Human Cu,Zn superoxide dismutase (SOD1) converts superoxide, a toxic byproduct of oxidative phosphorylation, into water and oxygen in all respiring cells. Tragically, mutations at dozens of positions in SOD1 cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. A prevailing hypothesis for the molecular mechanism of the disease involves the aggregation of marginally-soluble forms of SOD1 whose populations are increased by the ALS-inducing mutations. Biophysical analysis of the folding mechanism of this dimeric ?-barrel protein and quantitative assessment of the perturbations in the populations of monomeric forms induced by mutations, performed during the previous grant period, support this hypothesis. The overall goals of the present proposal are (1) to examine the structural, thermodynamic and kinetic properties of the rate-limiting monomer folding reaction in ALS-variants of SOD1 and (2) to develop and apply fluorescence-based optical methods to monitor directly the subsequent diffusion-limited subunit association reaction and the formation of oligomers during the early stages of the aggregation reaction. A combined denaturant and temperature analysis of the kinetic folding reaction will provide insights into the thermodynamic properties of the transition state ensemble (TSE) controlling the formation of the folded monomeric state. A complementary mutational analysis will highlight the side chains involved in defining the TSE and test the role of hydration in determining the barrier for this extraordinarily slow reaction. Small angle x-ray scattering experiments will probe the size and shape of monomeric ALS variants of SOD1. The resistance of main chain amide hydrogens to exchange with solvent will be monitored by mass spectrometry and NMR spectroscopy to probe for persistent secondary structure in partially-folded states of SOD1 and several ALS variants. The results will be compared with the protection patterns observed in aggregates of the ALS variants. Forster resonance energy transfer and fluorescence correlation spectroscopy will be used to monitor the subunit association reaction and the early stages of the aggregation reaction for several ALS variants labeled with extrinsic chromophores. The results will provide a quantitative framework for describing the effects of ALS-inducing mutations on SOD1 and insights into the mechanism by which they enhance its propensity to aggregate.

Public Health Relevance

Although inheritable mutations in SOD1 are responsible for only a small fraction of ALS cases, a detailed analysis of the mechanisms by which mutations in SOD1 enhance its aggregation propensity may provide a paradigm for other forms of ALS and other neurodegenerative diseases. The development and application of fluorescence correlation spectroscopy techniques to in vitro SOD1 folding and aggregation will enhance the understanding of the molecular basis of ALS and enable future applications to in vivo studies in cell-based systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054836-15
Application #
8266474
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
1996-07-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
15
Fiscal Year
2012
Total Cost
$332,208
Indirect Cost
$130,258
Name
University of Massachusetts Medical School Worcester
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Kathuria, Sagar V; Kayatekin, Can; Barrea, Raul et al. (2014) Microsecond barrier-limited chain collapse observed by time-resolved FRET and SAXS. J Mol Biol 426:1980-94
Mackness, Brian C; Tran, Meme T; McClain, Shannan P et al. (2014) Folding of the RNA recognition motif (RRM) domains of the amyotrophic lateral sclerosis (ALS)-linked protein TDP-43 reveals an intermediate state. J Biol Chem 289:8264-76
Kathuria, Sagar V; Guo, Liang; Graceffa, Rita et al. (2011) Minireview: structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering. Biopolymers 95:550-8
Svensson, Anna-Karin E; Bilsel, Osman; Kayatekin, Can et al. (2010) Metal-free ALS variants of dimeric human Cu,Zn-superoxide dismutase have enhanced populations of monomeric species. PLoS One 5:e10064
Kayatekin, Can; Zitzewitz, Jill A; Matthews, C Robert (2010) Disulfide-reduced ALS variants of Cu, Zn superoxide dismutase exhibit increased populations of unfolded species. J Mol Biol 398:320-31
Tiwari, Ashutosh; Liba, Amir; Sohn, Se Hui et al. (2009) Metal deficiency increases aberrant hydrophobicity of mutant superoxide dismutases that cause amyotrophic lateral sclerosis. J Biol Chem 284:27746-58
Noel, Amanda F; Bilsel, Osman; Kundu, Agnita et al. (2009) The folding free-energy surface of HIV-1 protease: insights into the thermodynamic basis for resistance to inhibitors. J Mol Biol 387:1002-16
Kayatekin, Can; Zitzewitz, Jill A; Matthews, C Robert (2008) Zinc binding modulates the entire folding free energy surface of human Cu,Zn superoxide dismutase. J Mol Biol 384:540-55
Svensson, Anna-Karin E; Bilsel, Osman; Kondrashkina, Elena et al. (2006) Mapping the folding free energy surface for metal-free human Cu,Zn superoxide dismutase. J Mol Biol 364:1084-102
Simler, B Robert; Levy, Yaakov; Onuchic, Jose N et al. (2006) The folding energy landscape of the dimerization domain of Escherichia coli Trp repressor: a joint experimental and theoretical investigation. J Mol Biol 363:262-78

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