Proteins structure-reactivity investigations

Certain bifunctional metal chelating agents have been used to investigate protein interactions by virtue of their ability to generate reactive oxygen species that affects protein structure in the immediate vicinity of their modification site. The following sections discuss two applications of such chelate labels, one of which cleaves peptide bonds while the other one causes covalent crosslinks to occur between interacting protein structures. 

The presence of a suitable chromophore, such as the PLP cofactor, makes it possible to investigate protein structure-function relationships that may be far removed from the chromophoric site. Tryptophan synthase is considered to be a prototype multienzyme complex in which metabolites are channeled directly between successive metabolic enzymes. As described above, allosteric interactions serve to coordinate catalytic events between the heterologous active sites in the complex. Such close interactions suggest that mutations in one enzyme may affect the reactivity of the other. We have found it possible to study the consequences of mutations in the a-subunit by looking for changes in the presteady state behavior of reactions catalyzed at the (3-site (88, 89). Since amino acid replacements in the a-subunit will not affect the primary amino acid sequence of the P-subunit, alterations in the reactivity of the a2P2 complex will be due primarily to differences in the reactivity of the a-subunit and/or aP-subunit interactions. 

There are three caveats when one forms structural hypotheses on the basis of the observed CIDNP signals. First, the polarization intensity of a residue is not simply a constant that is specific for that particular amino acid but is subject to a Stem-Volmer competition of all accessible residues for the excited dye molecules, so CIDNP of an accessible amino acid can be suppressed by other accessible amino acids that are more reactive that problem is most pronounced for histidine. Second, in one study surface accessibility as detected by CIDNP was found to depend not only on the location of the amino acid but also to some extent on the nature of the dye " no systematic investigation of this effect with a range of known protein structures has yet been attempted. Third, the radical cation of a tr)q3tophan or tyrosine residue could undergo electron transfer with a nearby tyrosine or tr)q)tophan that is located in the interior of the protein. If this pair substitution causes polarizations of the inner residue to develop, misinterpretations as to the protein structure might obviously result. This problem has attracted considerable attention. ° For lysozyme, such an intramolecular electron transfer appears to be more important in the denatured state than in the native state. ... 

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