structure-solution-fluorescent-protein-cerulean-using

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

Structural Biology Group

Univ. Grenoble Alpes, CNRS, CEA, IBS (Institut de Biologie Structurale)

The green fluorescent protein (GFP) from the jellyfish Aequoria victoria has been shown to dimerize at high concentrations, which could lead to artefacts in imaging experiments. To ensure a truly monomeric state, an A206K mutation has been introduced into most of its widely used variants, with minimal effect on the spectroscopic properties. Here, the first structure of one of these variants, the cyan fluorescent protein mTurquoise, is presented and compared with that of its dimeric version mTurquoise-K206A. No significant structural change is detected in the chromophore cavity, reinforcing the notion that this mutation is spectroscopically silent and validating that the structural analysis performed on dimeric mutants also applies to monomeric versions. Finally, it is explained why cyan versions of GFP containing the Y66W and N146I mutations do not require the A206K mutation to prevent dimerization at high concentrations.

Structure of a fluorescent protein from Aequorea victoria bearing the obligate-monomer mutation A206K.

Cryoprotection of a protein crystal by addition of small-molecule compounds may sometimes affect the structure of its active site. The spectroscopic and structural effects of the two cryoprotectants glycerol and ethylene glycol on the cyan fluorescent protein Cerulean were investigated. While glycerol had almost no noticeable effect, ethylene glycol was shown to induce a systematic red shift of the UV-vis absorption and fluorescence emission spectra. Additionally, ethylene glycol molecules were shown to enter the core of the protein, with one of them binding in close vicinity to the chromophore, which provides a sound explanation for the observed spectroscopic changes. These results highlight the need to systematically record spectroscopic data on crystals of light-absorbing proteins and reinforce the notion that fluorescent proteins must not been seen as rigid structures.

Alteration of fluorescent protein spectroscopic properties upon cryoprotection.

Glycosyltransferases (GTs) catalyse the sequential addition of monosaccharides to specific acceptor molecules and play major roles in key biological processes. GTs are classified into two main families depending on the inverted or retained stereochemistry of the glycosidic bond formed during the reaction. While the mechanism of inverting enzymes is well characterized, the precise nature of retaining GTs is still a matter of much debate. In an attempt to clarify this issue, studies were initiated to identify reaction-intermediate states by using a crystallographic approach based on caged substrates. In this paper, two distinct structures of AA(Gly)B, a dual-specificity blood group synthase, are described in complex with a UDP-galactose derivative in which the O6'' atom is protected by a 2-nitrobenzyl group. The distinct conformations of the caged substrate in both structures of the enzyme illustrate the highly dynamic nature of its active site. An attempt was also made to photolyse the caged compound at low temperature, which unfortunately is not possible without damaging the uracil group as well. These results pave the way for kinetic crystallography experiments aiming at trapping and characterizing reaction-intermediate states in the mechanism of enzymatic glycosyl transfer. 

Structures of a human blood group glycosyltransferase in complex with a photo-activatable UDP-Gal derivative reveal two different binding conformations.

Antoine Royant

European Synchrotron Radiation Facility

Structural Biology Group

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

Antoine Royant

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}European Synchrotron Radiation Facility

Structural Biology Group

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

European Synchrotron Radiation Facility