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

Author:

Zagotta William N1ORCID,Sim Brandon S1,Nhim Anthony K1,Raza Marium M1,Evans Eric GB1,Venkatesh Yarra2ORCID,Jones Chloe M23,Mehl Ryan A4ORCID,Petersson E James2ORCID,Gordon Sharona E1ORCID

Affiliation:

1. Department of Physiology and Biophysics, University of Washington

2. Department of Chemistry, University of Pennsylvania

3. Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania

4. Department of Biochemistry and Biophysics, Oregon State University

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.

Funder

National Eye Institute

National Institute of General Medical Sciences

National Science Foundation

National Institutes of Health

National Institute of Diabetes and Digestive and Kidney Diseases

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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