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We have used a tryptophan-requiiing Escherichia coli auxotroph to replace the three tryptophan residues of A cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved >95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-A cI are clearly distinct from A cI repressor and that the fluorescence of 5-OHTrp-A cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-A cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins wiil significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.Tryptophan fluorescence has been used widely to study conformation, function, dynamics, and intermolecular interactions of proteins (1). However, the presence of at least one and often several tryptophans in most proteins severely limits the utility oftryptophan fluorescence for studying one protein species in the company of others. For this reason, many fluorescence studies involving proteins and polypeptides make use of extrinsic probes, such as dansyl or fluorescein. Use of extrinsic probes, however, is complicated by the difficulty of specific placement and the risk that chemical modification can alter the functional and structural properties of the labeled protein.Clearly, it would be to great advantage to incorporate into a protein a fluorophore that is spectrally distinct from tryptophan while having none of the disadvantages of extrinsic probes. In this way, a spectrally enhanced protein (SEP) would be generated that would still have "intrinsic" fluorescence. To be useful for the generation of SEPs, a candidate tryptophan analogue mlist be readily incorporated by biosynthesis into the protein ofinterest, must be fluorescent and have spectral properties distinct from those oftryptophan, and must have little or no effect on the functional and structural properties of the protein into which it has been incorporated.An intriguing possibility for creation of SEPs is suggested by NMR studies in which tryptophan auxotrophs of Escherichia coli were used to incorporate fluorotryptophan derivatives into Salmonella typhimurium histidine binding protein J (2) and rat cellular retinol binding protein (3). The fluorotryptophan derivatives, however, are spectrally very similar to tryptophan. Thus, they do not have features that enhance fluorescence studies. By contrast, the absorbance spectra of another group of tryptophan derivatives, the hydroxytryptophans, are sufficiently red-shifted to allow selective excitation in the presence of tryptophan. In addition, several hydroxytryptophan derivatives have ...

Dementia has become an all-important disease because the population is aging rapidly and the cost of health care associated with dementia is ever increasing. In addition to cognitive function impairment, associated behavioral and psychological symptoms of dementia (BPSD) worsen patient’s quality of life and increase caregiver’s burden. Alzheimer’s disease is the most common type of dementia and both behavioral disturbance and cognitive impairment of Alzheimer’s disease are thought to be associated with the N-methyl-D-aspartate (NMDA) dysfunction as increasing evidence of dysfunctional glutamatergic neurotransmission had been reported in behavioral changes and cognitive decline in Alzheimer’s disease. We review the literature regarding dementia (especially Alzheimer’s disease), BPSD and relevant findings on glutamatergic and NMDA neurotransmission, including the effects of memantine, a NMDA receptor antagonist, and NMDA-enhancing agents, such as D-serine and D-cycloserine. Literatures suggest that behavioral disturbance and cognitive impairment of Alzheimer’s disease may be associated with excitatory neurotoxic effects which result in impairment of neuronal plasticity and degenerative processes. Memantine shows benefits in improving cognition, function, agitation/aggression and delusion in Alzheimer’s disease. On the other hand, some NMDA modulators which enhance NMDA function through the co-agonist binding site can also improve cognitive function and psychotic symptoms. We propose that modulating NMDA neurotransmission is effective in treating behavioral and psychological symptoms of Alzheimer’s disease. Prospective study using NMDA enhancers in patients with Alzheimer’s disease and associated behavioral disturbance is needed to verify this hypothesis.

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