Probing the secondary structure of a recombinant neuronal adaptor protein and its phosphotyrosine binding domains.
Rat brain Fe65 and its truncated forms corresponding to the combined PTB1 and PTB2 domains, as well as to the isolated PTB2 domain, were expressed in Escherichia coli and purified from inclusion bodies by affinity chromatography. The recombinant proteins were refolded and judged functionally active by their ability to interact with native APP. Limited proteolysis of recombinant Fe65 and PTB1-2 with trypsin, chymotrypsin and V8 proteases showed that the most sensitive proteoltytic sites were positioned at the level of the interdomain regions comprised between WW/PTB1 and PTB1/PTB2. Secondary structure of the recombinant proteins, evaluated by CD spectroscopy, showed a different degree of unordered structures, the PTB2 domain being the higher organised region. In addition, intrinsic fluorescence measurements of PTB2, indicated that a conformational transition of the protein can be induced by denaturating agents such as GuHCl. These data provide first evidences on the secondary structural levels of Fe65.
Lamberti
Annalisa
A
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Italy.
Longo
Olimpia
O
Del Vecchio
Pompea
P
Zambrano
Nicola
N
Barone
Guido
G
Russo
Tommaso
T
Arcari
Paolo
P
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology; D002942:Circular Dichroism; D015870:Gene Expression; D006868:Hydrolysis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
C486242:Apbb1 protein, rat; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine