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Acta Cryst. (2014). A70, C1164
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RNA aptamers are structured single-stranded oligonucleotides selected to bind tightly and specifically to a broad spectrum of biomolecular targets. The structural stability and diverse functionality of aptamers have enabled their use as diagnostic tools, inhibitors and potential therapeutic agents. However, since very few attempts at solving atomic structures of protein-aptamer complexes have succeeded, surprisingly little is known about how aptamers specifically bind to selected regions on the surface of proteins and cells. We show that aptamers can be effectively minimized for structural analysis using chemical mapping to experimentally define the secondary structure and identify tertiary contacts within the RNA and with the target protein. Ribonuclease and SHAPE mapping were used to determine the correct predicted secondary structure of a high affinity aptamer (Lys1) selected against lysozyme (KD ~ 30 nM). A deletion variant, minE (KD ~ 20 nM), was engineered to delete a long, apparently unstructured region. The lysozyme-minE complex was determined by x-ray crystallography at a 2.0 Å resolution, yielding a seventh RNA aptamer-protein structure. Solution hydroxyl-radical footprinting confirms the binding interface observed in the crystal. Although the minE aptamer interacts with a positively charged face of lysozyme, the electrostatic contribution to the binding free energy is minimal. The minE aptamer was found to inhibit the function of lysozyme in the standard cell-wall hydrolysis assay - a surprising result since the aptamer binding site is quite far from the catalytic site, and no structural differences between free lysozyme and that in complex with the aptamer could be detected. The long term goal of this study is to develop a systematic approach to aptamer minimization and use solved structures to probe the mechanisms by which RNA aptamers bind their targets and regulate catalytic activity and/or cellular function.

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Acta Cryst. (2014). A70, C1670
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Fluorescent proteins (FPs) have become valuable tools for molecular biology, biochemistry, medicine, and cancer research. Starting from parent green fluorescent protein (GFP), most challenging task of the FPs studies was the development of FPs with longer excitation/emission wavelength. This pursuit was motivated by advantages of so-called red-shifted FPs, namely, lower background of cellular autofluorescence in microscopy, lower light scattering and reduced tissue absorbance of longer wavelengths for in vivo imaging. In addition to FPs with regular spectral properties, there are proteins of other types available, including FPs with a large Stokes shift and photoconvertible FPs. These special kinds of FPs have become useful in super-resolution microscopy, imaging of enzyme activities, protein-protein interactions, photolabeling, and in vivo imaging. According to their emission wavelength, red-shifted FPs could be divided in the following groups: 520-540 nm yellow FPs (YFPs), 540-570 nm orange FPs (OFPs), 570-620 nm red FPs (RFPs), and > 620 nm far-RFPs. Red shift of the excitation/emission bands of these FPs is predominantly achieved by extension of the conjugated system of the chromophore and its protonation/deprotonation. The variety of spectral properties of FPs (excitation and emission wavelength, quantum yield, brightness, photo- and pH- stability, photoconversion, large Stokes shift, etc) results from the different chromophore structures and its interactions with surrounding amino acid residues. In this work we focus on structural studies and molecular mechanisms of FPs with orange emission.
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