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1.
Biochemistry ; 50(31): 6655-66, 2011 Aug 09.
Article in English | MEDLINE | ID: mdl-21714514

ABSTRACT

The sulfonylurea receptor 2A (SUR2A) is an ATP-binding cassette (ABC) protein that forms the regulatory subunit of ATP-sensitive potassium (K(ATP)) channels in the heart. ATP binding and hydrolysis at the SUR2A nucleotide binding domains (NBDs) control gating of K(ATP) channels, and mutations in the NBDs that affect ATP hydrolysis and cellular trafficking cause cardiovascular disorders. To date, there is limited information on the SUR2A NBDs and the effects of disease-causing mutations on their structure and interactions. Structural and biophysical studies of NBDs, especially from eukaryotic ABC proteins like SUR2A, have been hindered by low solubility of the isolated domains. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the precise definition of the domain boundaries. Putative boundaries of SUR2A NBD1 were identified by structure-based sequence alignments and subsequently tested by exploring the solubility of SUR2A NBD1 constructs with different N and C termini. We have determined boundaries of SUR2A NBD1 that allow for soluble heterologous expression of the protein, producing a folded domain with ATP binding activity. Surprisingly, our alignment and screening data indicate that SUR2A NBD1 contains two putative, previously unidentified, regulatory elements: a large insert within the ß-sheet subdomain and a C-terminal extension. Our approach, which combines the use of structure-based sequence alignments and predictions of disordered regions combined with biochemical and biophysical studies, may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , DNA-Binding Proteins/chemistry , Potassium Channels, Inwardly Rectifying/chemistry , Protein Folding , Receptors, Drug/chemistry , Regulatory Elements, Transcriptional , Response Elements , ATP-Binding Cassette Transporters/biosynthesis , ATP-Binding Cassette Transporters/genetics , Amino Acid Sequence , Animals , Crystallography, X-Ray , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , KATP Channels/chemistry , KATP Channels/genetics , KATP Channels/metabolism , Molecular Sequence Data , Potassium Channels, Inwardly Rectifying/biosynthesis , Potassium Channels, Inwardly Rectifying/genetics , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Rats , Receptors, Drug/biosynthesis , Receptors, Drug/genetics , Response Elements/genetics , Solubility , Sulfonylurea Receptors
2.
J Am Chem Soc ; 129(51): 15848-54, 2007 Dec 26.
Article in English | MEDLINE | ID: mdl-18052163

ABSTRACT

Aminoacylation of tRNA in cells involves activation of the amino acid as an aminoacyl adenylate, a mixed anhydride with AMP, which reacts with tRNA. We have now established that aminoacyl phosphate esters in the presence of lanthanide ions in water will acylate hydroxyls at the 3'-terminus of RNA or a simple nucleotide. By extension, this will permit synthetically aminoacylated tRNA to be produced in a single-step biomimetic process. The reactions of Boc-4-fluorophenylalanyl ethyl phosphate were followed by HPLC separation, MS, and 19F NMR analysis. In stoichiometric combination with lanthanum salts in aqueous buffer, Boc-4-fluorophenylalanyl ethyl phosphate rapidly produces 2'- and 3'-monoesters of cytidine and cytidine monophosphate. Reaction of the reagent with RNA in the presence of lanthanum and magnesium salts introduces a specifically detectable signal into the RNA, which is evidence of formation of the aminoacyl ester. When the same RNA is initially oxidized with periodate to convert the 3'-terminal vicinal diol to the cleaved dialdehyde, reaction with the aminoacyl phosphate no longer occurs as evidenced by the lack of a signal in the 19F NMR spectrum. The results are consistent with a requisite chelation mechanism in which lanthanum serves as a template for both the aminoacyl phosphate and the 3'-terminal diol of RNA and nucleotides. The coordinated diol will then react through specific base-catalyzed intramolecular addition of the alkoxide nucleophile to the acyl group of the aminoacyl phosphate. Assessment of the method with a single tRNA was also achieved using the fluorescent reagent N-dansyl-glycyl ethyl phosphate. Lanthanide-promoted aminoacylation at the 3'-terminus of tRNAPhe is detected by the introduction of fluorescence (detected directly and by antibody-enhanced emission). This does not occur if the 3'-terminus is converted to the dialdehyde by reaction with periodate.


Subject(s)
Lanthanum/chemistry , Molecular Mimicry , RNA/chemistry , Ribonucleotides/chemistry , Acylation , Chromatography, High Pressure Liquid , Esters , Nuclear Magnetic Resonance, Biomolecular
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