Palladium‐Mediated Cleavage of Proteins with Thiazolidine‐Modified Backbone in Live Cells

Chemical protein synthesis and biorthogonal modification chemistries allow production of unique proteins for a range of biological studies. Bond‐forming reactions for site‐selective protein labeling are commonly used in these endeavors. Selective bond‐cleavage reactions, however, are much less explored and still pose a great challenge. In addition, most of studies with modified proteins prepared by either total synthesis or semisynthesis have been applied mainly for in vitro experiments with very limited extension to live cells. Reported here is an approach for studying uniquely modified proteins containing a traceless cell delivery unit and palladium‐based cleavable element for chemical activation, and monitoring the effect of these proteins in live cells. This approach is demonstrated for the synthesis of a caged ubiquitin‐aldehyde, which was decaged for the inhibition of deubiquitinases in live cells.     

High‐Resolution Protein 3D Structure Determination in Living Eukaryotic Cells

Proteins in living cells interact specifically or nonspecifically with an enormous number of biomolecules. To understand the behavior of proteins under intracellular crowding conditions, it is indispensable to observe their three‐dimensional (3D) structures at the atomic level in a physiologically natural environment. We demonstrate the first de novo protein structure determinations in eukaryotes with the sf9 cell/baculovirus system using NMR data from living cells exclusively. The method was applied to five proteins, rat calmodulin, human HRas, human ubiquitin, T. thermophilus HB8 TTHA1718, and Streptococcus protein G B1 domain. In all cases, we could obtain structural information from well‐resolved in‐cell 3D nuclear Overhauser effect spectroscopy (NOESY) data, suggesting that our method can be a standard tool for protein structure determinations in living eukaryotic cells. For three proteins, we achieved well‐converged 3D structures. Among these, the in‐cell structure of protein G B1 domain was most accurately determined, demonstrating that a helix‐loop region is tilted away from a β‐sheet compared to the conformation in diluted solution.