Proteins, ODMR

Phosphorescence and ODMR are additional spectroscopies that can be used to investigate intramolecular interactions that affect tyrosine residues in proteins and polypeptides/215,216) An example is tyrosine and tyrosinate in horse liver alcohol dehydrogenase.(202) The same approach has been used to study the role of tyrosine in the mechanism of action of carboxypeptidase B.(21/,218) jn botli these proteins, as in other proteins which contain both... 

A striking example of the potential sensitivity of ODMR is the report by two groups of magnetic resonance detection in a single molecule of pentacene using FDMR. It should be pointed out, however, that this accomplishment was possible because of the rather special properties of the pentacene excited states comparable sensitivity should not be expected with biopolymers. Nonetheless, PDMR of Trp in proteins, for example, is carried out successfully at the picomole level without difficulty. 

In the next section we take a quick look at the triplet state and discuss the origin and significance of the ZFS. This is followed by an outline of some of the ODMR experiments that are in common use to determine static and dynamic triplet state properties in biopolymers. After a description of experimental equipment and methods used in ODMR spectroscopy, we conclude with some examples that illustrate the wealth of information provided by the application of ODMR to the study of tryptophan triplet states in proteins. Because this chapter is intended to focus on methods, we present neither a historical development of ODMR spectroscopy, nor an extensive review of its application to biopolymer studies. We refer the interested reader, instead, to some early reviews on the general methods and results of ODMR spectroscopy, as well as to a book and to additional reviews that deal more specifically with biological applications of ODMR. Applications to photosynthetic pigments and the reaction centers, in particular, have been reviewed thoroughly by Hoff. - ... 

Fig. 7. Wavelength dependence of the ODMR signal frequencies in the phosphorescence 0,0-band region for native E. coli Trp repressor protein ( ) and the single point mutants WI9F (A) and W99F ( ). Sample compositions and conditions of excitation are given in the legend for Fig. 4. The sample temperature is 1.2 K, the emission bandwidth is 1.5 nm, and all data are corrected for passage effects. [From M. R. Eftink, G. D. Ramsay, L. Bums, A. H. Maki, C. J. Mann, C. R. Matthews, and C. A. Ghiron, Biochemistry 32, 9189 (1993), with permission.]...

The Trp repressor protein is unusual in that characterization of the local environments of Trp-19 and Trp-99 based on phosphorescence and ODMR characteristics is at variance with that based on fluorescence and the X-ray crystal structure. The latter methods suggest considerably solvent exposure of both sites. In all other proteins studied to date where comparison is possible, the ODMR and phosphorescence are found to be consistent with the character of the local Trp environment as revealed by the X-ray structure. We think that the discrepancy in Trp repressor protein may be the result of a structural change induced by sample cooling that relocates Trp-99 in a buried, hydrophobic environment. 

To our knowledge the first appheation of ODMR to a molecule of biological importance was made by Kwiram who in 1970 reported the optical detection of magnetic resonance signals from the tryptophan moiety of lysozyme. A more recent report dealt with frozen glassy solutions of tryptophan, tyrosine, and the protein, bovine serum albumin (BSA) loob) See Fig. 10. Discrepancies between the lifetimes involved in the phosphorescence decay and the fast-passage ODMR responses... 

Thus far, ODMR measurements have been reported 9 -ioi,i78) only on the proteins bovine serum albumin, HLAD, lysoz mie, and lysoz3mie complexed with the inhibitor tri-N-acetylglucosamine (tri-NAG), and in these proteins only the tryptophan ODMR has been studied in detail. 

Rousslang and co-workers (1978, 1979) and Ross and co-workers (1980) used optically detected magnetic resonance (ODMR) of tryptophan triplet states to detect conformational variations in proteins and polypeptides. This method used in combination with phosphorescence appears to be an excellent probe for detection of conformational variations which affect the environment of tryptophan. 

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