NMR resonance assignments of RNase P protein from \u3cem\u3eThermotoga maritima\u3c/em\u3e

Ribonuclase P (RNase P) is an essential metallo-endonuclease that catalyzes 5′ precursor-tRNA (ptRNA) processing and exists as an RNA-based enzyme in bacteria, archaea, and eukaryotes. In bacteria, a large catalytic RNA and a small protein component assemble to recognize and accurately cleave ptRNA and tRNA-like molecular scaffolds. Substrate recognition of ptRNA by bacterial RNase P requires RNA-RNA shape complementarity, intermolecular base pairing, and a dynamic protein-ptRNA binding interface. To gain insight into the binding specificity and dynamics of the bacterial protein-ptRNA interface, we report the backbone and side chain 1H, 13C, and 15N resonance assignments of the hyperthermophilic Thermatoga maritima RNase P protein in solution at 318 K. Our data confirm the formation of a stable RNA recognition motif (RRM) with intrinsic heterogeneity at both the N- and C-terminus of the protein, consistent with available structural information. Comprehensive resonance assignments of the bacterial RNase P protein serve as an important first step in understanding how coupled RNA binding and protein-RNA conformational changes give rise to ribonucleoprotein function

Site-Specific Stabilization of DNA by a Tethered Major Groove Amine, 7‑Aminomethyl-7-deaza-2′-deoxyguanosine

A cationic 7-aminomethyl-7-deaza-2′-deoxyguanosine (7amG) was incorporated site-specifically into the self-complementary duplex d­(G¹A²­G³­A⁴­<u>X</u>⁵­C⁶­G⁷­C⁸­T⁹­C¹⁰­T¹¹C¹²)₂ (<u>X</u> = 7amG). This construct placed two positively charged amines adjacent to the major groove edges of two symmetry-related guanines, providing a model for probing how cation binding in the major groove modulates the structure and stability of DNA. Molecular dynamics calculations restrained by nuclear magnetic resonance (NMR) data revealed that the tethered cationic amines were in plane with the modified base pairs. The tethered amines did not form salt bridges to the phosphodiester backbone. There was also no indication of the amines being capable of hydrogen bonding to flanking DNA bases. NMR spectroscopy as a function of temperature revealed that the X⁵ imino resonance remained sharp at 55 °C. Additionally, two 5′-neighboring base pairs, A⁴:T⁹ and G³:C¹⁰, were stabilized with respect to the exchange of their imino protons with solvent. The equilibrium constant for base pair opening at the A⁴:T⁹ base pair determined by magnetization transfer from water in the absence and presence of added ammonia base catalyst decreased for the modified duplex compared to that of the A⁴:T⁹ base pair in the unmodified duplex, which confirmed that the overall fraction of the A⁴:T⁹ base pair in the open state of the modified duplex decreased. This was also observed for the G³:C¹⁰ base pair, where α<i>K</i>_op for the G³:C¹⁰ base pair in the modified duplex was 3.0 × 10⁶ versus 4.1 × 10⁶ for the same base pair in the unmodified duplex. In contrast, equilibrium constants for base pair opening at the X⁵:C⁸ and C⁶:G⁷ base pairs did not change at 15 °C. These results argue against the notion that electrostatic interactions with DNA are entirely entropic and suggest that major groove cations can stabilize DNA via enthalpic contributions to the free energy of duplex formation

Observation of Two Modes of Inhibition of Human Microsomal Prostaglandin E Synthase 1 by the Cyclopentenone 15-Deoxy-Δ^12,14-prostaglandin J₂

Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E₂ (PGE₂). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), a naturally occurring degradation product of prostaglandin D₂, is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ₂ can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH₂) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ₂ were determined. The question of whether the anti-inflammatory properties of 15d-PGJ₂ are due to inhibition of MPGES1 is discussed