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Genetically encoded sensors of glutamate concentration are based on FRET between cyan and yellow fluorescent proteins bracketing a bacterial glutamate-binding protein. Such sensors have yet to find quantitative applications in neurons, because of poor response amplitude in physiological buffers or when expressed on the neuronal cell surface. We have improved our glutamate-sensing fluorescent reporter (GluSnFR) by systematic optimization of linker sequences and glutamate affinities. Using SuperGluSnFR, which exhibits a 6.2-fold increase in response magnitude over the original GluSnFR, we demonstrate quantitative optical measurements of the time course of synaptic glutamate release, spillover, and reuptake in cultured hippocampal neurons with centisecond temporal and spine-sized spatial resolution. During burst firing, functionally significant spillover persists for hundreds of milliseconds. These glutamate levels appear sufficient to prime NMDA receptors, potentially affecting dendritic spike initiation and computation. Stimulation frequency-dependent modulation of spillover suggests a mechanism for nonsynaptic neuronal communication. fluorescence resonance energy transfer | hippocampal neurons | synaptic release

The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe here the properties of a glutamate photoswitch that controls an ion channel in cells. Affinity labeling and geometric constraints ensure that the photoswitch controls only the targeted channel, and enables spatial patterns of light to favor labeling in one location over another. Photoswitching to the activating state places a tethered glutamate at a high (millimolar) effective local concentration near the binding site. The fraction of active channels can be set in an analog manner by altering the photostationary state with different wavelengths. The bistable photoswitch can be turned on with millisecond-long pulses at one wavelength, remain on in the dark for minutes, and turned off with millisecond long pulses at the other wavelength, yielding sustained activation with minimal irradiation. The system provides rapid, reversible remote control of protein function that is selective without orthogonal chemistry. azobenzene | ion channel | photoisomerization | optical switch | remote control

The solution conformation of poly-L-lysine (PLL) and poly-L-glutamic acid (PGA) in their non-alpha-helical states is examined by using the UV resonance Raman (UVR) spectroscopy. A method is also developed to estimate the distribution of Psi Ramachandran angles for these conformations, which are used to estimate a Psi Ramachandran angle energy landscape.

The application of infrared scanning near-field microscopy to detect the spatial distribution of [alpha]-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid-type glutamate receptor clusters on hippocampal neurons is described. The results obtained showed that the combination of IR absorption and near-field scanning optical microscopy facilitates the detection of specific molecules at high spatial resolution without any photobleaching problem.

A combination of visible and infrared spectroscopy is used to explore the difference in the protonation state of a glutamic acid residue located at position 142 (Glu142) on the extracellular side of helix D. Glu142 in the green- and blue-absorbing proteorhodopsin (GPR and BPR) exhibits three distinctive photocycles depending upon the pH of its native marine environment and changes in pH might modulate its function in the cell.

The specific optical rotation of D- and L-enantiomers of alanine, aspartic acid, glutamic acid, glutamine, proline, threonine, and tryptophan are measured in aqueous solutions of a series of some monovalent electrolytes. Specific interactions of anions with the chiral shell are responsible for the effects and conformational changes in the chiral solutes are due to the presence of ionic species.

A study of the pH-dependent conformational changes in the well-characterized globular protein, bovine [beta]-lactoglobulin A (BLGA) which involves mostly the loop connecting strands E and F and deprotonation of a glutamic acid residue (Glu89), is carried out using fluorescence spectroscopy. It is concluded that the fluorescence of Trp 19 has the features of a more polar environment as compared to structural models, that a minimum of two unique rotamers of Trp19 contribute to the fluorescence of the protein and predicts the fluorescence lifetime of Trp61 in the unquenched form.

The diffusion coefficient (D) of poly-L-glutamic acid (PLG) at various pHs is investigated by the laser-induced transient-grating method with new photoreactive probe molecule. The pH dependence of D, which is compared with that of the helical content of PLG measured by circular dichroism, are very similar.