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Tools for investigating peptide–protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization

Nature Protocols volume 2, pages 3201–3209 (2007)Cite this article

Abstract

This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from these authors. In this protocol, the fluorophores are directly incorporated after solid-phase peptide synthesis (SPPS) via on-resin derivatization of peptides prepared using commercially available diamino acids, which are Alloc-protected on the side-chain amino group. The time required to complete the procedure depends on the size and number of peptides targeted. As an alternative to this approach, the corresponding fluorescent amino acids can be obtained in an Fmoc-protected form for convenient use as building blocks in SPPS. This option is described in a related protocol by these authors.

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Figure 1
Figure 2
Figure 3: General approaches for the insertion of the environment-sensitive fluorophores 6-dimethylaminonaphthalimide (6-DMN) or 4-dimethylaminophthalimide (4-DMAP) into peptides (illustrated here with 6-DMN insertion).
Figure 4
Figure 5: HPLC trace of Ac-ETPpYSHP(6-DMNA)G-NH2 monitored at 228 nm [YMC C18, ODS-A 5/120, 250 × 4.6 mm2; gradient: 5% acetonitrile (CH3CN) containing 0.1% trifluoroacetic acid (TFA) for 5 min followed by 5–35% CH3CN containing 0.1% TFA over 45 min in water containing 0.1% TFA at a flow rate of 1 ml min−1].
Figure 6: UV absorption spectra.
Figure 7: Normalized fluorescence emission spectra.
Figure 8: Comparison of maximal fluorescence intensity of environment-sensitive paxillin-derived peptides (20 μM) in PBS versus dioxane.

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Acknowledgements

This research was supported by NSF CHE-0414243 (to B.I.) and the Cell Migration Consortium (GM064346). The award of a Marie Curie Fellowship to M.S. is also gratefully acknowledged.

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139-4307, Massachusetts, USA

    Matthieu Sainlos & Barbara Imperiali

  2. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139-4307, Massachusetts, USA

    Barbara Imperiali

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Correspondence to Barbara Imperiali.

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Competing interests

The authors declare that a patent on the environment–sensitive fluorophores is pending: “Fluorescent Probes for Biological Studies” by Imperiali et al. US Patent Application serial No. 11/106,349, filed April 13, 2005, pending.

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Sainlos, M., Imperiali, B. Tools for investigating peptide–protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization. Nat Protoc 2, 3201–3209 (2007). https://doi.org/10.1038/nprot.2007.442

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