Slowly Relaxing Local Structure Analysis of 15N Relaxation from the Proteins p50 and Human Neutrophil Gelatinase-Associated Lipocalin: New Insights into the Dynamic Structure of ß-Barrel Proteins.

Nuclear magnetic resonance relaxation analysis is a powerful method for studying the internal mobility of proteins. We have developed for analysis the slowly relaxing local structure (SRLS) approach. SRLS is general in its nature in several respects, including the tensorial representation of the physical quantities comprising the dynamic model. By controlling tensor symmetry, a broad range of systems can be treated with physical relevance, typically with data-fitting techniques. In simple limits, SRLS yields the traditional model-free (MF) method. In the present context, MF simplicity means featuring the highest possible tensor symmetry. This renders MF-based data-fitting susceptible to the usage of fit parameters, yielding physically ill-defined results. A typical candidate is the Rex term, devised to represent ms-µs motions but often invoked by the fitting scheme just to improve the statistics. Here, we consider two such cases using the N-H bond as probe and the proteins p50 and human neutrophil gelatinase-associated lipocalin as paradigm systems. We illustrate the harm caused by the physically unjustified involvement of Rex in MF-based 15N relaxation analysis. Then, we show that forgoing the usage of Rex, SRLS analysis of the very same experimental data provides interesting new information.

Structural Dynamics from NMR Relaxation by SRLS Analysis: Local Geometry, Potential Energy Landscapes, and Spectral Densities.

We have developed the two-body coupled-rotator slowly relaxing local structure (SRLS) approach for elucidating protein dynamics by nuclear magnetic resonance (NMR) relaxation. The rotators are represented by diffusion tensors D1 for overall protein tumbling and D2 for locally ordered probe motion. D1 and D2 are coupled dynamically by a potential, u, typically given by linear combinations of the Wigner functions D002 and (D022 + D0-22). Until now, our SRLS analyses provided the tensors, D1 and D2, the potential, u, and the geometric link between SRLS and NMR. Here we enhance this description by also examining the SRLS spectral densities obtained by solving the SRLS Smoluchowski equation. In addition, we show that the form of u specified above complies with two NMR-detected potential energy landscapes representing preferential ordering along N-H or Cα-Cα. Pictorial illustrations thereof are provided. The extended SRLS analysis is applied to 15N-H relaxation from the carbohydrate recognition domain of galectin-3 (Gal3C) in complex with two diastereomeric ligands, S and R. We find that D2 is isotropic with a principal value, D2, of 1010 s-1 on average, and it is faster in the strands ß3, ß5, and ß8. The potential, u, is strong (∼20 kT); it is slightly rhombic when N-H is the main ordering axis and highly rhombic when Cα-Cα is the main ordering axis. Gal3C-S exhibits primarily preferential ordering along Cα-Cα; Gal3C-R exhibits both types of ordering. The binding-associated polypeptide chain segment of Gal3C-S is homogeneous, whereas that of Gal3C-R is diversified, with regard to D2 and ordering preference. We associate these features with the previously determined diminished binding constant of Gal3C-R in comparison with Gal3C-S. Thus, the present study enhances the SRLS analysis, in general, and provides new insights into the dynamic structure and binding properties of Gal3C-S and Gal3C-R, in particular.

Thiolate Spin Population of Type I Copper in Azurin Derived from 33S Hyperfine Coupling.

The electron transfer mediating properties of type I copper proteins stem from the intricate ligand coordination sphere of the Cu ion in their active site. These redox properties are in part due to unusual cysteine thiol coordination, which forms a highly covalent copper-sulfur (Cu-S) bond. The structure and electronic properties of type I copper have been the subject of many experimental and theoretical studies. The measurement of spin delocalization of the Cu(II) unpaired electron to neighboring ligands provides an elegant experimental way to probe the fine details of the electronic structure of type I copper. To date, the crucial parameter of electron delocalization to the sulfur atom of the cysteine ligand has not been directly determined experimentally. We have prepared 33S-enriched azurin and carried out W-band (95 GHz) electron paramagnetic resonance (EPR) and electron-electron double resonance detected NMR (EDNMR) measurements and, for the first time, recorded the 33S nuclear frequencies, from which the hyperfine coupling and the spin population on the sulfur of the thiolate ligand were derived. The overlapping 33S and 14N EDNMR signals were resolved using a recently introduced two-dimensional correlation technique, 2D-EDNMR. The 33S hyperfine tensor was determined by simulations of the EDNMR spectra using 33S hyperfine and quadrupolar tensors predicted by QM/MM DFT calculations as starting points for a manual spectral fit procedure. To reach a reasonable agreement with the experimental spectra, the 33S hyperfine principal value, Az, and one of the corresponding Euler angles had to be modified. The final values obtained gave an experimentally determined sulfur spin population of 29.8 ± 0.7%, significantly improving the wide range of 29-62% reported in the literature. Our direct, experimentally derived value now provides an important constraint for further theoretical work aimed at unravelling the unique electronic properties of this site.

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display.

Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with Ki of 1-4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors.