Table 3. Genetically incorporated probes used in VCF studies
ProbeIncorporation methodUsage notesReferences
TryptophanStandard mutagenesisEnvironmentally sensitive, naturally fluorescent amino acid that can also serve as a fluorescence quencher at distances >15 Å.Islas and Zagotta, 2006; Pantazis et al., 2014
Lanthanide binding tagShort tag sequence (DYNKDGWYEELE)Used to chelate lanthanides for distance measurements with LRET. Requires labeling with micromolar concentrations of Tb3+.Castillo et al., 2016; Carrasquel-Ursulaez et al., 2018
Fluorescent proteinsTagging with numerous available protein sequencesCommonly used in protein–protein interaction or domain rearrangements FRET studies. Large number of fluorescent proteins allow tuning of spectral properties (GFP, YFP, CFP, mCherry, Citrine, etc.) although tags are very large (>20 kD), limiting use.Siegel and Isacoff, 1997; Miranda et al., 2013
Halo tagTagging with a 295–amino acid sequenceLabeling achieved by incubation with Halo-specific label, with a wide range of labels available. Usage and limitations are similar to those of fluorescent proteins due to the large size.Not used to date
SNAP tagTagging with a 182–amino acid sequenceSimilar to Halo tag, with slightly smaller tag sequence (∼33 kD compared with 19.4 kD).Not used to date
Dihistidine motifIntroduction of two histidines into helical segment (HXXH or HXXXH)Coordinates transition metals for use as fluorescence quenchers for use in transition metal FRET to probe distance changes at short range (>20 Å).Taraska et al., 2009; Dai and Zagotta, 2017
ANAPNonsense codon suppressionSmall, environmentally sensitive fluorescent unnatural amino acid introduced into Xenopus oocytes by inserting Amber stop codon (TAG) and coinjecting with nonsense suppressor tRNA and ANAP-tRNA synthetase.Kalstrup and Blunck, 2013, 2018
CoumarinNonsense codon suppressionSmall, environmentally fluorescent unnatural amino acid similar to ANAP. Has been used in HEK cells via cotransfection of nonsense suppressor tRNA and coumarin-tRNA synthetase, and gene of interest with TAG codon inserted.Steinberg et al., 2017

SNAP, S-nitroso-N-acetylpenicillamine.