Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.

We have successfully designed and expressed a new fluorescent protein with improved second-order nonlinear optical properties. It is the first time that a fluorescent protein has been rationally altered for this particular characteristic. On the basis of the specific noncentrosymmetry requirements for second-order nonlinear optical effects, we had hypothesized that the surprisingly low first hyperpolarizability (β) of the enhanced yellow fluorescent protein (eYFP) could be explained by centrosymmetric stacking of the chromophoric Tyr66 and the neighboring Tyr203 residue. The inversion center was removed by mutating Tyr203 into Phe203, with minor changes in the linear optical properties and even an improved fluorescence quantum yield. Structure determination by X-ray crystallography as well as linear optical characterization corroborate a correct folding and maturation. Measurement of β by means of hyper-Rayleigh scattering (HRS) as well as their analysis using quantum chemistry calculations validate our hypothesis. This observation can eventually lead to improved red fluorescent proteins for even better performance. On the basis of the specific function (second-harmonic generation), the color of its emission, and in analogy with the "fruit" names, we propose SHardonnay as the name for this Tyr203Phe mutant of eYFP.

Author(s) of this paper may load this reprint on their own web site or institutional repository provided that this cover page is retained. Republication of this article or its storage in electronic databases other than as specified above is not permitted without prior permission in writing from the IUCr.For further information see http://journals.iucr.org/services/authorrights.html Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure-function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. Papers on crystallographic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Papers on the crystallization of biological molecules will be accepted providing that these focus on new methods or other features that are of general importance or applicability.Crystallography Journals Online is available from journals.iucr.org Acta Cryst. (2012 The crystal structure of the on-state of PDM1-4, a singlemutation variant of the photochromic fluorescent protein Dronpa, is reported at 1.95 Å resolution. PDM1-4 is a Dronpa variant that possesses a slower off-switching rate than Dronpa and thus can effectively increase the image resolution in subdiffraction optical microscopy, although the precise molecular basis for this change has not been elucidated. This work shows that the Lys145Asn mutation in PDM1-4 stabilizes the interface available for dimerization, facilitating oligomerization of the protein. No significant changes were observed in the chromophore environment of PDM1-4 compared with Dronpa, and the ensemble absorption and emission properties of PDM1-4 were highly similar to those of Dronpa. It is proposed that the slower off-switching rate in PDM1-4 is caused by a decrease in the potential flexibility of certain -strands caused by oligomerization along the AC interface.

The crystal structures of cis-dichlorido(ethylamine-κN)(piperidine-κN)platinum(II), [PtCl2(C2H7N)(C5H11N)], (I), cis-dichlorido(3-methoxyaniline-κN)(piperidine-κN)platinum(II), [PtCl2(C5H11N)(C7H9NO)], (II), and cis-dichlorido(piperidine-κN)(quinoline-κN)platinum(II), [PtCl2(C5H11N)(C9H7N)], (III), have been determined at 100 K in order to verify the influence of the nonpiperidine ligand on the geometry and crystal packing. The crystal packing is characterized by N-H...Cl hydrogen bonding, resulting in the formation of chains of molecules connected in a head-to-tail fashion. Hydrogen-bonding interactions play a major role in the packing of (I), where the chains further aggregate into planes, but less so in the case of (II) and (III), where π-π stacking interactions are of greater importance.

The crystal structures of three 5-alkenyl-2-arylthieno[3,2-b]thiophenes, namely 3,6-dibromo-5-(4-tert-butylstyryl)-2-(naphthalen-1-yl)thieno[3,2-b]thiophene, C(28)H(22)Br(2)S(2), (I), 3,6-dibromo-5-(4-methylstyryl)-2-(naphthalen-1-yl)thieno[3,2-b]thiophene, C(25)H(16)Br(2)S(2), (II), and 3,6-dibromo-2-(4-tert-butylphenyl)-5-(4-methylstyryl)thieno[3,2-b]thiophene, C(25)H(22)Br(2)S(2), (III), have been determined in order to evaluate the geometry of the molecules. The π-conjugated system containing the thieno[3,2-b]thiophene skeleton, the ethylene bridge and the phenyl rings is almost planar. The aromatic ring directly attached to the thieno[3,2-b]thiophene moiety is not coplanar with the thieno[3,2-b]thiophene moiety itself due to steric hindrance of the bromo substituent. The crystal packings are characterized by π-π stacking [only for (II)] and C-Br...π interactions. The long axes of the molecules in (I) are oriented in two directions; for the two other structures the long axis is oriented in one direction only.