Proteins, CIDNP applications

With protein CIDNP now being a universally accepted technique, the interest in its application is shifting more and more towards the denatured state of proteins and towards folding processes. A recent review is provided by Ref. 185. 

Photo-CIDNP spectra of amino acids are presented in the following section with the aim of establishing the ground rules for applications to protein systems. An illustrative example of protein CIDNP spectra is also given in this section. 

There has been a strong but very selective interest in CIDNP on amino acids, strong because of the importance for the application of CIDNP to proteins (see Section 6.9), and very selective because only three amino acids (tr)q)tophan 10, tyrosine 11, and histidine 12, compare Chart 12 for their CIDNP spectra, see Ref. 185) are routinely useable for that purpose while two others, cysteine 13 and methionine 14, have received attention because of their putative role for long-range electron transfer across cell membranes or oxidative damage of cell components. 

The determination of surface accessibilities in a protein is by now a well-established application of photo-ClDNP. Its operating principle is that a sensitizer in the bulk solution is photoexcited, forms a radical pair with an amino acid that is exposed to the solution, and so causes CIDNP to arise amino acids not accessible to the dye remain impolarized. To avoid disruptions of the structure the photore-action(s) must be cyclic. As this leads to exchange cancellation, one either observes the polarizations that remain because of relaxation in the free radicals or samples the geminate polarizations in a time-resolved CIDNP experiment. The latter appears preferable for quantitative conclusions as it also removes other artefacts, but cannot be applied to the pulse-labelling and related experiments described below. Commonly employed dyes are 2, 2 -dipyridyl 16 or flavins 17. As already mentioned in the preceding section, only tryptophan 10, tyrosine 11, and histidine 12 are polarizable. However, the reduction of the information content in a crowded protein spectrum by this selectivity is a much desired blessing rather than a drawback. 

Amino Acids, Peptides, and Proteins. The determination of the accessibility of amino acid residues is the standard application of CIDNP to proteins and larger peptides, the key idea being that only amino acids exposed to the surface can react with a photoexcited dye. The photoreactions must be reversible to avoid unwanted structural changes of the biopolymer that are induced by the experiment itself. This can be realized with cyclic electron-transfer (cf. Section V.A.2, Chart VI) or hydrogen-transfer reactions. Because of the photochemistry of amino acids, the only... 

CIDNP originates from magnetic Interactions In pairs of free radicals and Is observed In their reaction products (Kapteln, 1977). In spite of numerous applications of this effect In organic chemistry It has not thus far been observed In proteins or other biological... 

Combined with a difference technique the method results in substantial simplifications of the complex protein nmr spectra and in an effective increase in the spectral resolution for the polarized groups. This permits detailed studies of various interactions of proteins, such as the enzyme-inhibitor interactions discussed in sections 5,2 and 6.2 for RNase A and lysozyme. For both enzymes the photo-CIDNP spectrum proved to be very sensitive to the presence of inhibitors, albeit in conpletely different ways Examples of applications to the study of protein-nucleic acid interactions will be treated by Hilbers et al. elsewhere in this volume. We believe that in spite of its brief existence the photo-CIDNP method is potentially a powerful tool for the study of protein structure in solution. 

The application of the photo-CIDNP method in conjunction with the introduction of deuterated amino acids holds great promise for the study of protein-protein and protein-nucleic acid interactions with NMR. In this contribution it has been shown that this allows partial assignment of the aromatic part of the protein spectrum in a convenient way. Binding of DNA fragments affects both the resonances of the phenylalanyl residues as well as the resonances of the tyrosines at the surface of the protein. The disappearance of the emission signals of the latter provides direct evidence for their Involvement in the interaction with DNA. 

In another class of experiments, hyperpolarized states are generated by spin-sensitive chemical reactions. These include para-hydrogen-induced polarization (PHIP) [3-5] and chemically induced dynamic nuclear polarization (CIDNP) [6-8]. The latter involves non-equilibrium nuclear spin state populations that are produced in chemical reactions that proceed through radical pair intermediates. CIDNP s applicability has been focused towards the study of chemical reactions and the detection of surface exposed residues in proteins [9], but has so far remained limited to specialized chemical systems. 

---