Genetic Encoding of N⁶-(((Trimethylsilyl)methoxy)carbonyl)-l-lysine for NMR Studies of Protein–Protein and Protein–Ligand Interactions

Abstract

Trimethylsilyl (TMS) groups present outstanding NMR probes of biological macromolecules as they produce intense singlets in 1H NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of N 6-(((trimethylsilyl)­methoxy)­carbonyl)-l-lysine (TMSK) for site-specific incorporation into proteins. The system is based on pyrrolysyl-tRNA synthetase mutants, which deliver proteins with high yield and purity in vivo and in cell-free protein synthesis. As the TMS signal can readily be identified in 1D 1H NMR spectra of high-molecular weight systems without the need of isotopic labeling, TMSK delivers an excellent site-specific NMR probe for the study of protein structure and function, which is both inexpensive and convenient. We demonstrate the utility of TMSK to detect ligand binding, measure the rate of conformational change, and assess protein dimerization by paramagnetic relaxation enhancement. In addition, we present a system for dual incorporation of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) in the same protein in quantities sufficient for NMR spectroscopy. Close proximity of the TMS and tert-butyl groups was readily detected by nuclear Overhauser effects.

Ferritin Conjugates With Multiple Clickable Amino Acids Encoded by C-Terminal Engineered Pyrrolysyl-tRNA Synthetase

“…For instance, some potent in vivo bioorthogonal chemical reactions achieved through the expansion of genetic codes are copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted alkyne-azide cycloaddition (SPAAC), and strain-promoted inverse electron-demand Diels–Alder cycloaddition ( Lang and Chin, 2014 ). The bioorthogonal pyrrolysyl-tRNA synthetase (PylRS)•tRNA Pyl pairs from archaea Methanosarcina mazei (Mm) ( Yanagisawa et al, 2006 ; Kavran et al, 2007 ; Chen et al, 2009 ) and Methanosarcina barkeri ( Gautier et al, 2010 ), as well as eubacteria Desulfitobacterium hafniense (Dh) ( Lee et al, 2008 ; Nozawa et al, 2009 ), are widely engineered to incorporate non-canonical amino acids (ncAAs), mainly for lysine ( Cigler et al, 2017 ; Xuan et al, 2017 ; Abdelkader et al, 2021 ), phenylalanine (Phe, e.g., ncAAs 1–9 , Scheme 1 ) ( Wang et al, 2011 ; Wang et al, 2012 ; Wang et al, 2013 ), tryptophan ( Englert et al, 2015 ; Jiang et al, 2020b ), histidine ( Xiao et al, 2014 ; Sharma et al, 2016 ), cysteine ( Nguyen et al, 2014 ), homoarginine ( Mukai et al, 2015 ), and aspartic acid analogs ( Xuan et al, 2018 ) into proteins in vivo . Therefore, clickable ncAAs such as azide, propargyl, bi -cyclooctyne, trans -cyclooctene, or tetrazine functional groups can be developed through these pairs ( Wan et al, 2014 ; Tsai et al, 2015 ).…”

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR, EPR and Time‐Resolved Luminescence Studies

“…Paramagnetic lanthanide ions produce large effects in protein NMR spectra that present long-range structural restraints. Thus, paramagnetic relaxation enhancement (PRE) effects are observed for protons over 20 Å from a gadolinium(III) ion, [1] pseudocontact shifts (PCS) are observable for nuclear spins over 40 Å from the paramagnetic centre [2] and residual dipolar couplings (RDC) are observed throughout the entire molecule due to molecular alignment in the magnetic field conferred by a bound paramagnetic lanthanide ion (with the exception of Gd III ). [3] PCSs are of particular interest, as they are manifested in easy-to-measure changes in chemical shifts, which encode the position of the nuclear spin relative to the coordinate system defined by the magnetic susceptibility anisotropy (Δχ) tensor associated with the paramagnetic centre.…”

“…[3] PCSs are of particular interest, as they are manifested in easy-to-measure changes in chemical shifts, which encode the position of the nuclear spin relative to the coordinate system defined by the magnetic susceptibility anisotropy (Δχ) tensor associated with the paramagnetic centre. The PCS, δ PCS , of a nuclear spin is given by d PCS ¼ 1=ð12 pr 3 Þ½Dc ax ð3 cos 2 qÀ 1Þ þ 3 = 2 Dc rh sin 2 q cos 2�� (1) where δ PCS is measured as the difference in chemical shift measured in the presence and absence of the paramagnetic metal ion, Δχ ax and Δχ rh are the axial and rhombic components of the Δχ tensor, and r, θ and ϕ are the polar coordinates of the nuclear spin relative to the principal axes of the tensor. PCSs thus encode accurate long-range structural information that can be used for analysing protein conformations in solution and determining the structures of protein-protein and proteinligand complexes.…”

“…Paramagnetic lanthanide ions produce large effects in protein NMR spectra that present long‐range structural restraints. Thus, paramagnetic relaxation enhancement (PRE) effects are observed for protons over 20 Å from a gadolinium(III) ion, [1] pseudocontact shifts (PCS) are observable for nuclear spins over 40 Å from the paramagnetic centre [2] and residual dipolar couplings (RDC) are observed throughout the entire molecule due to molecular alignment in the magnetic field conferred by a bound paramagnetic lanthanide ion (with the exception of Gd III ). [3] …”

Genetic Encoding of Cyanopyridylalanine for In-Cell Protein Macrocyclization by the Nitrile-Aminothiol Click Reaction

“…Bioorthogonal reactions for site-specific protein conjugation with chemical and biochemical tags have a wide range of applications in the material, biological, and health sciences. [1] The siteselective modification of a target protein can be used to confer specific biophysical properties [2] and install labels for spectroscopic imaging and tracking of proteins by fluorescence, [3] nuclear magnetic resonance, [4] or electron paramagnetic resonance spectroscopy techniques. [5] Site-specific incorporation of noncanonical amino acids (ncAAs) by genetic encoding gives precise control of the sites, where new functional groups are installed in a target protein, [6] but there is only a very small number of established bioorthogonal reactions.…”