Triplet state of tryptophan in proteins. 2. Differentiation between tryptophan residues 62 and 108 in lysozyme

Abstract

We have used optically detected magnetic resonance (ODMR) to characterize the degree of solvent availability of the tryptophan residues in lysozyme that are likely to be responsible for the observed phosphorescence. From the phosphorescence spectra, ODMR zero-field splittings (zfs), and ODMR line widths, we concur with the X-ray structure [Blake, C. C., Mair, G. A., North, A. C. T., Phillips, D. C., & Sarma, V. R. (1967) Proc. R. Soc. London, ser. B 167, 365-377] that Trp-62 behaves as an exposed residue and Trp-108 is buried. In addition, we present evidence that ODMR can be used in conjunction with conventional phosphorescence to evaluate the degree of order in the microenvironments of tryptophan in a protein containing several tryptophans. By the specific modification of residues Trp-62 and Trp-108, we have identified those portions of the ODMR lines in the native enzyme that are due to those specific residues. Barring major enzyme conformational changes in the vicinity of unmodified tryptophan residues when Trp-62 or Trp-108 are selectively modified, we find that Trp-108 dominates both the phosphorescence and the ODMR signals in native lysozyme. The results are discussed in view of previous fluorescence findings.

Algorithm for the Analysis of Tryptophan Fluorescence Spectra and Their Correlation with Protein Structural Parameters

“…The methods of spectral analysis allow extraction of the fluorescence properties of individual tryptophan residues, thus creating an opportunity to investigate the spectral-structural relationship. More than 35 years ago the first attempts were made to reveal a correlation between fluorescence parameters and structural characteristics of the tryptophan fluorophore's environment based on the atomic structures of several proteins [54][55][56]. Since then rapid progress in X-ray crystallography and NMR spectroscopy methods have led to an increase in the number of works where the measured fluorescence properties of individual proteins were analyzed in relation to structural features [57][58][59][60][61][62][63][64][65][66].…”

Decomposition of Protein Tryptophan Fluorescence Spectra into Log-Normal Components. III. Correlation between Fluorescence and Microenvironment Parameters of Individual Tryptophan Residues

“…That is why one can observe the rousing interest in analyzing tryptophan location and its surrounding in proteins revealed by x-ray crystallography. More than 20 years ago the first attempts were done to reveal a correlation between emission spectral parameters and structural characteristics of tryptophan fluorophore environment based on atomic structures of several proteins (Pelley and Horowitz, 1976;Brown et al, 1977;Rousslang et al, 1979). The rapid progress in x-ray crystallography and NMR methods led to an increase in the amount of work on analyzing the measured fluorescence properties of individual proteins in relation to the structural features (for example, Turoverov et al, 1984Turoverov et al, , 1985Desie et al, 1986;Dolashka et al, 1992;Bhaskaran et al, 1996;Mely et al, 1997;Reshetnyak and Burstein, 1997a, b;Kuznetsova and Turoverov, 1998;Kuznetsova et al, 1999;Orlov et al, 1999;Turoverov, 1999).…”

Optically detected magnetic resonance of tryptophan triplet states in native and urea-denatured proteins and polypeptides

“…This is paper 3 in a series on the triplet state of tryptophan in proteins. Paper 2 is Rousslang et al (1979).…”