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.

Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids

“…MBP undergoes a well-characterized clamshell closure that is stabilized by the binding of maltose and other maltodextrin ligands. The structures and energetics of this conformational change have been subject to extensive study using numerous biophysical techniques, including DEER, , NMR, , FRET, − and X-ray crystallography. , We selected four residues in the MBP sequence (S211, E278, K295, and E322) for mutation either to cysteinefor labeling with the thiol-specific spin label MTSLor to Tet4 ncAA for labeling with sTCO-spin labels. In addition, we generated double-mutants with site-pairs 211/295 and 278/322 to probe the maltose-dependent conformational change of MBP using DEER (Figure A).…”

Ultra-Fast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids

“…MBP undergoes a well-characterized clamshell closure that is stabilized by the binding of maltose and other maltodextrin ligands. The structures and energetics of this conformational change have been subject to extensive study using numerous biophysical techniques, including DEER, 63,64 NMR, 65,66 FRET, [67][68][69][70] and X-ray crystallography. 71,72 We selected four residues in the MBP sequence (S211, E278, K295, and E322) for mutation either to cysteinefor labeling with the thiol-specific spin label MTSL-or to Tet4 ncAA for labeling with sTCOspin labels.…”

FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance‐dependent energy transfer

“…The relatively narrow excitation spectrum of BODIPY does not have a significant tail in the 400 nm region (unlike fluorescein, for example) which minimizes direct BODIPY excitation when irradiating the Acd donor (Speight et al, 2013). Recently, Acd has also been studied as fluorescent donor in a distance‐dependent quenching mechanism in proximity to a non‐emissive metal using transition metal FRET (TM‐FRET), particularly copper 1,4,7,10‐tetraazacyclododecane (TETAC) chelates (Zagotta et al, 2021). BODIPY and Cu TETAC can be selectively conjugated to cysteine as C Bdp and C CuT , respectively.…”