The pH dependence of 15N and l3C resonances of histidine, 95% 15N isotopically enriched in both imidazole nitrogens is studied. From the various couplings between the 13C, 15N, and nuclei a quantitative description of the tautomeric equilibrium of the deprotonated imidazole ring is possible. The chemical shift data and the coupling constants indicate an interaction of the -amino group and the lone electron pair of the imidazole -rr nitrogen in the pH range of 6.2-9.3. Owing to this interaction the conformation of the whole molecule can be determined from the couplings. It can be shown that also the tautomeric equilibrium of the deprotonated imidazole is influenced by this interaction. The analysis of the pH dependence of the spin-lattice relaxation times T\ and the NOE values reveals that histidine is associating around the pH value of the imidazole pK by forming possibly a dimeric structure.
A conformational analysis of the valine side chains of ribonuclease T1 (RNase T1) was performed using NMR spectroscopy, in particular homonuclear (1H, 1H and 13C, 13C) and heteronuclear (1H, 15N and 1H, 13C) vicinal spin-spin coupling constants as obtained from E.COSY-type NMR experiments. The coupling constants related to the chi 1 dihedral angle in valine, 3JH alpha H beta, 3JNH beta, 3JC'H beta, 3JH alpha C gamma 1, 3JH alpha C gamma 2, 3JC'C gamma 1, and 3JC'C gamma 2, were evaluated in a quantitative manner. The analysis of 3J data allowed for the stereospecific assignment of the valine methyl resonances. On the basis of various models for motional averaging of coupling constants, a fit of the torsion angles chi 1 to a set of the experimental 3J coupling constants (3JH alpha H beta, 3JNH beta, 3JC'H beta) was carried out. The resulting side-chain conformations were examined with respect to NOE distance informations. Single rotameric states emerged for Val16, Val67, Val79, and Val101, while conformational equilibria between staggered rotamers were found for Val33 and Val78. Using a different model approach, Val52 and Val89 are also likely to exhibit unimodal chi 1 angle distributions. The analysis was found to depend critically on the set of Karplus parameters used. Except for Val52 and Val78, the predominant rotamers obtained from 3J coupling informations agree with the conformation in the crystal structure of ribonuclease T1 (Martinez-Oyanedel et al., 1991).
Specific heats of aqueous solutions of various alkali halides (LiCl, KC1, KF, CsCl, and Csl) and alkylammonium salts (CH3NH3CI, C2H5NH3CI, and ÍM-C4H9) 4NBr) have been measured in the temperature range of 30-130°with an adiabatic calorimeter. The apparent molal heat capacities *CP evaluated from the heat capacity data are negative in the case of the investigated alkali halides and positive in the case of the alkylammonium salts.The temperature dependence of reveals a maximum at about 50-90°for all cases except (W-C4H9) 4NBr. Approximate values for the partial molal heat capacities CP2°c an be obtained by extrapolation of to infinite dilution. The influence of the dissolved salts on the solvent structure and the contribution of the solventsolute interaction to the partial molal heat capacities are discussed.
A complex between the lac repressor headpiece and a fully symmetric tight-binding 22 bp lac operator was studied by 2D N M R . Several 2D NOE spectra were recorded for the complex in both H 2 0 and 2H20. Many NOE cross-peaks between the headpiece and D N A could be identified, and changes in
There are an estimated 300-500 million cases of malaria and up to 3 million people die from this disease annually. Plasmodium falciparum is the causative agent of the most lethal and sever form of human malaria. Chemotherapy of malaria is available, but is complicated by both adverse effects and widespread resistance to most of the currently available anti-malaria drugs. The malaria parasite depends on de novo synthesis of pyrimidine nucleotides, whereas the human host has the ability to synthesize them by both de novo and salvage pathways. The de novo pathway contains six reaction steps. In the final two steps, uridine 5'-monophosphate (UMP) requires the addition of a ribose phosphate moiety from 5-phosphoribosyl-1-pyrophosphate to orotate by orotate phosphoribosyltransferase (OPRT) to form orotidine 5'-monophosphate (OMP) and pyrophosphate (PPi), and the subsequently decarboxylation of OMP to form UMP, by OMP decarboxylase (OMPDC). Here, we report the X-ray analysis of OMP or UMP-complex forms of OMPDC from Plasmodium falciparum (PfOMPDC) at 2.65 Å resolution. The structural analysis provides the substrate recognition mechanism with dynamic structural changes. And anti-malaria drugs design by using the structure of OMPDC is in progress. Plants provide nourishment for animals and other heterotrophs as the sole primary producer in the food chain. Glutamine synthetase (GS), one of the essential enzymes for plant autotrophy catalyzes the incorporation of ammonia into glutamate to produce glutamine with concomitant hydrolysis of ATP, and plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. We have determined the crystal structures of maize glutamine synthetase in complexes with three kinds of substrate analogues (J. Biol. Chem., 281, 29287-29296(2006)). From these structures we found a unique decameric structure of the enzyme which is significantly different from the bacterial glutamine synthetase and proposed a phosphate transfer reaction mechanism of ATP. In this study, we aims at gaining insights how the enzyme recognizes the substrate glutamic acid by the methods of mutagenesis and X-ray crystal structure analysis and we prepared several mutant enzymes. We determined new two crystal structures. One structure (WT/ PPT/AMPPNP) is a wild type (WT) enzyme in complex with phosphinothricin (PPT) and AMPPNP (ATP analog). The other (H249A/MetSox-P/ADP) is an H249A mutant enzyme in complex with methionine sulfoximine phosphate (MetSox-P) and ADP. Crystal structures of the two complexes were determined at 3.06 and 2.97Å resolutions, respectively. In comparison to the previously reported structures, PPT in the WT/PPT/AMPPNP complex is closer to the γ-phosphate group than MetSox in the WT/MetSox/AMPPNP complex. It is expected that the phosphotransfer energy to PPT is smaller than that of MetSox. It was confirmed that the H249A/ MetSox-P/ADP complex contains three manganese ions. The calcium-dependent phosphotriesteras...
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