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

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln^III tag, referred to as C12 , is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with Tb^III or Tm^III ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron–electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the Tb^III complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.     

Installation of a Rigid EDTA‐Like Motif into a Protein α‐Helix for Paramagnetic NMR Spectroscopy with Cobalt(II) Ions

Coupling two copies of an iminodiacetic acid–cysteine hybrid ligand to a pair of cysteine residues positioned in an i, i+4 arrangement within a protein α‐helix leads to generation of an EDTA‐like metal ion‐binding motif. Rigid binding of a Co^II ion by this motif produces pseudo‐contact shifts suitable for paramagnetic NMR structural studies.     

Site‐Specific Labeling of Proteins with a Chemically Stable,High‐Affinity Tag for Protein Study

Site‐specific labeling of proteins with paramagnetic lanthanides offers unique opportunities by virtue of NMR spectroscopy in structural biology. In particular, these paramagnetic data, generated by the anisotropic paramagnetism including pseudocontact shifts (PCS), residual dipolar couplings (RDC), and paramagnetic relaxation enhancement (PRE), are highly valuable in structure determination and mobility studies of proteins and protein–ligand complexes. Herein, we present a new way to label proteins in a site‐specific manner with a high‐affinity and chemically stable tag, 4‐vinyl(pyridine‐2,6‐diyl)bismethylenenitrilo tetrakis(acetic acid) (4VPyMTA), through thiol alkylation. Its performance has been demonstrated in G47C and E64C mutants of human ubiquitin both in vitro and in a crowded environment. In comparison with the published tags, 4VPyMTA has several interesting features: 1) it has a very high binding affinity for lanthanides (higher than EDTA), 2) there is no heterogeneity in complexes with lanthanides, 3) the derivatized protein is stable and potentially applicable to the in situ analysis of proteins.     

“Invisible” Conformers of an Antifungal Disulfide Protein Revealed by Constrained Cold and Heat Unfolding,CEST‐NMR Experiments,and Molecular Dynamics Calculations

Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two‐ and three‐state analysis of thermal unfolding, that the population of hidden states may weight 20–40 % at 298 K in a disulfide‐rich protein. In addition, sensitive ¹⁵N‐CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR “dark matter”. Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction.     

Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

Magic‐angle spinning solid‐state NMR spectroscopy has been applied to study the dynamics of CBM3b–Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146‐residue protein has a nine‐stranded β‐sandwich fold, in which 35 % of the residues are in the β‐sheet and the remainder are composed of loops and turns. Dynamically averaged ¹H‐¹³C dipolar coupling order parameters were extracted in a site‐specific manner by using a pseudo‐three‐dimensional constant‐time recoupled separated‐local‐field experiment (dipolar‐chemical shift correlation experiment; DIPSHIFT). The backbone‐Cα and Cβ order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water‐mediated contacts, whereas other crystal contacts remain rigid.