1: J Biol Chem. 2006 Nov 3;281(44):33697-703. Epub 2006 Aug 14. Sequence-specific interactions in the RNA-binding domain of Escherichia coli transcription termination factor Rho. Hitchens TK, Zhan Y, Richardson LV, Richardson JP, Rule GS. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA. Rho factor is an essential protein that causes termination of transcription in a wide variety of bacteria by an RNA-dependent helicase activity. Rho is activated by transcripts that contain a high proportion of cytidine residues. The interaction between Rho and two adjacent cytidine residues within the bound RNA has been identified by previous crystallographic studies (Skordalakes, E., and Berger, J. M. (2003) Cell 114, 135-146). In this study, NMR methods were used to investigate the sequence dependence of the binding of oligonucleotides to the RNA-binding domain of Rho protein (rho130). A comparison of the NMR spectra obtained for rho130 bound to single-stranded oligonucleotides ACTTCCA or ATTTCCA showed that the 5'-cytidine residue interacts with Rho at a site that is distinct from the CC binding site identified by crystallographic studies. Two amino acid residues within this new cytidine binding site, Arg(88) and Phe(89), were altered to Glu and Ser, respectively. These mutant forms of Rho were defective in transcriptional termination, suggesting that those residues play an important role in the activation of Rho by bound RNA. Publication Types: Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't PMID: 16908525 [PubMed - indexed for MEDLINE] 2: J Am Chem Soc. 2005 Nov 16;127(45):15714-5. Stable triplet of uracil-uracil basepairs in a small antisense RNA. Zhan Y, Rule GS. Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA. Publication Types: Research Support, Non-U.S. Gov't PMID: 16277508 [PubMed - indexed for MEDLINE] 3: J Chromatogr B Analyt Technol Biomed Life Sci. 2004 Sep 25;809(1):75-80. Validation and application of an automated 96 -well solid-phase extraction liquid chromatography-tandem mass spectrometry method for the quantitative determination of SCH 201781 in human plasma. Yang L, Wu N, Rule GS, Campbell DA, Perkins JR, Rudewicz PJ. Schering-Plough Research Institute, Kenilworth, NJ 07033-1300, USA. SCH 201781 is a direct thrombin inhibitor recently under study in clinical trials to determine its safety and efficacy for the treatment of venous and arterial thrombosis. In aqueous solution, SCH 201781 exists as three forms, a ring-opened hydrated form and two ring-closed diastereomers. An automated solid-phase extraction LC-MS/MS method that chromatographically separates and measures each form was developed and validated from 1 to 1000 ng/mL in human plasma. For calibration curve standards, within- and between-run precision (%CV) ranged from 0.6 to 13.7%, while accuracy (%bias) ranged from -4.8 to 13.1%. For quality control samples, within- and between-run %CV ranged from 1.5 to 9.9% while %bias ranged from -9.1 to 4.9%. The method requires a sample volume of 0.8 ml and utilizes 2H6-labeled SCH 201781 as the internal standard. For sample processing, an Isolute C-8 96-well solid phase extraction plate and a Tomtec Quadra 96 sample processor is employed. Separation of the three forms of SCH 201781 is achieved using a 5 microm, 2 mm x 100 mm Asahipak C8 HPLC column and gradient elution. A Sciex API 365 equipped with a turbo ionspray source is used in the selected reaction monitoring mode for detection. The validated method was used to support clinical studies. Publication Types: Validation Studies PMID: 15354313 [PubMed - indexed for MEDLINE] 4: Biochemistry. 2004 Jun 15;43(23):7244-54. Glutathione induces helical formation in the carboxy terminus of human glutathione transferase A1-1. Zhan Y, Rule GS. Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA. The structure and dynamic properties of the C-terminal region of the human class alpha glutathione transferase A1-1 have been investigated with high-resolution NMR methods. On the basis of crystallographic and fluorescence measurements, this 13-residue segment of the enzyme is presumed to be disordered in the unliganded enzyme. When the product or product analogue is bound, a C-terminal alpha-helix is observed in crystal structures. Conflicting data exists regarding the structure of this region when one of the substrates, glutathione (GSH), is bound. The NMR studies presented here show that in the unliganded protein, this region of the protein samples different conformations, most likely an ensemble of helix-like structures. Addition of either GSH or the conjugate between GSH and ethacrynic acid (EASG) causes this segment to become a stable alpha-helix. In the GSH complex, the ends of this helix exhibit dynamic behavior on both the millisecond and nanosecond time scales. In contrast, there is no evidence of millisecond dynamics in the EASG complex. The ligand-induced ordering of the enzyme reduces the intrinsic affinity of the enzyme for its product, facilitating enzymatic turnover. Publication Types: Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. PMID: 15182170 [PubMed - indexed for MEDLINE]