Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells

Abstract

Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-crosslinking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.

Efficient Covalent Bond Formation in Gas-Phase Peptide–Peptide Ion Complexes with the Photoleucine Stapler

“…This was motivated by the steric similarity of the L* and L residues [6,32], so that GL*L*L*K can be used as a surrogate for GL*LLK, GLL*LK, and GLLL*K peptides in their respective complexes. At the same time, molecular dynamics trajectory calculations of [GLLLK + GL*L*L*K + H] + allow one to simultaneously track the behavior of all three L* residues in a comprehensive and economical fashion.…”

“…Photochemical crosslinking, in particular, utilizes highly reactive intermediates such as radicals, nitrenes [2], or carbenes [3] produced by photolysis of a chemically stable chromophore that is incorporated into the protein or ligand [4,5]. With the introduction of photolabile amino acid residues photoleucine and photomethionine, it became possible to achieve photochemical crosslinking in complexes closely resembling native systems, such as membrane protein [6] and protein-peptide complexes [7]. Photoleucine (L-2-amino-4,4-azi-pentanoic acid, abbreviated as L*) contains a diazirine ring that is a specific chromophore absorbing at 350-370 nm where the native peptide chromophores are transparent and are not photoactivated.…”

Mesencephalic Astrocyte-derived Neurotrophic Factor Protects the Heart from Ischemic Damage and Is Selectively Secreted upon Sarco/endoplasmic Reticulum Calcium Depletion

“…To do this, we employed zero distance photo cross-linking using incorporation of photo-activatable forms of the amino acids, methionine and leucine, into proteins in live cells (30). Accordingly, cells were co-transfected, as described above, and then subjected to UV irradiation prior to extraction and IP/IB.…”

Photo‐Cross‐Linkers Incorporated into G‐Protein‐Coupled Receptors in Mammalian Cells: A Ligand Comparison

“…A faint radioactive band was detectable in the lane of the wt receptor irradiated in the presence of Ast*, probably due to minimal direct crosslinking not mediated by Azi. [18] However, based on the relative expression level, the amount of wt receptor loaded on this gel was at least 20 times higher than that of the mutants.…”