Mechanism and thermodynamics of guanidinium chloride-induced denaturation of ALS-associated mutant Cu,Zn superoxide dismutases.


Abstract

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. Study holds ProTherm entries: 18920, 18921, 18922, 18923, 18924, 18925, 18926, 18927, 18928, 18929, 18930, 18931, 18932, 18933, 18934, 18935, 18936, 18937, 18938 Extra Details: dG_H2O data are in units of kcal/mol.dimer; m in kcal/mol.dimer/M superoxide dismutase; amyotrophic lateral sclerosis; protein stability and folding; dimeric protein; guanidine hydrochloride denaturation

Submission Details

ID: YvZdZji63

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Rumfeldt JA;Stathopulos PB;Chakrabarrty A;Lepock JR;Meiering EM,J. Mol. Biol. (2006) Mechanism and thermodynamics of guanidinium chloride-induced denaturation of ALS-associated mutant Cu,Zn superoxide dismutases. PMID:16307756
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2MP3 2015-05-20 Truncated L126Z-sod1 in DPC micelle
1L3N 2002-05-08 The Solution Structure of Reduced Dimeric Copper Zinc SOD: the Structural Effects of Dimerization
1KMG 2002-10-02 The Solution Structure Of Monomeric Copper-free Superoxide Dismutase
2LU5 2012-06-27 Structure and chemical shifts of Cu(I),Zn(II) superoxide dismutase by solid-state NMR
1DSW 2000-03-22 THE SOLUTION STRUCTURE OF A MONOMERIC, REDUCED FORM OF HUMAN COPPER, ZINC SUPEROXIDE DISMUTASE BEARING THE SAME CHARGE AS THE NATIVE PROTEIN
1BA9 1998-09-16 THE SOLUTION STRUCTURE OF REDUCED MONOMERIC SUPEROXIDE DISMUTASE, NMR, 36 STRUCTURES
2AF2 2005-11-15 Solution structure of disulfide reduced and copper depleted Human Superoxide Dismutase
1RK7 2003-12-02 Solution structure of apo Cu,Zn Superoxide Dismutase: role of metal ions in protein folding
2NAM 2016-12-14 Full-length WT SOD1 in DPC MICELLE
4A7U 2012-11-28 0.98 Structure of human I113T SOD1 complexed with adrenaline in the p21 space group.
2WYT 2010-10-27 1.0 1.0 A resolution structure of L38V SOD1 mutant
4A7V 2012-11-28 1.0 Structure of human I113T SOD1 mutant complexed with dopamine in the p21 space group
1MFM 1999-04-21 1.02 MONOMERIC HUMAN SOD MUTANT F50E/G51E/E133Q AT ATOMIC RESOLUTION
4A7S 2012-12-05 1.06 Structure of human I113T SOD1 mutant complexed with 5-Fluorouridine in the p21 space group
2C9V 2005-12-20 1.07 Atomic resolution structure of Cu-Zn Human Superoxide dismutase
2V0A 2007-06-19 1.15 Atomic resolution crystal structure of Human Superoxide Dismutase
4A7Q 2012-10-24 1.22 Structure of human I113T SOD1 mutant complexed with 4-(4-methyl-1,4- diazepan-1-yl)quinazoline in the p21 space group.
4A7G 2012-10-24 1.24 Structure of human I113T SOD1 mutant complexed with 4-methylpiperazin- 1-yl)quinazoline in the p21 space group.
2C9S 2005-12-15 1.24 1.24 Angstroms resolution structure of Zn-Zn Human Superoxide dismutase
2C9U 2005-12-16 1.24 1.24 Angstroms resolution structure of as-isolated Cu-Zn Human Superoxide dismutase
5J0F 2017-02-01 1.25 Monomeric Human Cu,Zn Superoxide dismutase, loops IV and VII deleted, apo form, circular permutant P4/5
4BCY 2013-02-27 1.27 Monomeric Human Cu,Zn Superoxide dismutase, mutation H43F
4NIO 2013-12-04 1.3 GVTGIAQ segment 147-153 from Human Superoxide Dismutase with I149T mutation associated with a familial form of amyotrophic lateral sclerosis
2VR6 2008-04-15 1.3 Crystal Structure of G85R ALS mutant of Human Cu,Zn Superoxide Dismutase (CuZnSOD) at 1.3 A resolution
1OZU 2003-05-27 1.3 Crystal Structure of Familial ALS Mutant S134N of human Cu,Zn Superoxide Dismutase (CuZnSOD) to 1.3A resolution
5O3Y 2018-06-13 1.3 SOD1 bound to Ebsulfur
2VR8 2008-04-08 1.36 Crystal Structure of G85R ALS mutant of Human Cu,Zn Superoxide Dismutase (CuZnSOD) at 1.36 A resolution
1P1V 2003-08-26 1.4 Crystal Structure of FALS-associated human Copper-Zinc Superoxide Dismutase (CuZnSOD) Mutant D125H to 1.4A
5WMJ 2018-03-28 1.4 KVWGSI segment from Superoxide Dismutase 1,residues 30-35
4NIN 2013-12-04 1.4 DSVISLS segment 101-107 from Human Superoxide Dismutase
3GZQ 2009-10-13 1.4 HUMAN SOD1 A4V Metal-free Variant
4A7T 2012-11-28 1.45 Structure of human I113T SOD1 mutant complexed with isoproteranol in the p21 space group
2XJK 2010-09-01 1.45 Monomeric Human Cu,Zn Superoxide dismutase
2WZ5 2010-12-08 1.5 L38V SOD1 mutant complexed with L-methionine.
5O40 2018-06-13 1.5 SOD1 bound to Ebselen
2XJL 2010-09-01 1.55 Monomeric Human Cu,Zn Superoxide dismutase without Cu ligands
2WZ6 2010-12-08 1.55 G93A SOD1 mutant complexed with Quinazoline.
3H2P 2010-05-05 1.55 Human SOD1 D124V Variant
2VR7 2008-04-15 1.58 Crystal Structure of G85R ALS mutant of Human Cu,Zn Superoxide Dismutase (CuZnSOD) at 1.58 A resolution
5J0C 2017-02-01 1.6 Monomeric Human Cu,Zn Superoxide dismutase, loops IV and VII deleted, apo form, circular permutant P2/3
1UXL 2004-03-19 1.6 I113T mutant of human SOD1
3QQD 2011-03-09 1.65 Human SOD1 H80R variant, P212121 crystal form
1PU0 2003-09-09 1.7 Structure of Human Cu,Zn Superoxide Dismutase
2GBT 2007-01-02 1.7 C6A/C111A CuZn Superoxide dismutase
2WYZ 2010-10-27 1.7 L38V SOD1 mutant complexed with UMP
2WZ0 2010-12-08 1.72 L38V SOD1 mutant complexed with aniline.
3K91 2010-01-19 1.75 Polysulfane Bridge in Cu-Zn Superoxide Dismutase
6FLH 2018-11-07 1.79 Monomeric Human Cu,Zn Superoxide dismutase, SOD1 7+7, apo form
6B79 2018-05-30 1.8 Curved pair of sheets formed from SOD1 residues 28-38 with familial mutation G37R.
1HL5 2003-05-08 1.8 The Structure of Holo Type Human Cu, Zn Superoxide Dismutase
1PTZ 2003-09-09 1.8 Crystal structure of the human CU, Zn Superoxide Dismutase, Familial Amyotrophic Lateral Sclerosis (FALS) Mutant H43R
3T5W 2012-08-01 1.8 2ME modified human SOD1
1HL4 2003-05-08 1.82 The Structure of Apo Type Human Cu, Zn Superoxide Dismutase
3H2Q 2010-05-05 1.85 Human SOD1 H80R variant, P21 crystal form
1N19 2002-11-27 1.86 Structure of the HSOD A4V mutant
3ECV 2009-05-19 1.9 Crystal structure of the ALS-related pathological mutant I113T of human apo Cu,Zn Superoxide Dismutase (SOD1)
5YTO 2018-11-21 1.9 Crystal Structure of human Superoxide Dismutase I (hSOD1) in complex with a napthalene-catechol linked compound.
4NIP 2013-12-04 1.9 GVIGIAQ segment 147-153 from Human Superoxide Dismutase
2ZKW 2009-03-24 1.9 Crystal structure of human Cu-Zn superoxide dismutase mutant G85R in space group P21
5YUL 2018-11-21 1.9 Native Structure of hSOD1 in P6322 space group
5YTU 2018-11-21 1.9 Structure of human SOD1 complexed with isoproteranol in C2221 space group
1AZV 1998-02-25 1.9 FAMILIAL ALS MUTANT G37R CUZNSOD (HUMAN)
3ECU 2009-05-19 1.9 Crystal structure of human apo Cu,Zn Superoxide Dismutase (SOD1)
3CQQ 2008-04-29 1.9 Human SOD1 G85R Variant, Structure II
1UXM 2004-03-19 1.9 A4V mutant of human SOD1
4BCZ 2013-02-27 1.93 Monomeric Human Cu,Zn Superoxide dismutase, loops IV and VII deleted, apo form.
6FFK 2018-11-28 1.94 Human apo-SOD1 bound to PtCl2(1R,2R-1,4-DACH
3CQP 2008-04-29 1.95 Human SOD1 G85R Variant, Structure I
2WKO 2009-11-24 1.97 Structure of metal loaded Pathogenic SOD1 Mutant G93A.
5K02 2016-11-23 1.99 Structure of human SOD1 with T2D mutation
5J07 2017-02-01 2.0 Monomeric Human Cu,Zn Superoxide dismutase, loops IV and VII deleted, apo form, circular permutant P1/2
2GBU 2007-01-02 2.0 C6A/C111A/C57A/C146A apo CuZn Superoxide dismutase
6DTK 2019-06-19 2.0 Heterodimers of FALS mutant SOD enzyme
5IIW 2017-06-28 2.0 Corkscrew assembly of SOD1 residues 28-38 without potassium iodide
1N18 2002-11-27 2.0 Thermostable mutant of Human Superoxide Dismutase, C6A, C111S
6FOI 2019-01-30 2.0 Human Cys57/156Ala superoxide dismutase-1 (SOD1), as isolated.
2GBV 2007-01-02 2.0 C6A/C111A/C57A/C146A holo CuZn Superoxide dismutase
2R27 2007-12-11 2.0 Constitutively zinc-deficient mutant of human superoxide dismutase (SOD), C6A, H80S, H83S, C111S
5DLI 2016-09-14 2.1 Corkscrew assembly of SOD1 residues 28-38
3GZO 2009-10-13 2.1 HUMAN SOD1 G93A Variant
4B3E 2012-09-26 2.15 Structure of copper-zinc superoxide dismutase complexed with bicarbonate.
3ECW 2009-05-19 2.15 Crystal structure of the ALS-related pathological mutant T54R of human apo Cu,Zn Superoxide Dismutase (SOD1)
1OEZ 2003-05-29 2.15 Zn His46Arg mutant of Human Cu, Zn Superoxide Dismutase
3HFF 2009-06-16 2.2 Monomeric human Cu,Zn Superoxide dismutase without Zn ligands
4MCM 2014-08-27 2.2 Human SOD1 C57S Mutant, As-isolated
3GQF 2009-04-07 2.2 Structural and Biophysical Properties of the Pathogenic SOD1 Variant H46R/H48Q
3GTV 2010-09-08 2.2 Human-mouse SOD1 chimera
3RE0 2012-04-25 2.28 Crystal structure of human apo Cu,Zn superoxide dismutase (SOD1) complexed with cisplatin
2NNX 2006-11-07 2.3 Crystal Structure of the H46R, H48Q double mutant of human [Cu-Zn] Superoxide Dismutase
5U9M 2017-05-31 2.35 Copper-Zinc Superoxide Dismutase is Activated through a Sulfenic Acid Intermediate at a Copper-ion Entry Site
4OH2 2014-10-15 2.38 Crystal Structure of Cu/Zn Superoxide Dismutase I149T
2ZKY 2009-03-24 2.4 Crystal structure of human Cu-Zn superoxide dismutase mutant G93A
1SPD 1994-04-30 2.4 AMYOTROPHIC LATERAL SCLEROSIS AND STRUCTURAL DEFECTS IN CU,ZN SUPEROXIDE DISMUTASE
3LTV 2010-09-08 2.45 Mouse-human sod1 chimera
5J0G 2017-02-01 2.5 Monomeric Human Cu,Zn Superoxide dismutase, loops IV and VII deleted, apo form, circular permutant P7/8
1OZT 2003-05-27 2.5 Crystal Structure of apo-H46R Familial ALS Mutant human Cu,Zn Superoxide Dismutase (CuZnSOD) to 2.5A resolution
1SOS 1993-04-15 2.5 ATOMIC STRUCTURES OF WILD-TYPE AND THERMOSTABLE MUTANT RECOMBINANT HUMAN CU, ZN SUPEROXIDE DISMUTASE
6A9O 2019-07-17 2.5 Rational discovery of a SOD1 tryptophan oxidation inhibitor with therapeutic potential for amyotrophic lateral sclerosis
4FF9 2013-09-04 2.5 Crystal Structure of cysteinylated WT SOD1.
6FOL 2019-01-30 2.55 Domain II of the human copper chaperone in complex with human Cu,Zn superoxide dismutase
4MCN 2014-08-27 2.6 Human SOD1 C57S Mutant, Metal-free
2ZKX 2009-03-24 2.72 Crystal structure of human Cu-Zn superoxide dismutase mutant G85R in space group I212121
5WOR 2018-05-30 2.77 Corkscrew assembly of SOD1 residues 28-38 with familial mutation G37R
4BD4 2013-02-27 2.78 Monomeric Human Cu,Zn Superoxide dismutase, loops IV and VII deleted, apo form, mutant H43F
1FUN 1999-07-23 2.85 SUPEROXIDE DISMUTASE MUTANT WITH LYS 136 REPLACED BY GLU, CYS 6 REPLACED BY ALA AND CYS 111 REPLACED BY SER (K136E, C6A, C111S)
6FP6 2019-01-30 3.0 Complex of human Cu,Zn SOD1 with the human copper chaperone for SOD1 in a compact conformation
6FON 2019-01-30 3.05 Elongated conformer of the human copper chaperone for SOD1 complexed with human SOD1
3GZP 2009-10-13 3.1 HUMAN SOD1 G93A Metal-free Variant
3KH3 2010-08-11 3.5 Crystal structure of human Cu/Zn superoxide dismutase recombinantly produced in Leishmania tarantolae; P212121 crystal form containing 12 chains in the asymmetric unit
3KH4 2010-08-11 3.5 Crystal structure of human Cu/Zn superoxide dismutase recombinantly produced in Leishmania tarantolae; P6522 crystal form containing 6 chains in the asymmetric unit
4XCR 2016-01-20 3.6 Monomeric Human Cu,Zn Superoxide dismutase, loops IV and VII deleted, apo form, mutant I35A

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.6 Superoxide dismutase [Cu-Zn] Q8HXQ0 SODC_MACMU
91.6 Superoxide dismutase [Cu-Zn] Q8HXQ2 SODC_MACFU
91.6 Superoxide dismutase [Cu-Zn] Q8HXQ1 SODC_MACFA
92.2 Superoxide dismutase [Cu-Zn] Q8HXP8 SODC_CALJA
93.5 Superoxide dismutase [Cu-Zn] Q8HXP9 SODC_SAPAP
94.2 Superoxide dismutase [Cu-Zn] Q8HXQ3 SODC_HYLLA
95.5 Superoxide dismutase [Cu-Zn] Q8HXQ4 SODC_PONPY
100.0 Superoxide dismutase [Cu-Zn] P60052 SODC_PANTR
100.0 Superoxide dismutase [Cu-Zn] P00441 SODC_HUMAN