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Proceedings Paper

T4 phage lysozyme: a protein designed for understanding tryptophan photophysics
Author(s): Bruce S. Hudson; Dan Harris
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Paper Abstract

Bacteriophage T4 lysozyme in its wild type form contains three tryptophan residues (at sequence postions 126, 138 and 158). These three residues are in rather different environments in the protein: 126 and 158 are near the protein surface while residue 138 is more buried. T4 lysozyme has been genetically engineered to prepare all possible variants in which one or more of the tryptophan residues have been replaced by tyrosine. The available data supports the hypothesis that this substitution has, at most, a very minor effect on the structure of the protein. The three species with single tryptophan residues have been investigated in detail. The surface location of residue 126 compared to the buried location of residue 138 is reflected in the difference in collisional quenching observed with added potassium iodide. It is found that the spectral and radiative properties of the three proteins are very similar but that their radiationless decay properties are quite distinct. This is apparently due to short-range collisional quenching by neighboring side chains. Comparison with solution quenching measurements permits the identification of the specific quenching groups involved for each tryptophan residue and provides a semi-quantitative rationale for the radiationless decay rate. This collisional quenching interpretation is supported by mutational effects on fluorescence quantum yield. This simple picture of the behavior of these single-tryptophan proteins is clearly revealed in this particular case because of the unambiguous choice of collisional quenching groups. The time dependence of the fluorescence decay of each of these single-tryptophan proteins is quite complex. Several methods of analysis are presented and discussed in terms of their underlying physical basis. Internal collisional quenching, as suggested from the comparative studies, is expected to lead to non-exponential behavior. This is consistent with the observed time dependence. Analysis of the temporal nature of the fluorescence as a function of emission wavelength is also revealing. Such data can be used to test discrete component, distribution and relaxation models of the time decay. It is found, in agreement with previous studies for other proteins, that the average lifetime for the emission increases with increasing emission wavelength. Analysis of the overall emission wavelength dependence of the time dependent data in a global sense based on a discrete population model shows acceptable agreement with the data in only one of the three cases. Application of several continuous distribution models to this data at each emission wavelength reveals that as the emission is moved to the red, a negative component appears in the distribution of decay components. This is a characteristic feature of relaxation behavior resulting in emission from kinetic species that are not present at the time of excitation. This negative preexponential character is not revealed by discrete component analyses since these do not have sufficient flexibility to describe the underlying complexity of the relaxing distribution. Finally, examination of the three proteins containing two tryptophan residues indicates that there is energy transfer between these residues in these cases and in the wild type protein. The order of energy transfer is in accord with the variation of the magnitude of the ratio k2/R6 controlling the efficiency of Forster energy transfer.

Paper Details

Date Published: 1 May 1990
PDF: 12 pages
Proc. SPIE 1204, Time-Resolved Laser Spectroscopy in Biochemistry II, (1 May 1990); doi: 10.1117/12.17690
Show Author Affiliations
Bruce S. Hudson, Univ. of Oregon (United States)
Dan Harris, Univ. of Oregon (United States)


Published in SPIE Proceedings Vol. 1204:
Time-Resolved Laser Spectroscopy in Biochemistry II
Joseph R. Lakowicz, Editor(s)

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