RESUMO
An NMR weakly-aligning polymer gel has been prepared by copolymerization of acrylonitrile and 2-acrylamide-2-methyl-1-propanesulfonic acid in the presence of 1,4-butanediol diacrylate as a cross-linker. The polymer readily swells in water in a large range of temperatures, although the swelling ratio is decreased in saline solutions. The swollen gel can be mechanically compressed, in a reversible way, generating anisotropy, as easily shown in 2 Hâ NMR experiments, and allowing measurement of 1 DCH residual dipolar couplings (RDCs) through F1-coupled HSQC experiments. The performance of this gel as a NMR alignment medium was evaluated in several water-soluble organic molecules and, while it provided RDCs of proper size for sucrose and even such as small molecule as 5-norbornen-2-ol, in the case of azidothymidine and cefuroxime sodium salt the strong interaction of these molecules with the gel prevented successful extraction of the RDCs.
RESUMO
Cytochrome P450cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1H-15N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed.
Assuntos
Cânfora/química , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Hidroxilação , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
The 1 H and 13 C NMR spectra of 17-α-ethinylestradiol (EE2), a well-known contraceptive, including diastereotopic methylene groups, were fully assigned with the help of residual dipolar couplings (RDC) measured in the recently developed grafted graphene oxide orienting medium. RDC analysis, which included all 1 DCH couplings and the long-range 2 DCH1 H-C≡13 C coupling, also pointed to the presence of a minor conformation arising from pseudo-rotation of the steroid B ring. Saturation-transfer difference (STD) measurements revealed that the most likely interaction between EE2 and orienting medium occurred on the C and D ring. Copyright © 2016 John Wiley & Sons, Ltd.