Pseudocontact shifts (PCS) from paramagnetic lanthanide ions present powerful long-range structural restraints for structural biology by NMR spectroscopy, but site-specific tagging of proteins with ...lanthanides remains a challenge, as most of the available lanthanide tags require proteins with single cysteine residues. We show that cyclen-based paramagnetic lanthanide tags can be attached to proteins in a site-specific manner by Cu(I)-catalyzed azide–alkyne cycloaddition to a genetically encoded p-azido-l-phenylalanine residue with a tether that proved sufficiently short and rigid for the observation of PCSs in several proteins. Despite the sterically demanding conditions associated with bulky tags and reactions close to the protein surface, ligation yields consistently above 50% and approaching 100% were obtained with the help of the Cu(I)-stabilizing ligand BTTAA. The yields were high independent of the presence of cysteine residues, thereby avoiding the need for cysteine mutations associated with conventional lanthanide-tagging strategies.
It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron‐electron ...resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide‐DOTA‐Gd(III) in the N‐ and C‐terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo‐CaM and holo‐CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo‐ and holo‐CaM bind to target proteins in a similar way as apo‐ and holo‐CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in‐cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in‐cell components. These results show‐case the importance of in‐cell analyses of protein structures.
Double electron‐electron resonance distance measurements of calmodulin with Gd(III) spin labels reveal different conformations and conformational changes in HeLa cells compared to in vitro measurements and studies on HeLa cell extracts.
High‐frequency double electron‐electron resonance (DEER) distance measurements using different Gd3+ tags (Gd‐DOTA and Gd‐C1) were carried out on transmembrane helical peptides (ca. 0.15 nmol; WALP ...peptides) in a model membrane. The ability to pick up small distance variations, the chemical flexibility of the tags, and the remarkable absolute sensitivity, make this approach attractive for studies of membrane proteins.
Paramagnetic metal ions with fast-relaxing electrons generate pseudocontact shifts (PCSs), residual dipolar couplings (RDCs), paramagnetic relaxation enhancements (PREs) and cross-correlated ...relaxation (CCR) in the nuclear magnetic resonance (NMR) spectra of the molecules they bind to. These effects offer long-range structural information in molecules equipped with binding sites for such metal ions. Here we present the new open-source software Paramagpy, which has been written in Python 3 with a graphic user interface. Paramagpy combines the functionalities of different currently available programs to support the fitting of magnetic susceptibility tensors using PCS, RDC, PRE and CCR data and molecular coordinates in Protein Data Bank (PDB) format, including a convenient graphical user interface. Paramagpy uses efficient fitting algorithms to avoid local minima and supports corrections to back-calculated PCS and PRE data arising from cross-correlation effects with chemical shift tensors. The source code is available from 10.5281/zenodo.3594568 .
Narrow 1H NMR linewidths can be obtained for fully protonated protein samples in the solid state by using ultrafast magic‐angle spinning (60 kHz). Medium‐size microcrystalline and noncrystalline ...proteins can be analyzed without any need for deuteration of the protein sample. This approach provides assignments of the backbone 1H, 15N, 13Cα, and 13CO resonances and yields information about 1H–1H proximities.
NMR studies of ligand binding Nitsche, Christoph; Otting, Gottfried
Current opinion in structural biology,
February 2018, 2018-02-00, 20180201, Letnik:
48
Journal Article
Recenzirano
Odprti dostop
Display omitted
•NMR studies of ligand binding are a frequently reviewed topic.•NMR plays an important role in fragment-based drug discovery.•New labelling techniques allow determination of ligand ...binding modes in stable protein–ligand complexes.•Paramagnetic metal tags enable a GPS approach to analyse ligand binding modes.
NMR spectroscopy is an established tool in drug discovery, but its strength is commonly regarded to be largely confined to the early stages of hit discovery and fragment based drug design, where NMR offers unique capabilities of characterizing the binding modes of ligand molecules that bind sufficiently weakly to be in rapid exchange between bound and free state. Here we, first, provide a meta-review of recent reviews on NMR studies of ligand binding and, second, review recent progress towards NMR characterization of the ligand binding mode in stable protein–ligand complexes, with particular emphasis on the global positioning system (GPS) approach enabled by paramagnetic lanthanide tags.
Double‐arm cyclen‐based Gd3+ tags are shown to produce accurate nanometer scale Gd3+–Gd3+ distance measurements in double electron–electron resonance (DEER) experiments by confining the space ...accessible to the metal ion. The results show excellent agreement with predictions both for the maximum and width of the measured distance distributions. For distance measurements in proteins, the tags can be attached to two cysteine residues located in positions i and i+4, or i and i+8, of an α‐helix. In the latter case, an additional mutation introducing an aspartic acid at position i+4 achieves particularly narrow distribution widths. The concept is demonstrated with cysteine mutants of T4 lysozyme and maltose binding protein. We report the narrowest Gd3+–Gd3+ distance distributions observed to date for a protein. By limiting the contribution of tag mobility to the distances measured, double‐arm Gd3+ tags open new opportunities to study the conformational landscape of proteins in solution with high sensitivity.
Metal on a tether: Double‐arm cyclen‐based Gd3+ tags confine the space accessible to the metal ion (see figure) and produce accurate nanometer scale Gd3+–Gd3+ distance measurements by double electron‐electron resonance (DEER) experiments, which show excellent agreement with predictions both for the maximum and width of the measured distance distributions. This research opens new opportunities to study the conformational landscape of proteins in solution.
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. The reaction proceeds in two steps, where the first step involves the conversion of an amide bond of substrate peptide into a thioester intermediate with the enzyme. Here we show that the free energy required for this step is matched by an about 30-fold increase in binding affinity of a calcium ion at the calcium binding site of SrtA, which is remote from the thioester bond. The magnitude of this allosteric effect highlights the importance of calcium for the activity of SrtA. The increase in calcium binding affinity upon binding of substrate not only achieves catalytic formation of an energy-rich intermediate in the absence of nucleotide triphosphates or any tight non-covalent enzyme-substrate interactions, but is also accompanied by accumulation of the labile thioester intermediate, which makes it directly observable in nuclear magnetic resonance (NMR) spectra.
Pseudocontact shift (PCS) effects induced by a paramagnetic lanthanide bound to a protein have become increasingly popular in NMR spectroscopy as they yield a complementary set of orientational and ...long-range structural restraints. PCS are a manifestation of the χ-tensor anisotropy, the Δχ-tensor, which in turn can be determined from the PCS. Once the Δχ-tensor has been determined, PCS become powerful long-range restraints for the study of protein structure and protein-ligand complexes. Here we present the newly developed package Numbat (New User-friendly Method Built for Automatic Δχ-Tensor determination). With a Graphical User Interface (GUI) that allows a high degree of interactivity, Numbat is specifically designed for the computation of the complete set of Δχ-tensor parameters (including shape, location and orientation with respect to the protein) from a set of experimentally measured PCS and the protein structure coordinates. Use of the program for Linux and Windows operating systems is illustrated by building a model of the complex between the E. coli DNA polymerase III subunits ε186 and θ using PCS.
Cell-free synthesis. Display omitted
► Cell-free synthesis affords high protein yields with unnatural amino acids. ► 4-(Trifluoromethyl)phenylalanine incorporated by a polyspecific synthetase. ► ...Truncation at amber stop codons can be suppressed almost completely. ► Two unnatural amino acids can readily be incorporated at two different sites.
Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNACUAopt and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.
PDF
