Analysis of conformational changes in WASP using a split YFP.
WASP (Wiskott-Aldrich syndrome protein) has been proposed to adopt a closed conformation (autoinhibited conformation) due to interaction between the carboxy terminal and the GTPase binding domain. Various WASP-interacting proteins have been suggested to relieve this autoinhibition. We have used the split YFP (Yellow Fluorescent Protein) to analyze the conformation of WASP. Saccharomyces cerevisiae cells expressing YFP1-154-WASP-YFP155-238 were found to exhibit YFP fluorescence while cells expressing of YFP1-154-WASP and WASP-YFP155-238 did not suggesting an intramolecular complementation of the YFP molecule. The fluorescence signal of YFP1-154-WASP-YFP155-238 was enhanced in the presence of WIP (WASP-interacting protein) however this is not due to the increased stability of YFP1-154-WASP-YFP155-238. Expression of Toca-1 and Nck1 reduced the YFP fluorescence from YFP1-154-WASP-YFP155-238 even in the presence of WIP suggesting that binding of Toca-1 or Nck1 altered the conformation of YFP1-154-WASP-YFP155-238. Thus both Nck1 and Toca-1 can relieve the autoinhibition of the WASP molecule.
Lim
Rina Pei Zhi
RP
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore.
Misra
Ashish
A
Wu
Zhihao
Z
Thanabalu
Thirumaran
T
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence / Q000379:methods; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000648:ultrastructure*; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000737:chemistry* / Q000648:ultrastructure*