title: Stress-induced aggregation profiles of GST-alpha-synuclein fusion proteins: role of the C-terminal acidic tail of alpha-synuclein in protein thermosolubility and stability.
abstract: Alpha-synuclein is a well-known heat-resistant protein that does not aggregate upon heat treatment, whereas glutathione S-transferase (GST) is a heat-labile protein that easily precipitates as a result of thermal stress. This paper reports the role of the C-terminal acidic tail of alpha-synuclein in protein thermosolubility and stability. The region of alpha-synuclein that is responsible for the heat resistance was initially investigated using a series of deletion mutants, and the C-terminal acidic tail (residues 96-140) was found to be crucial for the thermosolubility of alpha-synuclein. The thermal behavior of the GST-alpha-synuclein fusion protein was next investigated, and the fusion protein was seen to be extremely heat-resistant. Using a series of GST-synuclein deletion mutants, the C-terminal acidic tail of alpha-synuclein was shown to play a critical role in conferring the heat resistance of the fusion proteins. Furthermore, the acidic tail appeared to protect the fusion protein from pH- and metal-induced protein aggregation, suggesting that the acidic tail can increase the virtual stability of the protein by protecting it from the aggregation induced by environmental stresses. Interestingly, the acidic tail also appeared to protect the GST enzyme from the thermal inactivation to a considerable extent. However, CD analysis of the heat-induced secondary structural changes of the GST-alpha-synuclein fusion protein revealed that the fusion protein is irreversibly denatured by heat treatment with a slightly lowered melting temperature (Tm). Thus, the results demonstrate that introducing an acidic tail into GST promotes the thermosolubility and virtual stability of the fusion protein, although it might be unfavorable for its intrinsic stability.
authors.affiliation: Department of Microbiology and Brain Korea 21 Project of Medical Sciences, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Sang Myun
authors.initials: SM
authors.lastname: Park
authors.forename: Han Young
authors.initials: HY
authors.lastname: Jung
authors.forename: Kwang Chul
authors.initials: KC
authors.lastname: Chung
authors.forename: Hyangshuk
authors.initials: H
authors.lastname: Rhim
authors.forename: Jeon Han
authors.initials: JH
authors.lastname: Park
authors.forename: Jongsun
authors.initials: J
authors.lastname: Kim
mesh_terms: D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D005982:Glutathione Transferase / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D051843:Synucleins; D051844:alpha-Synuclein
chemical_list: D017931:DNA Primers; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D051843:Synucleins; D051844:alpha-Synuclein; D005982:Glutathione Transferase