There is fundamental gap in the understanding of the factors causing quenched and blue-shifted fluorescence emission of tryptophan in proteins. The long term goal is to define a set of spectroscopic markers such that any tryptophan environment and structure can be readily deciphered and quantified. The objective of this proposal is to understand how the disparate factors of pH, in the case of short tryptophan peptides, and hydrophobicity and steric hindrance, in the case of proteinaceous tryptophans, both result in blue-shifted fluorescence emission spectra with reduced emission intensity. The central hypothesis of this application is that UV resonance Raman vibrational bands for tryptophan can be used to decipher the underlying structural factors that cause the atypical blue fluorescence features seen in both proteinaceous tryptophan and tryptophan peptide fluorescence emission spectra. UV resonance Raman bands are sharper, and better-resolved than fluorescence emission bands, and many have already been associated with specific tryptophan features such as hydrogen bonding at the indole amine. The rationale for the proposed research is that once the underlying structural features and environment responsible for the quenched, blue fluorescence of tryptophan is made explicit, protein characterization by fluorescence emission spectroscopy will yield more useful and explicit information: so-called structural markers. In the context of strong preliminary data, we plan to test our central hypothesis and accomplish the objectives of this application by pursuing the following two specific aims: 1) Determine the structural motifs common to blue-fluorescing tryptophan peptides and proteins;and 2) Resolve the overlap of 1Bb, 1La and 1Lb transition dipole moment contributions to the absorption bands of blue- fluorescing tryptophan peptides and proteins. Under the first aim, a set of blue-fluorescing peptides, a complementary set of red-shifted peptides and blue-fluorescing proteins will be studied by UV resonance Raman spectroscopy. Guided by known UV resonance Raman band-structure associations, a pattern of factors contributing to the blue-shift of fluorescence are expected from analysis. Under the second aim, fluorescence excitation anisotropy measurements on the tryptophan-containing peptides will be used to resolve the contribution of the 1Bb, 1La and 1Lb transition dipole moments to the 220 nm and 280 nm absorption bands. This proposal is innovative because UV resonance Raman results have not been applied systematically to the study of blue-fluorescing proteins. Also, the energy profile and position of the high energy 1Bb transition dipole moment has not previously been included in studies of the 1La and 1Lb absorption band profile. The proposed research is significant because a higher level of understanding of the features seen in the fluorescence emission spectra of tryptophan-bearing proteins will result. Therefore, this study will have a positive impact on future studies of normal and mutant protein structure, and therefore influence drug design to combat disease resulting from aberrant proteins.
Mutant and misfolded proteins are disease-causing agents in humans. In order to design drugs to treat protein-related diseases, the structure and function of the aberrant proteins must be first understood. This study aims to advance the understanding of disease-related proteins through development of the technique, fluorescence emission spectroscopy, which probes the structure and environment of the naturally-occurring fluorescent amino acid, tryptophan.
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|Eisenberg, Azaria Solomon; Juszczak, Laura J (2013) Relating Trp-Glu dipeptide fluorescence to molecular conformation: the role of the discrete Chi 1 and Chi 2 angles. J Comput Chem 34:1549-60|
|Meng, Xianwei; Harricharran, Trisheena; Juszczak, Laura J (2013) A spectroscopic survey of substituted indoles reveals consequences of a stabilized 1Lb transition. Photochem Photobiol 89:40-50|
|Eisenberg, Azaria Solomon; Juszczak, Laura J (2012) Correlation of TrpGly and GlyTrp Rotamer Structure with W7 and W10 UV Resonance Raman Modes and Fluorescence Emission Shifts. J Amino Acids 2012:735076|