C O M M U N I C A T I O N S
hydrophobicity, photo-leucine likely forms intimate contacts with
Sec61R in a manner similar to the leucine side chain of 1. Second,
the short lifetime (nanoseconds)21 of the carbene derived from 2
(Figure 1) ensures that molecules not bound to Sec61R are rapidly
quenched by intramolecular rearrangement, solvent, or membrane
lipids. Despite the short lifetime, the photo-cross-linking yield of
2 to Sec61R was estimated to be ∼23% (Figure S4). Thus,
depending on the specific application, alkyl diazirines may offer
advantages over the widely used benzophenone cross-linker. The
modular cyclodepsipeptide scaffold of 1 should facilitate installation
of a diazirine at multiple positions. High-resolution mapping of
the cross-linking site(s) by mass spectrometry will likely shed light
on the mechanism by which these compounds selectively inhibit
cotranslational translocation.
Acknowledgment. This work was supported by the NIH
(GM81644), the intramural research program of the NICHD, and
the Scleroderma Research Foundation.
Supporting Information Available: Detailed experimental section,
synthesis, and spectral characterization of new compounds. This material
Figure 2. (A) Photo-cross-linking of 2 in the presence of ER microsomes,
followed by click chemistry with a rhodamine-azide reporter. An asterisk
marks the major photo-cross-linked protein. (B) Sec61R antibody or rabbit
serum (control) was added to photolysis/click reactions, and immuno-
precipitates (IP) analyzed by in-gel fluorescent scanning. (C) Proteolipo-
somes derived from control extracts (lane 3), Sec61-depleted extracts (lane
1), or depleted extracts replenished with purified Sec61 (lane 2) were
photolyzed in the presence of 2 and analyzed by click chemistry/in-gel
fluorescent scanning. The major cross-linked protein at ∼50 kDa is shown.
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