A Strongly Absorbing Class of Non-Natural Labels for Probing Protein Electrostatics and Solvation with FTIR and 2D IR Spectroscopies

TitleA Strongly Absorbing Class of Non-Natural Labels for Probing Protein Electrostatics and Solvation with FTIR and 2D IR Spectroscopies
Publication TypeJournal Article
Year of Publication2013
AuthorsWoys AM, Mukherjee S, Skoff DR, Moran SD, Zanni MT
Secondary TitleThe Journal of Physical Chemistry B
Volume117
Issue17
Pagination5009-5018
Date Published03/2013
Abstract

A series of non-natural infrared probes is reported that consist of a metal-tricarbonyl modified with a −(CH2)n– linker and cysteine-specific leaving group. They can be site-specifically attached to proteins using mutagenesis and similar protocols for EPR spin labels, which have the same leaving group. We characterize the label’s frequencies and lifetimes using 2D IR spectroscopy in solvents of varying dielectric. The frequency range spans 10 cm–1, and the variation in lifetimes ranges from 6 to 19 ps, indicating that these probes are very sensitive to their environments. Also, we attached probes with −(CH2)–, −(CH2)3–, and −(CH2)4– linkers to ubiquitin at positions 6 and 63 and collected spectra in aqueous buffer. The frequencies and lifetimes were correlated for 3C and 4C linkers, as they were in the solvents, but did not correlate for the 1C linker. We conclude that lifetime measures solvation, whereas frequency reflects the electrostatics of the environment, which in the case of the 1C linker is a measure of the protein electrostatic field. We also labeled V71C α-synuclein in buffer and membrane-bound. Unlike most other infrared labels, this label has extremely strong cross sections and thus can be measured with 2D IR spectroscopy at sub-millimolar concentrations. We expect that these labels will find use in studying the structure and dynamics of membrane-bound, aggregated, and kinetically evolving proteins for which high signal-to-noise at low protein concentrations is imperative.

URLhttp://www.ncbi.nlm.nih.gov/pubmed/23537223
DOI10.1021/jp402946c.
Short TitleJ. Phys. Chem. B