A new high sensitivity ¹⁹F probe for labeling cysteine groups of proteins

Abstract

A novel iodoacetamide label 4-perfluoro-tert-butyl-phenyliodoacetamide (PFP) containing nine fluorine atoms in equivalent positions has been synthesized. It provides a homogeneous 19F NMR resonance line which can be detected with high sensitivity when coupled to proteins. As an example, the sulfhydryl groups of actin have been labeled with PFP; < 100 nmol of this medium sized protein (corresponding to 2.5 mL of a 40 microM solution) can be detected easily in a single scan at 470 MHz.

Fluorine-19 NMR of integral membrane proteins illustrated with studies of GPCRs

“…This can be especially useful for membrane proteins, which are often poorly expressed even in eukaryotic cell lines. Two preferred reagents for membrane protein studies are 2,2,2-trifluoroethanethiol (TET) [14,15,18 •• ,20 • ], and 3-bromo-1,1,1-trifluoroacetone (BTFA) [16,19 • ,21 • ], and 4-(perfluoro-tert-butyl)-phenyliodocetamide (PFP) [34] and S-ethyl-trifluorothioacetate (SETFA) [35] have also been used. TET and BTFA are commercially available, exhibit high labeling efficiency, and cause minimal structural perturbations.…”

A comparison of chemical shift sensitivity of trifluoromethyl tags: optimizing resolution in 19F NMR studies of proteins

“…19 F NMR studies of large proteins, such as G protein coupled receptors (GPCRs), are further complicated by both slow tumbling of the protein of interest, intermediate timescale exchange, and low sample concentrations which necessitates the use of CF 3 tags rather than potential mono- or difluorinated species. It is also possible, for purposes of further enhancing the signal to noise ratio, to resort to sulfhydryl specific probes, consisting of higher numbers of equivalent fluorine nuclei, such as perfluoro tert-butyl moieties (Kalbitzer et al 1992) and even larger structures (Janjic and Ahrens 2009). In this case, the challenge becomes avoiding perturbation to the states or conformations of interest in the protein.…”

Solution NMR Studies of Anesthetic Interactions with Ion Channels

“…2,2,2-Trifluoroethanethiol (TET) and 3-bromo-1,1,1-trifluoroacetone (BTFA) have been used as 19 F-labeling reagents for ion channels (Kinde, Bondarenko, et al, 2016; Kinde, Bu, et al, 2016; Kinde et al, 2015). 4-(perfluoro-tert-butyl)-phenyliodoacetamide (PFP) and S-ethyl-trifluorothioacetate (SETFA) have also been used for 19 F-labeling proteins (Kalbitzer et al, 1992; Mehta, Kulkarni, Mason, Constantinescu, & Antich, 1994). In order to have site-specific labeling, it is ideal to use an ion channel construct that contains a single cysteine residue.…”