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. 2007 Jul 4;129(26):8068-9.
doi: 10.1021/ja072013j. Epub 2007 Jun 13.

Covalent capture of phospho-dependent protein oligomerization by site-specific incorporation of a diazirine photo-cross-linker

Miquel Vila-Perelló  1 , Matthew R Pratt, Frej Tulin, Tom W Muir
Affiliations

Covalent capture of phospho-dependent protein oligomerization by site-specific incorporation of a diazirine photo-cross-linker

Miquel Vila-Perelló et al. J Am Chem Soc. .
No abstract available

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Figures

Figure 1
Figure 1
Semi-synthesis of Smad2-MH2-CSpSM-photo-Met (9). (a) Fmoc-SPPS. (b) Cleavage. (c) EPL reaction with peptide 7 and HA-MH2 thioester (red, PDB code 1DEV) bound to SARA-SBD (in yellow) 8. (d) Trimerization of ligation product 9 and release of SARA-SBD. Structural model is based on the crystal structure of the MH2 homo-trimer (PDB code 1KHX). Inset: hypothetical protein–protein interface with the tail of one MH2 subunit shown in sticks (with the diazirine moiety in yellow) and the surface of the adjacent subunit rendered in blue.
Figure 2
Figure 2
Cross-linking experiments. (A) Anti-HA western blot of semi-synthetic proteins 9 and 10 with and without UV irradiation, # indicates cross-links. (B) Effect of CIP treatment or addition of excess of SARA-SBD on the cross-linking efficiency of proteins 9 and 10. Shown is the normalized cross-linking efficiency (change of the ratio between oligomer and monomer bands before and after UV irradiation, ±SD, n = 3).
Scheme 1
Scheme 1
a Conditions: (a) N-methylmorpholine, isobutyl chloroformate, N,O-dimethylhydroxylamine, CH2Cl2, 2 h, 99%; (b) Me–MgBr, toluene, 3 h, 67%; (c) (i) NH3, hydroxylamine-O-sulfonic acid, (ii) TEA, I2, MeOH, 49%; (d) 4 M HCl, THF, 20 h, 99%; (e) NaHCO3, Fmoc-OSu, 1,4-dioxane, 20 h, 97%.
See this image and copyright information in PMC

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