-
Article
Open AccessImproved spectral resolution of [13C,1H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive 13C-labelled precursors
Cell-free protein synthesis using eCells allows production of amino acids from inexpensive 13C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amin...
-
Article
Open AccessAltered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme
Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equall...
-
Article
Open AccessConversion of an amide to a high-energy thioester by Staphylococcus aureus sortase A is powered by variable binding affinity for calcium
Thioesters are key intermediates in biology, which often are generated from less energy-rich amide precursors. Staphylococcus aureus sortase A (SrtA) is an enzyme widely used in biotechnology for peptide ligation...
-
Article
Genetically encoded amino acids with tert-butyl and trimethylsilyl groups for site-selective studies of proteins by NMR spectroscopy
The amino acids 4-(tert-butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and site-specifically incorporate...
-
Article
Trimethylsilyl tag for probing protein–ligand interactions by NMR
Protein–ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined ...
-
Article
Structure restraints from heteronuclear pseudocontact shifts generated by lanthanide tags at two different sites
Pseudocontact shifts (PCS) encode long-range information on 3D structures of protein backbones and side-chains. The level of structural detail that can be obtained increases with the number of different sites ...
-
Protocol
3D Computational Modeling of Proteins Using Sparse Paramagnetic NMR Data
Computational modeling of proteins using evolutionary or de novo approaches offers rapid structural characterization, but often suffers from low success rates in generating high quality models comparable to th...
-
Article
Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags
Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is k...
-
Article
Selective 15N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy
The side-chain amide groups of asparagine and glutamine play important roles in stabilizing the structural fold of proteins, participating in hydrogen-bonding networks and protein interactions. Selective 15N-labe...
-
Article
How reliable are pseudocontact shifts induced in proteins and ligands by mobile paramagnetic metal tags? A modelling study
The anisotropic component of the magnetic susceptibility tensor (Δχ tensor) associated with various paramagnetic metal ions can induce pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) in prote...
-
Article
A systematic study of labelling an α-helix in a protein with a lanthanide using IDA-SH or NTA-SH tags
The previously published IDA-SH and NTA-SH tags are small synthetic lanthanide-binding tags derived from cysteine, which afford site-specific lanthanide labelling by disulfide-bond formation with a cysteine re...
-
Article
Biosynthetically directed 2H labelling for stereospecific resonance assignments of glycine methylene groups
Stereospecific resonance assignments of the α-protons of glycine are often difficult to obtain by measurements of scalar coupling constants or nuclear Overhauser effects. Here we show that these stereospecific...
-
Article
Erratum to: Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
-
Article
Engineering [Ln(DPA)3]3− binding sites in proteins: a widely applicable method for tagging proteins with lanthanide ions
Paramagnetic relaxation enhancements from unpaired electrons observed in nuclear magnetic resonance (NMR) spectra present powerful long-range distance restraints. The most frequently used paramagnetic tags, ho...
-
Article
Erratum to: Paramagnetic labelling of proteins and oligonucleotides for NMR
-
Article
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope ...
-
Article
Tunable paramagnetic relaxation enhancements by [Gd(DPA)3]3− for protein structure analysis
Paramagnetic relaxation enhancements (PRE) present a powerful source of structural information in nuclear magnetic resonance (NMR) studies of proteins and protein–ligand complexes. In contrast to conventional ...
-
Article
Paramagnetic labelling of proteins and oligonucleotides for NMR
Paramagnetic effects offer a rich source of long-range structural restraints. Here we review current methods for site-specific tagging of proteins and oligonucleotides with paramagnetic molecules. The paramagn...
-
Article
Glutarate and N-acetyl-l-glutamate buffers for cell-free synthesis of selectively 15N-labelled proteins
Cell-free protein synthesis provides rapid and economical access to selectively 15N-labelled proteins, greatly facilitating the assignment of 15N-HSQC spectra. While the best yields are usually obtained with buff...
-
Article
Prospects for lanthanides in structural biology by NMR
The advent of different lanthanide-binding reagents has made site-specific labelling of proteins with paramagnetic lanthanides a viable proposition. This brings many powerful techniques originally established ...
