Carol A. Caperelli

Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy

NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is ~40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.

Enantiospecific Synthesis of Carbocyclic Aminoimidazole Carboxamide Ribonucleotide (C-AICAR), Succinoaminoimidazole Carboxamide Ribonucleotide (C-SAICAR), and a New Intermediate for SAICAR Analogs

Abstract The (-)-enantiomer of the carbocyclic analogs of aminoimidazole carboxamide ribonucleotide (C-AICAR1, 7), and succinoaminoimidazole carboxamide ribonucleotide (C-SAICAR, 14) have been prepared. En route, a new intermediate (19) for the preparation of SAICAR analogs was developed.

1H, 13C and 15N resonance assignment of the N-terminal domain of human lysyl aminoacyl tRNA synthetase

Human lysyl aminoacyl tRNA synthetase (hLysRS) is integral to a variety of different functions ranging from protein biosynthesis, initiation of a proinflammatory response as well as signal transduction. Another important, non-canonical function of hLysRS is that it chaperones tRNALys,3, the HIV-1 reverse transcription primer molecule into new HIV-1 particles. Since the N-terminal domain of hLysRS has been shown to be essential for such primer uptake, NMR studies of this domain are being conducted to obtain a better understanding of how hLysRS interacts with the primer tRNA. In order to study the RNA binding behavior of this domain, we are studying its complex with a fragment of the cognate tRNA corresponding to the tRNA anticodon loop. We report herein the backbone and side chain NMR resonance assignments of uniformly 15N-, 13C-labeled hLysRS N-terminal domain alone, as well as complexed to RNA.

Synthesis of the Optically Active Carbocyclic Analogs of the Four 2′-Deoxyribonucleoside Monophosphates

Abstract The (+)-enantiomer of the carbocyclic analogs of the four 2′-deoxyribonucleoside monophosphate constituents of DNA, C-dAMP2 (1: A), C-dGMP (1: G), C-dCMP (1: C), and C-TMP (1: T) have been synthesized via the Mitsunobu coupling reaction. Two new N3-protected thymines were developed en route.

Exploring the Hexokinase Glucose Binding Site Through Correlation Analysis and Molecular Modeling of Glucosamine Inhibitors

A series of N-substituted glucosamines has been designed, synthesized, and tested as inhibitors of yeast hexokinase. All derivatives exhibited competitive inhibition kinetics with respect to glucose. Quantitative structure-activity relationships were derived from the resulting inhibition data. The most significant equation demonstrated the existence of highly specific steric effects for the seven meta-substituted benzoylglucosamines included in the relationship. Molecular modeling of potential complexes between the inhibitors and the hexokinase substrate binding site strongly suggests that the steric effects arise from potential contacts with two amino acid residues lying in the region occupied by the amide substituents.

SYNTHESIS OF CHIRAL CARBOCYCLIC RIBONUCLEOTIDES

The carbocyclic analogs of CMP, UMP, GMP, IMP, and ribo-TMP, of the same absolute configuration as the naturally occurring beta-D-ribofuranose-based ribonucleoside monophosphates, have been synthesized. The synthetic route employed Mitsunobu coupling of the heterocycles, appropriately protected where necessary, with a differentially protected, chiral carbocyclic core.

CARBOCYCLIC SUBSTRATES AND INHIBITORS FOR THE BIFUNCTIONAL LYASE OF PURINE NUCLEOTIDE BIOSYNTHESIS

Abstract The carbocyclic analogs of succinoaminoimidazole carboxamide ribonucleotide (SAICAR) and adenylosuccinate (SAMP) are substrates for the bifunctional lyase of purine biosynthesis, which catalyzes the elimination of fumarate from both SAICAR and SAMP to generate aminoimidazole carboxamide ribonucleotide (AICAR) and AMP, respectively. The glutamate analogs of both ribo- and carbo-SAICAR are inhibitors.

PHOSPHONATE ANALOGS OF CARBOCYCLIC PHOSPHORIBOSYLAMINE AND CARBOCYCLIC GLYCINAMIDE RIBONUCLEOTIDE

Abstract Analogs of intermediates in the de novo purine nucleotide biosynthetic pathway were synthesized to study the binding requirements of the corresponding enzymes. Because of the instability of the natural stubstrates, such as phosphoribosylamine, the use of the structurally stable phosphonate moiety and the carbocyclic ribose yields ideal analogs for these studies. In addition, these analogs can act as potential inhibitors of the de novo pathway leading to the design of anticancer agents. Enzyme studies with GAR synthetase and GAR transformylase reveal that the title compounds can act as substrates or inhibitors of the de novo enzymes.

Glycinamide ribonucleotide analogue probes for glycinamide ribonucleotide transformylase

Abstract Glycinamide ribonucleotide (GAR) transformylase catalyzes the conversion of glycinamide ribonucleotide and 10-formyltetrahydrofolate to formylglycinamide ribonucleotide and tetrahydrofolate. This reaction constitutes the third step in de novo purine biosynthesis. A series of glycinamide ribonucleotide analogues, in which the glycinamide side chain (R = CH 2 NH 2 ) has been replaced by R = CH 2 Br, CH 2 Cl, CH 2 CN, CHN 2 , CHClCH 2 NH 2 , and , has been prepared. All of these analogues were inhibitors of GAR transformylase, competitive against GAR, but none of these proved to be enzyme inactivators. Neither R = CHClCH 2 NH 2 nor served as substrates for the enzyme-catalyzed transformylation reaction.