In contrast, the 1H and 15N chemical shifts of N 1H in the PNP Hx binds to PNP as the N 7H tautomer, and the N 7H 1H and 15N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. Hypoxanthine (Hx) with specific 15N labels has been used to probe active site interactions with PNP by NMR spectroscopy ( Deng et al., 2004). Ab initio chemical shift calculations on various model systems that contain the ligand and its surrounding environment will then be performed to interpret the experimental observations as shown below. To characterize enzyme active contacts based on ab initio analysis of NMR chemical shifts of bound ligands, we used an approach that is similar to our ab initio analysis of vibrational data: first, 15N and 1H chemical shift changes of a ligand are determined by isotope-edited NMR methods when the ligand is transferred from solution to enzyme. Previous ab initio 1H chemical shift calculations also suggest that the effect of hydrogen bonding on 1H N/ 1H O chemical shift is particularly significant ( de Dios et al., 1993 Westler, Weinhold, & Markley, 2002). In order to reproduce the experimentally observed 15N chemical shifts, it is essential to include the hydrogen bonding partners in the calculation models ( Facelli & deDios, 1999 Hu, Facelli, Alderman, Pugmire, & Grant, 1998 Laxer, Major, Gottlieb, & Fischer, 2001). It has been found that 15N H chemical shifts are sensitive to the molecular conformation as well as to hydrogen bonding and electrostatics ( de Dios et al., 1993 Facelli & deDios, 1999). The effects of molecular structure and environment on the chemical shift values of CH ( Martin, AllenIII, & Moore, 2000 Rablen, Pearlman, & Finkbiner, 1999 Wang, Hinton, & Pulay, 2002) and OH ( Lampert, Mikenda, Karpfen, & Kählig, 1997) protons in small molecules have also been studied using ab initio proton chemical shift calculations. Since it has been shown that it is possible to extract protein structural information from NMR chemical shift data by ab initio calculations ( de Dios, Pearson, & Oldfield, 1993), 13C and 15N chemical shift calculations have been used extensively for interpretation of NMR data to solve a variety of problems (for recent reviews, see Facelli, 2004 Lodewyk, Siebert, & Tantillo, 2012 Mulder & Filatov, 2010). Hua Deng, in Advances in Protein Chemistry and Structural Biology, 2013 4 Active Site Interactions by NMR and Ab Initio Calculations
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