Conformational Kinetics Study of MP-11 Using TIMS-MS and Molecular Dynamics

In the present work, we studied the conformational kinetics of microperoxidase 11 (MP-11), a digest fragment of cytochrome C that contains 11 amino acids with a covalently attached heme group. In particular, a novel technique recently developed at FIU in collaboration with Bruker Daltonics Inc. combined with theoretical calculation was used for the characterization of MP- 11 conformational space [1-3]. Accurate ion-neutral collision cross sections were measured for all MP-11 generated charge states. Since MP-11 (like cytochrome C) undergoes conformational changes as a function of the solvent state, MP-11 ions were produced by electrospray ionization (ESI) in order to preserve the initial solution state structure and analyzed on the basis of size-to-charge, inside the Trapped Ion Mobility Spectrometer (TIMS) followed by mass identification using a time-of-flight mass analyzer (MS) [4-5]. TIMS-MS has the advantage that molecular ions can be trapped for several seconds which allow us to study the kinetics and stability of various isomers as a function of time, initial pH value (6.1, 4.5, 3.1), and molecular ion temperature. Results showed that MP-11 conformations vary with pH levels and trapping time, and multiple interconversion pathways were observed for [M+2H]+2 and [M+3H]+3 charge states. Candidate structures were proposed for each conformation observed and main molecular interactions responsible for the conformational changes are discussed

Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. In this paper we report the synthesis of a type of unique fluorescence-labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 carrying a 42 nt. AT sequence with fluorescein and dabcyl labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905. Since the fluorescence intensity of pAB1_FL905 is dependent on its supercoiling status, pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high-throughput screening assays to identify inhibitors that target various DNA topoisomerases

Strain Promoted Click Chemistry of 2- or 8-Azidopurine and 5-Azidopyrimidine Nucleosides and 8-Azidoadenosine Triphosphate with Cyclooctynes. Application to Living Cell Fluorescent Imaging

Strain-promoted click chemistry of nucleosides and nucleotides with an azido group directly attached to the purine and pyrimidine rings with various cyclooctynes in aqueous solution at ambient temperature resulted in efficient formation (3 min to 3 h) of fluorescent, light-up, triazole products. The 2- and 8-azidoadenine nucleosides reacted with fused cyclopropyl cyclooctyne, dibenzylcyclooctyne, or monofluorocyclooctyne to produce click products functionalized with hydroxyl, amino, N-hydroxysuccinimide, or biotin moieties. The 5-azidouridine and 5-azido-2\u27-deoxyuridine were similarly converted to the analogous triazole products in quantitative yields in less than 5 min. The 8-azido-ATP quantitatively afforded the triazole product with fused cyclopropyl cyclooctyne in aqueous acetonitrile (3 h). The novel triazole adducts at the 2- or 8-position of adenine or 5-position of uracil rings induce fluorescence properties which were used for direct imaging in MCF-7 cancer cells without the need for traditional fluorogenic reporters. FLIM of the triazole click adducts demonstrated their potential utility for dynamic measuring and tracking of signaling events inside single living cancer cells