Engineering a Thermo-Stable Superoxide Dismutase Functional at Sub-Zero to >50 C, Which

Engineering a thermo-stable superoxide dismutase functional at sub-zero to >50 °C, which also tolerates autoclaving

Arun Kumar1, Som Dutt1, Ganesh Bagler1, Paramvir Singh Ahuja1, Sanjay Kumar1*

1Biotechnology Division, CSIR- Institute of Himalayan Bioresource Technology (Council of Scientific and Industrial Research), Palampur-176 061, Himachal Pradesh, India. Tel,: +91-1894- 233339; Fax: +91-1894-230433.

*Correspondence and requests for materials should be addressed to S.K.: [email protected]; [email protected].

1 Supplementary Figure S1. Effect of mutation and autoclaving on the activity of superoxide dismutase. Un-autoclaved and autoclaved protein samples of WT and mutants were loaded onto

10% native-polyacrylamide gel and stained for activity. Names of the protein samples are shown at the top of each panel whereas specific activities are given at the bottom of the panel. Enzyme activity was expressed as unit/mg of protein. Values were mean ± SE of three separate replicates.

ND, not detectable. Mutants were created as described essentially in the materials and methods section, except the primers. The details of primers and PCR conditions used for creating mutants are given in Supplementary Table S4. WT, wild type; G4A, glycine at position 4 substituted with alanine; G4I, glycine at position 4 substituted with isoleucine; C56A, cysteine at position 56 substituted with alanine; C95A, cysteine at position 95 substituted with alanine; L132E, leucine at position 132 substituted with glutamic acid; S135K, serine at position 135 substituted with lysine; C145A, cysteine at position 145 substituted with alanine. 2 Supplementary Figure S2. POLYVIEW analysis with details of chemical property, secondary structure, relative solvent accessibility, protein complex interface etc. for 2Q2L

(chain A, shown in panel a; chain B shown in panel b) of native (wild type, WT) superoxide dismutase. Prediction of interface residues with the POLYVIEW with a threshold of 4% relative difference in surface exposed area using Relative Solvent Accessibility (RSA) index. The numerical labels associated with grey scale bar indicate completely buried state, 0 (for 0-9%

RSA) and fully exposed state, 9 (for 90-100% RSA).

3 Supplementary Figure S3. Structure of native superoxide dismutase depicting the interface residues (shown as magenta and yellow dotted spheres) and Cys-95 residue (depicted as a red sphere). Cys-95 emerged as a critical residue in the interface residue interaction sub- network (cluster) via long- and short-range interactions.

4 Supplementary Figure S4. Structure of native superoxide dismutase illustrating the residue interaction network (in chain A) and the ‘Critical Interaction Residue Clique (CIRC)’ identified by the graph-theoretical analysis. The CIRC is highlighted as spheres. Interactions among the CIRC residues, with long-range inter-secondary structure interactions, emerge critical for holding the tertiary structure together.

5 Supplementary Figure S5. Activity staining for SOD as analyzed as in Fig. 1a. SOD analyzed as in Fig. 1a was stained for SOD activity for which SDS-PAGE was performed essentially as described except that protein mixed with the gel loading buffer was not boiled.

Methods section has all the details. The name of sample is given above top of each panel. SOD activity was not detected for C56A and C145A. WT, wild type; C56A, cysteine at position 56 substituted with alanine; C95A, cysteine at position 95 substituted with alanine; C145A, cysteine at position 145 substituted with alanine. Induced proteins are marked by arrow.

6 Supplementary Figure S6. Mass spectrometric analysis of the monomeric SOD. Purified monomer fraction (Fig. 1b), as also analyzed in Fig. 1c, was used for mass analysis as described in Supplementary Methods section. Data evidently showed the presence of a protein with only one molecular mass of 17.39 kDa.

7 Supplementary Figure S7. Expression of WT, C95A, C56A and C145A in E.coli. Proteins were induced using 1mM IPTG as described in Methods section and analyzed on SDS-PAGE.

Samples were harvested at 0h, 1h, 2h, 3h, 4h, and 5h of induction as shown on each panel. M, molecular weight marker procured from Amersham, UK; the protein markers were- phosphorylase b (97 000 Da), albumin (66 000 Da), ovalbumin, 45 000 Da), carbonic anhydrase

(30 000 Da), Trypsin inhibitor (20 100 Da), and α-lactalbumin (14 400 Da). WT, wild type;

C56A, cysteine at position 56 substituted with alanine; C95A, cysteine at position 95 substituted with alanine; C145A, cysteine at position 145 substituted with alanine. Induced proteins are marked by arrow.

8 Supplementary Figure S8. Effect of pH on SOD activity. Per cent relative activity with respect to maximum at pH 7.8 was plotted. SOD activity was not detected for C56A and C145A, and hence corresponding activity curves are not shown. Data represents mean ± standard error of mean of three individual replicates. WT, wild type; C95A, cysteine at position 95 substituted with alanine.

9 Supplementary Figure S9. Effect of incubation of SOD with proteolytic enzymes. SOD was incubated in the absence (-) and presence (+) of 1/20 w/w of trypsin, chymotrypsin or papain in

50 mM potassium phosphate buffer, pH 7.8, and 37 ºC for 1, 2, 3h for WT, and for 1, 2, 3, and

24h for C95A, C56A, and C145A and run on a SDS-PAGE. Panel “a” shows the full-length gels used for Fig. 4a in which data for 24 h was not shown, and hence the lane (24h) was cropped. M,

10 molecular weight marker procured from Fermentas, USA; the protein markers were- β- galactosidase (11 6000 Da), bovine serum albumin (66 200 Da), Ovalbumin, 45 000 Da), lactate dehydrogenase (35 000 Da), REase Bsp981 (21 000), β- lactoglobulin (18 400) and lysozyme

(14 400Da)].

Activity staining of protease treated SODs is shown in panel “b”. Protease treated SODs were run on native rather than SDS containing poly-acrylamide gel. Panel “b” shows the full-length gels used for Fig. 4b in which data for 24 h was not shown, and hence the lane (24h) was cropped. Abbreviation “T” represents trypsin.

11 Supplementary Table S1. Thermal inactivation rate constant (kd), half-time of thermal

2 inactivation (t1/2), linear correlation coefficient (r), and regression coefficient (R ) values of

WT and C95A. The enzymes were incubated at 80 °C for 160 min as detailed in methods section entitled “Thermal inactivation assay”. The section also details on calculation of parameters related to first order thermal inactivation kinetics. The kinetic data supplement Fig. 3.

Values are mean ± SE of three separate replicates. WT, wild type; C95A, cysteine at position 95 substituted with alanine.

-1 2 Enzyme kd (min ) t1/2 (min) r R WT 4.6 ± 0.18 × 10-3 151 ± 6 - 0.97 0.98 C95A 2.4 ± 0.31 × 10-3 345 ± 36 - 0.98 0.96

12 Supplementary Table S2. Content (in %) of secondary structural elements in WT and mutant enzymes at 4 °C. CD spectra for un-autoclaved and autoclaved WT and mutant enzymes were determined at a concentration of 0.2 mg/ml in 50 mM potassium phosphate buffer

(pH, 7.8) with a Jasco-815 spectropolarimeter at 4 °C. Un, un-autoclaved protein samples;

Auto, autoclaved samples. Values in parentheses represent percent loss (-)/gain (+) in secondary structure with respect to their un-autoclaved samples. ND represents those cases wherein initial secondary structural elements were zero. WT, wild type; C56A, cysteine at position 56 substituted with alanine; C95A, cysteine at position 95 substituted with alanine; C145A, cysteine at position 145 substituted with alanine.

Sample % α-helix % β-sheets % turns % randomness Un Auto Un Auto Un Auto Un Auto WT 10.3 0 (- 100) 57 45.5 (- 20.2) 0 8.3 (ND) 32.7 46.2 (+ 29.2) C56A 1.4 0 (- 100) 61.7 40.3 (- 34.7) 1.3 12.3 (+ 89.4) 35.6 47.4 (+ 24.9) C95A 9.2 0.9 (- 90.2) 55.8 57.2 (+ 2.5) 0 2.1 (ND) 35.1 39.9 (+ 12.0) C145A 4.8 0 (- 100) 51.3 31.8 (- 38.0) 6.2 15.6 (+ 60.3) 37.7 52.6 (+ 28.3)

13 Supplementary Table S3. Energy values observed using Build Mutant tool of Discovery

Studio suite (Accelerys, USA). Total 15 models were built for each protein. The energy values given in the table are of the model with the lowest values for each protein. The parameters used are: Number of models, 15; start model index, 1; optimization level high, use dope score method, true; cut radius, 4.5. PDF, probability density function; DOPE, Discrete Optimized Protein

Energy. Values in parentheses are per cent change with respect to WT values. Positive and negative values show increase and decrease, respectively. WT, wild type; C56A, cysteine at position 56 substituted with alanine; C95A, cysteine at position 95 substituted with alanine;

C145A, cysteine at position 145 substituted with alanine.

Protein PDF total Energy PDF Physical Energy DOPE Score WT 6331.73 7675.76 28660.05

C56A 8405.05 (32.74) 7699.31 (0.31) 28732 (0.25)

C95A 6285.34 (-0.73) 7636.06 (-0.52) 28413.20 (-0.86)

C145A 8403.69 (32.72) 7686.26 (0.14) 28660.57 (0.00)

14 Supplementary Table S4. Oligonucleotide sequences and PCR conditions used in the present work. aPrimers with suffix “F” and “R” represents forward and reverse primers, respectively. bNucleotides in lower case letters represent the site of mutation.

Namea Sequence (5’ – 3’)b PCR conditions

C56A F 5’-GACACAACCAATGGTgcCATGTCAACTGGACC-3’ Initial denaturation: 95 C56A R 5’-GGTCCAGTTGACATGgcACCATTGGTTGTGTC-3’ C95A F 5’-GATGACGGAACTGCTgcCTTCACAATTGTTGAC-3’ ºC, 30 s followed by 16 C95A R 5’-GTCAACAATTGTGAAGgcAGCAGTTCCGTCATC-3’ C145A F 5’-GTGGCAGGATAGCTgcTGGTATTATTGGCCTTC-3’ cycles of: 95ºC, 30 s; C145A R 5’-GAAGGCCAATAATACCAgcAGCTATCCTGCCAC-3’ 65ºC, 30 s; 68 ºC, 9 min. G4A F 5’-ATGGCAAAGgccGTTGCTGTACTTAGCTCC-3’ G4A R 5’-GGAGCTAAGTACAGCAACggcCTTTGCCAT-3’ Final extension was G4I F 5’-ATGGCAAAGatcGTTGCTGTACTTAGCTCCAGT-3’ G4I R 5’-ACTGGAGCTAAGTACAGCAACgatCTTTGCCAT-3’ performed at 68 ºC for L132E F 5’-AAGGGTGGACATGAGgaaAGCAAATCCACTGGA-3’ L132E R 5’-TCCAGTGGATTTGCTttcCTCATGTCCACCCTT-3’ 14 min. S135K F 5’-CATGAGCTTAGCAAAaagACTGGAAATGCTGGTGGC-3’ S135K R 5’-GCCACCAGCATTTCCAGTcttTTTGCTAAGCTCATG-3’

Supplementary Methods:

Construction of coarse-grained residue interaction graph (RIG) and long-range interaction network (LIN) Models:

Coarse-grained RIG model1, representing a network of residues that are in contact (proximity) with each other in 3D space, was constructed for the monomer A of WT native structure (PDB

ID: 2Q2L). A threshold of 8Å, a distance in optimal range that encapsulates noncovalent interactions within the structure, was used as contact threshold. Further a LIN model, that depicts the tertiary contacts between residues that are distant (|i-j|>=12) along the backbone, was built.

Identification of “Critical interface residue clique (CIRC)”:

CIRC, a subset of residues that form cliques (a group of well-connected and interlinked residues), was identified in the LIN model. First, the residues making maximum number of long- range spatial contacts were identified. Thereafter, clique of these rich interacting residues, potentially important for structure, was elucidated. 15 Identification of interface residues:

POLYVIEW server2 was used for identification of residues at the interface of 2Q2L dimer complex. Interface residues were identified using a threshold of 4% relative difference in surface exposed area with Relative Solvent Accessibility (RSA) index.

Amino terminal sequencing: The purified protein was subjected to electrophoresis on 15%

SDS-PAGE. Protein bands in the gel were transferred to a polyvinylidene fluoride membrane, stained with Coomassie Brilliant Blue R-250 and subjected for N-terminal sequencing by automated Edman degradation3 using protein sequencer (Procise Model 491 from Applied

Biosystems, Switzerland) following the Manufacturer’s instructions.

Mass determination: Purified monomer fraction (Fig. 1 c), as also analyzed in Fig. 1d, was used for mass analysis. The sample was processed using ZipTip pipette tips (Millipore, USA) and spotted along with 3,5-dimethoxy-4-hydroxycinnamic acid matrix (10 mg/ml solution with

60% acetonitrile and 0.1% trifluoroacetic acid) on to the target plate as per the manufacturer’s instruction. Analysis was performed using a MALDI-ToF, AXIMA-CFR Plus spectrometer

(Shimadzu-Kratos, UK) in linear mode. A mixture of calibration proteins standards (CAL-1;

Sigma, USA) was used for external calibration. The molecular weight was also determined in silico by using free online software, www.expasy.org.

Determination of pH optima. In order to determine pH optima of enzymes, separate reaction mixtures for carrying out enzyme assay were prepared by using different buffers of pH values: potassium phosphate buffer (pH 6, 7, 7.5, 7.8), or 0.05 carbonate - bicarbonate buffer (pH 9).

Enzyme assay was carried out using different pH mixtures at 4 °C. Separate controls were used for each pH values where, all the components were same except equal amount of buffer was added to reaction medium instead of enzyme. Enzyme activity was calculated as percent

16 inhibition in reduction of nitro blue tetrazolium (NBT) with respect to the control at respective pH.

Supplementary References

1. Gromiha, M. M. & Selvaraj, S. Inter-residue interactions in protein folding and stability.

Prog. Biophys. Mol. Biol. 86, 235-277 (2004).

2. Porollo, A., Adamczak, R. & Meller, J. POLYVIEW: a flexible visualization tool for

structural and functional annotations of proteins. Bioinformatics 20, 2460-2462 (2004).

3. Oury, T. D., Crapo, J. D., Valnickova, Z. & Enghild, J. J. Human extracellular superoxide

dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-

yield purification of extracellular superoxide dismutase. Biochem. J. 317, 51-57 (1996).