Microsecond Time-Scale Conformational Exchange in Proteins: Using Long Molecular Dynamics Trajectory To Simulate NMR Relaxation Dispersion Data

With the advent of ultra-long MD simulations it becomes possible to model microsecond time-scale protein dynamics and, in particular, the exchange broadening effects (<i>R</i>^ex) as probed by NMR relaxation dispersion measurements. This new approach allows one to identify the exchanging species, including the elusive “excited states”. It further helps to map out the exchange network, which is potentially far more complex than the commonly assumed 2- or 3-site schemes. Under fast exchange conditions, this method can be useful for separating the populations of exchanging species from their respective chemical shift differences, thus paving the way for structural analyses. In this study, recent millisecond-long MD trajectory of protein BPTI (Shaw et al. <i>Science</i> <b>2010</b>, <i>330</i>, 341) is employed to simulate the time variation of amide ¹⁵N chemical shifts. The results are used to predict the exchange broadening of ¹⁵N lines and, more generally, the outcome of the relaxation dispersion measurements using Carr–Purcell–Meiboom–Gill sequence. The simulated <i>R</i>^ex effect stems from the fast (∼10–100 μs) isomerization of the C14–C38 disulfide bond, in agreement with the prior experimental findings (Grey et al. <i>J. Am. Chem. Soc.</i> <b>2003</b>, <i>125</i>, 14324)

Coordination to Imidazole Ring Switches on Phosphorescence of Platinum Cyclometalated Complexes: The Route to Selective Labeling of Peptides and Proteins via Histidine Residues

In this study, we have shown that substitution of chloride ligand for imidazole (Im) ring in the cyclometalated platinum complex Pt­(phpy)­(PPh₃)Cl (<b>1</b>; phpy, 2-phenylpyridine; PPh₃, triphenylphosphine), which is nonemissive in solution, switches on phosphorescence of the resulting compound. Crystallographic and nuclear magnetic resonance (NMR) spectroscopic studies of the substitution product showed that the luminescence ignition is a result of Im coordination to give the [Pt­(phpy)­(Im)­(PPh₃)]Cl complex. The other imidazole-containing biomolecules, such as histidine and histidine-containing peptides and proteins, also trigger luminescence of the substitution products. The complex <b>1</b> proved to be highly selective toward the imidazole ring coordination that allows site-specific labeling of peptides and proteins with <b>1</b> using the route, which is orthogonal to the common bioconjugation schemes via lysine, aspartic and glutamic acids, or cysteine and does not require any preliminary modification of a biomolecule. The utility of this approach was demonstrated on (i) site-specific modification of the ubiquitin, a small protein that contains only one His residue in its sequence, and (ii) preparation of nonaggregated HSA-based Pt phosphorescent probe. The latter particles easily internalize into the live HeLa cells and display a high potential for live-cell phosphorescence lifetime imaging (PLIM) as well as for advanced correlation PLIM and FLIM experiments