Conformational behavior of human alpha-synuclein is modulated by familial Parkinson's disease point mutations A30P and A53T.
Structural properties and response to changes in the environment of wild-type (WT), A30P and A53T alpha-synucleins, as well as their propensity to aggregate orform fibrils, were compared by a variety of biophysical methods, including far-UV CD, FTIR, SAXS, static light scattering and Thioflavin T (TFT) fluorescence. All three proteins were natively unfolded under physiological conditions but adopted identical partially-folded conformations under conditions of acidic pH or high temperature. The initial kinetic event in the fibrillation of all three alpha-synucleins was shown to be the formation of a partially-folded intermediate with properties close to those described for these proteins at acidic pH or at high temperatures. Both mutants showed a greater propensity to form non-fibrillar aggregates than wild-type protein. All three proteins formed fibrils faster in the presence of heparin, although substantially higher concentrations were required for the A30P mutant. In contrast to the wild-type and A53T proteins, in which fibrillation was further accelerated by the presence of the pesticide diethyldithiocarbamate (DDC), the A30P mutant was inhibited by DDC. The mutant proteins had significantly lower affinity for DDC than the WT. A model of the effect of mutations on the aggregation behavior of alpha-synuclein is proposed, which explains the different effects of exogenous agents on the three proteins, based on different kinetic partitioning along pathways leading to fibrils and to non-fibrillar aggregates.
Li
Jie
J
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
Uversky
Vladimir N
VN
Fink
Anthony L
AL
D000465:Algorithms; D002942:Circular Dichroism; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010300:Parkinson Disease / Q000235:genetics* / Q000378:metabolism*; D010575:Pesticides / Q000737:chemistry; D017354:Point Mutation / Q000235:genetics*; D011487:Protein Conformation; D017510:Protein Folding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D051843:Synucleins; D014965:X-Rays; D051844:alpha-Synuclein
D007202:Indicators and Reagents; D009419:Nerve Tissue Proteins; D010575:Pesticides; C497604:SNCA protein, human; D051843:Synucleins; D051844:alpha-Synuclein