An improved fluorescent noncanonical amino acid for measuring conformational distributions using time-resolved transition metal ion FRET

Abstract

With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor, to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.

Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands

“…MBP lacks native cysteine residues, allowing us to introduce single cysteines to introduce site-specific labels. To probe the conformational rearrangement in MBP, we used amber codon suppression to introduce Acd as a donor and Cu 2+ bound to the cysteine-reactive chelator cyclen as an acceptor (3,5). Using the molecular modeling software chiLife (14), together with the structures of apo and holo MBP (pdb 1omp and 1anf) (21,22), we modeled the rotameric ensembles of Acd and cysteine-reactive chelators to estimate the donor–acceptor distance distributions in the resting and active states (Figure 1C).…”

“…MBP with a C-terminal twin-strep tag in the pETM11 plasmid and the AcdA9 aminoacyl tRNA synthetase and its cognate tRNA in the pDule2 plasmid (24) were co-transformed into BL-21(DE3) cells using electroporation and grown until single colonies were apparent an plates with appropriate antibiotics as previously described (3). A single colony was grown overnight in 5 mL of Luria Broth and 60 mL of Terrific Broth was inoculated with 60 µL of starter culture and induced when the optical density was 1 with 1 mM IPTG.…”

“…We recently developed a novel system for FRET to improve its ability to resolve state-dependent distance changes in proteins (3)(4)(5). We used gene c code expansion to introduce the noncanonical amino acid acridonylalanine (Acd; Figure 1A), which is only slightly bigger than a tryptophan and has no addi onal linker to the protein backbone (6).…”