Cloning, expression, purification, and spectroscopic analysis of the fragment 57-102 of human alpha-synuclein.
The protein alpha-synuclein plays an important role in many neurodegenerative disorders, referred to as alpha-synucleinopathies, that include, among others, Parkinson's and Alzheimer's diseases. The central region of the wild type protein, known as the non-Abeta component of amyloid plaques (NAC, amino acids 61-95), seems to be responsible for its aggregation process. To structurally characterize this fragment by nuclear magnetic resonance, we produced it by DNA recombinant technology. This technique, unlike chemical synthesis, allows the production of labeled samples (13C, 15N) required for NMR studies. Because the NAC region is very sparingly soluble in aqueous buffer, we cloned a slightly larger portion of alpha-synuclein, alphasyn57-102, with the presence of several charged residues in both extremities of the NAC region. The conformational preferences of purified alphasyn57-102, in solution and bound to SDS micelles, was studied. Our results indicate that the protein is largely unfolded in solution but exhibits a helical conformation in the lipid-associated state. The methodology that we have used in this work for the cloning, expression, and purification of alphasyn57-102 can be easily applied to most small proteins, thus representing a powerful tool for structural NMR analysis of labeled peptides.
Bisaglia
Marco
M
Department of Chemical Sciences, University of Padova, Via F. Marzolo 1, 35131, Padova, Italy.
Trolio
Alessandra
A
Tessari
Isabella
I
Bubacco
Luigi
L
Mammi
Stefano
S
Bergantino
Elisabetta
E
D003001:Cloning, Molecular; D005822:Genetic Vectors; D009682:Magnetic Resonance Spectroscopy; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids; D051843:Synucleins; D051844:alpha-Synuclein
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D051843:Synucleins; D051844:alpha-Synuclein