Search

The histidine protein kinase DcuS of Escherichia coli senses [C.sub.4]-dicarboxylates and citrate by a periplasmic domain. The closely related sensor kinase CitA binds citrate, but no [C.sub.4]-dicarboxylates, by a homologous periplasmic domain. CitA is known to bind the three carboxylate and the hydroxyl groups of citrate by sites C1, C2, C3, and H. DcuS requires the same sites for [C.sub.4]-dicarboxylate sensing, but only C2 and C3 are highly conserved. It is shown here that sensing of citrate by DcuS required the same sites. Binding of citrate to DcuS, therefore, was similar to binding of [C.sub.4]-dicarboxylates but different from that of citrate binding in CitA. DcuS could be converted to a [C.sub.4]-dicarboxylate-specific sensor (Dcu[S.sub.DC]) by mutating residues of sites C1 and C3 or of some DcuS-subtype specific residues. Mutations around site C1 aimed at increasing the size and accessibility of the site converted DcuS to a citrate-specific sensor (Dcu[S.sub.Cit]). Dcu[S.sub.DC] and Dcu[S.sub.Cit] had complementary effector specificities and responded either to [C.sub.4]-dicarboxylates or to citrate and mesaconate. The results imply that DcuS binds citrate (similar to the [C.sub.4]-dicarboxylates) via the [C.sub.4]-dicarboxylate part of the molecule. Sites C2 and C3 are essential for binding of two carboxylic groups of citrate or of [C.sub.4]-dicarboxylates; sites C1 and H are required for other essential purposes.

Cross-related relaxation rates linearly depend on the correlation time. Thus, they can be utilized as nuclear Overhauser effects in transfer-type experiments. The technique has been applied to the determination of sugar puckers in aminoacylated tRNA-mimetics which are weakly bound to EF-Tu. The results showed that transfer cross-correlated relaxation can provide projected restraints of the bound conformation of a ligand when it is in fast exchange with the free form.

1H, 13C and the geminal homonuclear 1H,1H dipolar interactions are the main mechanism for relaxation of transvers 13C and 1H magnetization in 13C-labeled macromolecules. Two pulse sequences were employed to show the sensitivity enhancement achieved through multiple-quantum coherences rathe than single-quantum to correlate 1H,13C resonances.

The small 2J(C4',H5') heteronuclear coupling constant for uniformly 13C labeled 19-mer RNA hairpin indicates a gauche+ rotamer conformation about the backbone angle. In the oligonucleotides, the stereospecific assignment of the H5' diastereotopic protons depends on the signs of the 2J(C4',H5') coupling constants. For majority regions in the hairpin, the H5'(super (pro-R)) protons resonate downfield of the H5'(super (pro-S)) protons. A sensitive NMR method is proposed for estimating the homonuclear 3J(H4',H5') and heteronuclear 2J(C4',H5') coupling constants in the 13C labeled RNA.

Antamanide reveals the occurrence of 1ValNH and 6phe NH, sites for quick hydrogen bond exchange dynamics that are emphasized by the T1rho calculation and the structural location. These methods occur along with modifications in the local backbone dihedral angle. The conformational modifications, associated to syn-m and anti-pairs at the two residues, help in identifying the two pairs due to their recognition as dynamic exchange systems. Antamanide also reveals the possible occurrence of other couple geometries.

The effects of subnanosecond internal motions on proton-proton cross relaxation are investigated using the cyclic decapeptide antamanide (-Val1-Pro2-Pro3-Ala4-Phe5-Phe6-Pro7-Pro8-Phe9-Phe10-) in chloroform. An 800-ps molecular dynamics simulation of the antamanide shows that most of these effects may be illustrated by using a product approximation which distinguishes between radial and angular order parameters and an internal correlation time.