Halogen bonding is a recently rediscovered secondary interaction that shows potential to become a complementary molecular tool to hydrogen bonding in rational drug design and in material sciences. Whereas hydrogen bond symmetry has been the subject of systematic studies for decades, the understanding of the analogous three-center halogen bonds is yet in its infancy. The isotopic perturbation of equilibrium (IPE) technique with (13)C NMR detection was applied to regioselectively deuterated pyridine complexes to investigate the symmetry of [N-I-N](+) and [N-Br-N](+) halogen bonding in solution. Preference for a symmetric arrangement was observed for both a freely adjustable and for a conformationally restricted [N-X-N](+) model system, as also confirmed by computation on the DFT level. A closely attached counterion is shown to be compatible with the preferred symmetric arrangement. The experimental observations and computational predictions reveal a high energetic gain upon formation of symmetric, three-center four-electron halogen bonding. Whereas hydrogen bonds are generally asymmetric in solution and symmetric in the crystalline state, the analogous bromine and iodine centered halogen bonds prefer symmetric arrangement in solution.

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Site-specific 13C labeling offers a desirable means of eliminating unwanted relaxation pathways and coherent magnetization transfer in NMR relaxation experiments. Here we use [1-13C]-glucose as the sole carbon source in the growth media for protein overexpression in Escherichia coli. The approach results in specific incorporation of 13C at isolated positions in the side chains of aromatic amino acids, which greatly simplifies the measurements and interpretation of 13C relaxation rates in these spin systems. The method is well suited for characterization of chemical exchange by CPMG or spin-lock relaxation methods. We validated the method by acquiring 13C rotating-frame relaxation dispersion data on the E140Q mutant of the C-terminal domain of calmodulin, which reveal conformational exchange dynamics with a time constant of 71 mus for Y138.

Transverse relaxation dispersion NMR spectroscopy can provide atom-specific information about time scales, populations, and the extent of structural reorganization in proteins under equilibrium conditions. A method is described that uses side-chain methyl groups as local reporters for conformational transitions taking place in the microsecond regime. The experiment measures carbon nuclear spin relaxation rates in the presence of continuous wave off-resonance irradiation, in proteins uniformly enriched with 13C, and partially randomly labeled with 2H. The method was applied to human FK-506 binding protein (FKBP12), which uses a common surface for binding substrates in its dual role as both an immunophilin and folding assistant. Conformational dynamics on a time scale of approximately 130 micros were detected for methyl groups located in the substrate binding pocket, demonstrating its plasticity in the absence of substrate. The spatial arrangement of affected side-chain atoms suggests that substrate recognition involves the rapid relative movement of the subdomain comprising residues Ala81-Thr96 and that the observed dynamics play an important role in facilitating the interaction of this protein with its many partners, including calcineurin.