site) and Q146E-apoMnSOD prepared by denaturation followed by reconstitution with Mn (0.13 ±.01 Mn/site). Samples contained 15 μm of Q146E-SOD (dimers) in 5 mm potassium phosphate buffer at ph 7.4 with 0.8 M GdmCl to prevent protein aggregation, and the temperature was increased from 20 to 100 at 1 /min. For data collected every 5 degrees, four spectra were collected and averaged at each temperature. For data collected every one degrees, θ 222 was measured with a time constant of 1 s. S 9

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