A Genetically Encoded Two‐Dimensional Infrared Probe for Enzyme Active‐Site Dynamics

Abstract

While two-dimensional infrared (2D-IR) spectroscopy is uniquely suitable for monitoring femtosecond (fs) to picosecond (ps) water dynamics around static protein structures, its utility for probing enzyme active-site dynamics is limited due to the lack of site-specific 2D-IR probes. We demonstrate the genetic incorporation of a novel 2D-IR probe, m-azido-l-tyrosine (N3Y) in the active-site of DddK, an irondependent enzyme that catalyzes the conversion of dimethylsulfoniopropionate to dimethylsulphide. Our results show that both the oxidation of active-site iron to Fe III , and the addition of denaturation reagents, result in significant decrease in enzyme activity and active-site water motion confinement. As tyrosine residues play important roles, including as general acids and bases, and electron transfer agents in many key enzymes, the genetically encoded 2D-IR probe N3Y should be broadly applicable to investigate how the enzyme active-site motions at the fs-ps time scale direct reaction pathways to facilitating specific chemical reactions.

A Genetically Encoded F-19 NMR Probe Reveals the Allosteric Modulation Mechanism of Cannabinoid Receptor 1

“…Allosteric modulators offer important therapeutic applications owing to their receptor subtype selectivity and spatial and temporal coupling with tissue-specific endogenous ligands. The pharmacological signaling profile of Org27569 is fairly unique, in that it increases the binding of CB1 agonists as a positive allosteric modulator (PAM) but inhibits G protein-dependent signaling as a negative allosteric modulator (NAM). , However, much is still to be learned about the unique mechanisms of allosteric ligand participation in CB1 activation. − …”

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

“…As shown (Figure 19), their experimental and computational results reveled that VET between opposite βstrands is dominated by contact pathways and that transfer over a H-bond shortcut is about as efficient as transport over a stretch of 3−4 amino acids units in the backbone. Furthermore, Wang and co-workers 229 demonstrated the spectroscopic utility of the azido stretching vibration of mazido-L-tyrosine (N3Y) in the study of enzyme active-site dynamics. Specifically, they genetically incorporated N3Y into an iron-dependent enzyme, DddK, that catalyzes the conversion of dimethylsulfoniopropionate into dimethylsulphide, and used 2D IR spectroscopy to measure the dynamics of the azido vibrational mode.…”

“…Furthermore, Wang and co-workers demonstrated the spectroscopic utility of the azido stretching vibration of m -azido- l -tyrosine (N3Y) in the study of enzyme active-site dynamics. Specifically, they genetically incorporated N3Y into an iron-dependent enzyme, DddK, that catalyzes the conversion of dimethylsulfoniopropionate into dimethylsulphide, and used 2D IR spectroscopy to measure the dynamics of the azido vibrational mode.…”

Expanding the functionality of proteins with genetically encoded dibenzo[b,f][1,4,5]thiadiazepine: a photo-transducer for photo-click decoration

“…Genetic code expansion (GCE) with noncanonical amino acids (ncAAs, Scheme 1a ) has emerged as an extremely powerful tool to incorporate artificial functionalities into engineered proteins in living systems. 17 Harbouring the unique chemical or physical features of exogenous ncAAs at specific residues, the encoded proteins have been exploited for mapping protein–protein interactions (proximity dependent photo-crosslinking), 18 probing and tuning protein structures 19 and the construction of artificial conjugates on demand. 20 In particular, it is of great interest to utilize GCE to encode RILDs ( Scheme 1c ) because its exceptional biocompatibility and the photo-chemical energy transduction can enhance the chemoselectivity of the photo-click chemistry.…”