Wednesday, January 23, 2008

Hiyam's Journal Update

J. Am. Chem. Soc., 130 (4), 1236 -1244, 2008. 10.1021/ja075584k S0002-7863(07)05584-9 Web Release Date: January 5, 2008 Copyright © 2008 American Chemical Society
Solid-State NMR Spectroscopy Detects Interactions between Tryptophan Residues of the E. coli Sugar Transporter GalP and the -Anomer of the D-Glucose Substrate
Simon G. Patching, Peter J. F. Henderson

An experimental approach is described in which high resolution 13C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 C indicated that the - and -anomers of D-[1-13C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 C showed a selective interaction between the -anomer of D-[1-13C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the -anomer of D-glucose to within 6 Å of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the -isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-13C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.

J. Am. Chem. Soc., 130 (4), 1264 -1273, 2008. 10.1021/ja0759949 S0002-7863(07)05994-X Web Release Date: January 4, 2008 Copyright © 2008 American Chemical Society
NMR Investigations of the Static and Dynamic Structures of Bisphosphonates on Human Bone: a Molecular Model
Sujoy Mukherjee, Yongcheng Song, and Eric Oldfield*

We report the results of an investigation of the binding of a series of bisphosphonate drugs to human bone using 2H, 13C, 15N, and 31P nuclear magnetic resonance spectroscopy. The 31P NMR results show that the bisphosphonate groups bind irrotationally to bone, displacing orthophosphate from the bone mineral matrix. Binding of pamidronate is well described by a Langmuir-like isotherm, from which we deduce an ~30-38 Å2 surface area per pamidronate molecule and a G = -4.3 kcal mol-1. TEDOR of [13C3, 15N] pamidronate on bone shows that the bisphosphonate binds in a gauche [N-C(1)] conformation. The results of 31P as well as 15N shift and cross-polarization measurements indicate that risedronate binds weakly, since it has a primarily neutral pyridine side chain, whereas zoledronate (with an imidazole ring) binds more strongly, since the ring is partially protonated. The results of 2H NMR measurements of side-chain 2H-labeled pamidronate, alendronate, zoledronate, and risedronate on bone show that all side chains undergo fast but restricted motions. In pamidronate, the motion is well simulated by a gauche+/gauche- hopping motion of the terminal -CH2-NH3+ group, due to jumps from one anionic surface group to another. The results of double-cross polarization experiments indicate that the NH3+-terminus of pamidronate is close to the bone mineral surface, and a detailed model is proposed in which the gauche side-chain hops between two bone PO43- sites.

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