Observation of NMR signals from proteins introduced into living mammalian cells by reversible membrane permeabilization using a pore-forming toxin, streptolysin O.
We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.
Ogino
Shinji
S
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Kubo
Satoshi
S
Umemoto
Ryo
R
Huang
Shuxian
S
Nishida
Noritaka
N
Shimada
Ichio
I
D001426:Bacterial Proteins / Q000494:pharmacology; D002118:Calcium; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects*; D006801:Humans; D008722:Methods; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000493:pharmacokinetics; D013301:Streptolysins / Q000494:pharmacology*; D013947:Thymosin / Q000737:chemistry / Q000493:pharmacokinetics; D000099201:Thymosin beta(4)
D001426:Bacterial Proteins; D002118:Calcium; D009587:Nitrogen Isotopes; D011506:Proteins; D013301:Streptolysins; D013947:Thymosin; D000099201:Thymosin beta(4); C025964:streptolysin O