INTRINSIC FLUORESCENCE OF PROTEINS

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A limitation of intrinsic protein absorbance and fluorescence is the inability to distinguish a specific protein or polypeptide against a background of spectrally similar molecules. We have shown that a tryptophan auxotroph of E coli can utilize 5-hydroxytryptophan (5-OHTrp) to replace tryptophan in vivo, generating a spectrally enhanced protein (SEP). This metabolically engineered protein, bacteriophage lambda cI repressor, is functionally indistinguishable from wild-type protein both in terms of protein-protein interactions and cooperative binding to operator DNA. Using this SEP to study the effects of DNA on self-assembly of repressor, we have shown that the high order oligomeric unit is an octamer that is competent to bind operator DNA. Thus, the currently - accepted hypothesis for the mechanism of control of cellular development by this important DNA-binding protein in response to external signals needs to be re-evaluated. The first aim of this proposal is to investigate the role of protein-protein and protein-DNA interactions in the function of lambda cI repressor. We will use wild-type and mutant repressor, containing tryptophan or tryptophan analogues, such as 5-OHTrp, to generate SEPS. Specific DNA interactions will be investigated using the N-terminal domain of the repressor for which an x-ray crystal structure is known. Cooperative interactions will be investigated using full-length repressor. RNA polymerase - lambda cI repressor interactions will also be examined. A multi-disciplinary approach will be used. Homomeric and heteromeric protein-DNA assemblies will be studied by fluorescence resonance energy transfer, fluorescence anisotropy, and ultracentrifugation. Footprint analysis will be used to examine the thermodynamics of DNA binding by full-length repressors, while fluorescence methods will assess binding by N-terminal domain constructs. Data interpretation and analysis of specific N-terminal domain mutations, including SEPs, will be assisted by molecular modeling. The second aim of this proposal is to continue our studies on the photophysics of the aromatic amino acids and to apply this knowledge to the characterization of specific protein systems, such as lambda cI repressor. We will study the fluorescence intensity decay of the aromatic amino acids and the tryptophan analogues used for SEPs in the context of model compounds, small peptides, and larger polypeptides of known tertiary structure including the N-terminal domain of the repressor. Hypotheses, including the side chain rotamer model, will be evaluated with respect to the data, structure, and photophysics. NMR experiments will provide independent information about side chain conformations and populations. These studies will permit us to fully interpret the data obtained on the repressor and its SEP analogues (aim 1).