35 research outputs found
Distal unfolding of ferricytochrome C induced by the F82K mutation
It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82\u2014conserved amino acid and a key actor in cytochrome c intermolecular interactions\u2014with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by1 H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization
Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR
Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer
Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein–protein complex structure determination
Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein–protein and protein–ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination
Accurate Structure and Dynamics of the Metal-Site of Paramagnetic Metalloproteins from NMR Parameters Using Natural Bond Orbitals
Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts
Lanthanide Tags for Site-Specific Ligation to an Unnatural Amino Acid and Generation of Pseudocontact Shifts in Proteins
Superoxide biosensing with engineered cytochrome c
Several mutation positions have been chosen for introducing positively charged lysines in human cytochrome c (cyt c) with the aim of increasing the reaction rate with superoxide radicals (SO) and thus, the sensitivity of an electrochemical cyt c based SO biosensor. The impact of the mutations on structural and redox properties as well as on the reaction rate with SO are verified. Four mutants show a higher reaction rate with the radical compared to the wild type. These mutants are used for the construction of SO sensors based on thiol-modified gold electrodes and covalently fixed proteins. The E66K mutant electrode has a clearly higher sensitivity in comparison to the wildtype based sensor