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1.  Structural basis for activity of highly efficient RNA mimics of green fluorescent protein 
Green fluorescent protein (GFP) and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro evolved RNA mimic of GFP, which as genetically encoded fusions, makes possible live-cell, real-time imaging of biological RNAs, without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we have solved its co-crystal structure bound to its cognate exogenous chromophore, revealing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex, and an unpaired guanine. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs, and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure has guided the design of a miniaturized 'Baby Spinach', and provides the foundation for structure-driven design and tuning of fluorescent RNAs.
PMCID: PMC4143336  PMID: 25026079
2.  In-Vitro and Ex-Vivo Investigations of the Microtubule Binding Drug Targetin on Angiogenesis 
Intervention aimed at disrupting or inhibiting newly formed vascular network is highly desired to attenuate the progression of angiogenesis-dependent diseases. In cancer, this is tightly associated with the generation of VEGF by hypoxia inducible factor-1α following its activation by hypoxia. In light of the multiple cellular roles played by microtubules and their involvement in the processing of the hypoxia inducible factor-1α transcript, modulation of microtubule dynamics is emerging as a logical approach to suppress tumor reliance on angiogenesis. Targetin is a novel noscapinoid that interferes with microtubule dynamicity and inhibits the growth of cell lines from many types of cancers.
Methods and Results
Utilizing in-vitro and ex-vivo angiogenic models, we discovered the vascular disrupting and anti-angiogenic properties of Targetin. Targetin disrupted pre-assembled capillary-like networks of human endothelial cells by severing cell-cell junctions, inhibiting endothelial cell proliferation and metabolic activity in the presence and absence of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Furthermore, we show that Targetin significantly inhibits the formation of neovasculature network sprouting from rat aortic explants stimulated with proangiogenic stimuli, namely VEGF or bFGF.
We conclude that Targetin is a potential clinically promising anti-angiogenic agent for the treatment of many diseases including cancers.
PMCID: PMC3991473  PMID: 24749126
Drug discovery; angiogenesis; microtubule; opioid
3.  Extending molecular-replacement solutions with SHELXE  
Under favourable circumstances, density modification and polyalanine tracing with SHELXE can be used to improve and validate potential solutions from molecular replacement.
Although the program SHELXE was originally intended for the experimental phasing of macromolecules, it can also prove useful for expanding a small protein fragment to an almost complete polyalanine trace of the structure, given a favourable combination of native data resolution (better than about 2.1 Å) and solvent content. A correlation coefficient (CC) of more than 25% between the native structure factors and those calculated from the polyalanine trace appears to be a reliable indicator of success and has already been exploited in a number of pipelines. Here, a more detailed account of this usage of SHELXE for molecular-replacement solutions is given.
PMCID: PMC3817699  PMID: 24189237
molecular replacement; density modification; autotracing; SHELX
4.  Structure and activity of the only human RNase T2 
Nucleic Acids Research  2012;40(17):8733-8742.
Mutations in the gene of human RNase T2 are associated with white matter disease of the human brain. Although brain abnormalities (bilateral temporal lobe cysts and multifocal white matter lesions) and clinical symptoms (psychomotor impairments, spasticity and epilepsy) are well characterized, the pathomechanism of RNase T2 deficiency remains unclear. RNase T2 is the only member of the Rh/T2/S family of acidic hydrolases in humans. In recent years, new functions such as tumor suppressing properties of RNase T2 have been reported that are independent of its catalytic activity. We determined the X-ray structure of human RNase T2 at 1.6 Å resolution. The α+β core fold shows high similarity to those of known T2 RNase structures from plants, while, in contrast, the external loop regions show distinct structural differences. The catalytic features of RNase T2 in presence of bivalent cations were analyzed and the structural consequences of known clinical mutations were investigated. Our data provide further insight into the function of human RNase T2 and may prove useful in understanding its mode of action independent of its enzymatic activity.
PMCID: PMC3458558  PMID: 22735700
5.  Enhanced rigid-bond restraints 
An extension is proposed to the rigid-bond description of atomic thermal motion in crystals.
The rigid-bond model [Hirshfeld (1976 ▶). Acta Cryst. A32, 239–244] states that the mean-square displacements of two atoms are equal in the direction of the bond joining them. This criterion is widely used for verification (as intended by Hirshfeld) and also as a restraint in structure refinement as suggested by Rollett [Crystallographic Computing (1970 ▶), edited by F. R. Ahmed et al., pp. 167–181. Copenhagen: Munksgaard]. By reformulating this condition, so that the relative motion of the two atoms is required to be perpendicular to the bond, the number of restraints that can be applied per anisotropic atom is increased from about one to about three. Application of this condition to 1,3-distances in addition to the 1,2-distances means that on average just over six restraints can be applied to the six anisotropic displacement parameters of each atom. This concept is tested against very high resolution data of a small peptide and employed as a restraint for protein refinement at more modest resolution (e.g. 1.7 Å).
PMCID: PMC3377366
rigid-bond test; refinement restraints; anisotropic displacement parameters
6.  ANODE: anomalous and heavy-atom density calculation 
Journal of Applied Crystallography  2011;44(Pt 6):1285-1287.
The program ANODE determines anomalous (or heavy-atom) densities by reversing the usual procedure for experimental phase determination. Instead of adding a phase shift to the heavy-atom phases to obtain a starting value for the native protein phase, this phase shift is subtracted from the native phase to obtain the heavy-atom substructure phase.
The new program ANODE estimates anomalous or heavy-atom density by reversing the usual procedure for experimental phase determination by methods such as single- and multiple-wavelength anomalous diffraction and single isomorphous replacement anomalous scattering. Instead of adding a phase shift to the heavy-atom phases to obtain a starting value for the native protein phase, this phase shift is subtracted from the native phase to obtain the heavy-atom substructure phase. The required native phase is calculated from the information in a Protein Data Bank file of the structure. The resulting density enables even very weak anomalous scatterers such as sulfur to be located. Potential applications include the identification of unknown atoms and the validation of molecular replacement solutions.
PMCID: PMC3246834  PMID: 22477786
anomalous density; heavy-atom density; experimental phasing; computer programs

Results 1-6 (6)