Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein.

A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.

Zinc coordination geometry and ligand binding affinity: the structural and kinetic analysis of the second-shell serine 228 residue and the methionine 180 residue of the aminopeptidase from Vibrio proteolyticus.

The chemical properties of zinc make it an ideal metal to study the role of coordination strain in enzymatic rate enhancement. The zinc ion and the protein residues that are bound directly to the zinc ion represent a functional charge/dipole complex, and polarization of this complex, which translates to coordination distortion, may tune electrophilicity, and hence, reactivity. Conserved protein residues outside of the charge/dipole complex, such as second-shell residues, may play a role in supporting the electronic strain produced as a consequence of functional polarization. To test the correlation between charge/dipole polarity and ligand binding affinity, structure-function studies were carried out on the dizinc aminopeptidase from Vibrio proteolyticus. Alanine substitutions of S228 and M180 resulted in catalytically diminished enzymes whose crystal structures show very little change in the positions of the metal ions and the protein residues. However, more detailed inspections of the crystal structures show small positional changes that account for differences in the zinc ion coordination geometry. Measurements of the binding affinity of leucine phosphonic acid, a transition state analogue, and leucine, a product, show a correlation between coordination geometry and ligand binding affinity. These results suggest that the coordination number and polarity may tune the electrophilicity of zinc. This may have provided the evolving enzyme with the ability to discriminate between reaction coordinate species.