Trichloroethylene (TCE) is the most frequently detected groundwater contaminant, and 1-naphthol is an important chemical manufacturing intermediate. Directed evolution was used to increase the activity of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 for both chlorinated ethenes and naphthalene oxidation. When expressed in Escherichia coli, the variant TOM-Green degraded TCE (2.5...

Time-resolved fluorescence dynamics are investigated in two mutants of a thermophilic alcohol dehydrogenase (ht-ADH): Y25A (at the dimer interface) and V260A (at the cofactor-binding domain). These residues, ca. 32 Å apart, are shown to exhibit opposing low-temperature effects on the hydride tunneling step. Using single-tryptophan constructs at the active site (Trp87) and a remote, surface-expo...

Considerable evidence has accumulated that structural changes in dendritic spines and their synapses are associated with adaptive functional changes in cortical circuits, such as during circuit refinement in young animals and in learning and memory in adults. Understanding the mechanisms of circuit plasticity requires detailed investigation of the structural dynamics of dendritic spines and how...

I discovered the green fluorescent protein GFP from the jellyfish Aequorea aequorea in 1961 as a byproduct of the Ca-sensitive photoprotein aequorin (Shimomura et al., 1962; Johnson et al., 1962), and identified its chromophore in 1979 (Shimomura, 1979). GFP was a beautiful protein but it remained useless for the next 30 years after the discovery. My story begins in 1945, the year the city of N...

The green fluorescent protein (GFP) from the jellyfish Aequorea Victoria forms an intrinsic chromophore through cyclization and oxidation of an internal tripeptide motif [Prasher, D. C., et al. (1992) Gene 111, 229-233; Cody, C. E., et al. (1993) Biochemistry 32, 1212-1218]. We monitored the formation of the chromophore in vitro using the S65T-GFP chromophore mutant. S65T-GFP recovered from inc...

In the few years since its gene was first cloned, the Aequorea victoria green fluorescent protein (GFP) has become a powerful tool in cell biology, functioning as a marker for gene expression, protein localization and protein dynamics in living cells. GFP variants with improved fluorescence intensity and altered spectral characteristics have been identified, but additional GFP variants are stil...

A computer-based modeling and bench-top experiments are combined to understand the fluorescence of GFP. Random octapeptides are inserted into individual loops of the GFP. Amino acid sequences and fluorescence levels of clones from each loop are determined. The effect of peptide insertions into the loop regions of GFP are studied computationally using quantum mechanics and molecular dynamics cal...

We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long a...