Ivan S. Podkorytov

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Site‐directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as 1HN is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean‐force potential (Smoluchowski equation); (iv) explicit‐atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the 1HN residue number and static magnetic field strength; the results are appreciably different from the Gillespie–Shortle model. At the same time, the Gillespie–Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between 1HN spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin‐labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme—using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates—lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random‐coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random‐coil protein in good solvent/poor solvent.

The structure and dynamic behaviour of disubstituted derivatives of [Rh6(CO)16] containing bidentate phosphorus ligands ☆

The solution structure and dynamic behaviour of [Rh6(CO)14(m,h 2 -dppm)] (1), [Rh6(CO)14(m,h 2 -dppe)] (2) and [Rh6(CO)14(m,h 2 dppe f )] (3) containing bridging diphosphine ligands have been examined using 1D 13 C, 31 P and 2D 13 C/{ 103 Rh}, 31 P/{ 103 Rh} HMQC and 13 C EXSY NMR techniques. It has been shown that the solid state structure of these clusters remains unchanged in solution, including the close non-bonding intramolecular interactions of the perfluorinated phenyl rings with adjacent terminal carbonyls. In solution, two different dynamic processes have been found: for 2 and 3, the P/CH2/CH2/P chain of the coordinated dppe and dppe f is non-rigid and this leads to the interchange of the two enantiomeric forms of the clusters through a ‘rocking’ motion of the bridging diphosphine; the rate of this exchange depends strongly on the non-bonding van der Waal’s interactions between the phosphorus substituents and adjacent carbonyl ligands which results in substantially slower dynamics for 3 because the steric requirements of the fluorinated phenyl rings considerably hinder this racemization. The second type of dynamics found in 1/3 involves exchange of terminal/face-bridging CO’s associated with the unsubstituted rhodium atoms. Regioselectivity of these exchanges is essentially similar for all three clusters, whereas the rates of these CO-exchanges are substantially higher in 3, which may be due to the lower net donicity of the per-fluorinated diphosphine. # 2003 Elsevier B.V. All rights reserved.

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 (1; 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 1 proved to be highly selective toward the imidazole ring coordination that allows site-specific labeling of peptides and proteins with 1 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.

Origin of anti-tumor activity of the cysteine-containing GO peptides and further optimization of their cytotoxic properties

Antitumor GO peptides have been designed as dimerization inhibitors of prominent oncoprotein mucin 1. In this study we demonstrate that activity of GO peptides is independent of the level of cellular expression of mucin 1. Furthermore, these peptides prove to be broadly cytotoxic, causing cell death also in normal cells such as dermal fibroblasts and endometrial mesenchymal stem cells. To explore molecular mechanism of their cytotoxicity, we have designed and tested a number of new peptide sequences containing the key CxC or CxxC motifs. Of note, these sequences bear no similarity to mucin 1 except that they also contain a pair of proximal cysteines. Several of the new peptides turned out to be significantly more potent than their GO prototypes. The results suggest that cytotoxicity of these peptides stems from their (moderate) activity as disulfide oxidoreductases. It is expected that such peptides, which we have termed DO peptides, are involved in disulfide-dithiol exchange reaction, resulting in formation of adventitious disulfide bridges in cell proteins. In turn, this leads to a partial loss of protein function and rapid onset of apoptosis. We anticipate that coupling DO sequences with tumor-homing transduction domains can create a potentially valuable new class of tumoricidal peptides.