Journal Article

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

Gordon, Sharona E, Raza, Marium M, Petersson, E James, Zagotta, William N, Mehl, Ryan A, Sim, Brandon S, Evans, Eric GB, Nhim, Anthony K, Jones, Chloe M, Venkatesh, Yarra

2021

Overview

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.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Amino Acid Sequence / Amino acids / beta-Alanine - analogs & derivatives / beta-Alanine - chemistry / Binding sites / Biochemistry and Chemical Biology / Copper - chemistry / Efficiency / Energy transfer / Fluorescence / fluorescence lifetime / Fluorescence Resonance Energy Transfer / Fluorometry / FRET / HEK293 Cells / Humans / Maltose / Maltose-binding protein / Maltose-Binding Proteins - chemistry / Maltose-Binding Proteins - genetics / Maltose-Binding Proteins - metabolism / Mammalian cells / Membrane proteins / Metal ions / Methods / Models, Chemical / Mutation / NMR / noncanonical amino acid / Nuclear magnetic resonance / Protein Conformation, alpha-Helical / protein dynamics / Proteins / Structural Biology and Molecular Biophysics / Structure-Activity Relationship / Time Factors / Tools and Resources / Transfer RNA