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Fluorescent and colored trinitrophenylated (TNP) analogs of ATP and GTP can interact with nucleotide-requiring enzymes and proteins as a substitute for the parent nucleotide. These analogs have strong binding affinities for most nucleotide-requiring systems. Their bindings are easily detected by absorption and fluorescence changes in the visible region. Recent years have seen dramatic developments in the application of the TNP nucleotide analogs as spectroscopic probes for the study on the nucleotide-interacting properties of various enzymes and proteins including their mutants. This review is intended as a broad overview of currently extensively used applications of the nucleotide analogs in various biological systems.

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One of the putative actin-binding sites of Dictyostelium myosin II is the ␤-strand-turn-␤-strand structure (Ile 398 -Leu-Ala-Gly-Arg-Asp 403 -Leu-Val 405 ), the "myopathy loop," which is located at the distal end of the upper 50-kDa subdomain and next to the conserved arginine (Arg 397 ), whose mutation in human cardiac myosin results in familial hypertrophic cardiomyopathy. The myopathy loop contains the TEDS residue (Asp 403 ), which is a target of the heavy-chain kinase in myosin I. Moreover, the loop contains a cluster of hydrophobic residues (Ile 398 , Leu 399 , Leu 404 , and Val 405 ), whose side chains are fully exposed to the solvent. In our study, the myopathy loop was deleted from Dictyostelium myosin II to investigate its functional roles. The mutation abolished hydrophobic interactions of actin and myosin in the strong binding state during the ATPase cycle. Association of the mutant myosin and actin was maintained only through ionic interactions under these conditions. Without strong hydrophobic interactions, the mutant myosin still exhibited motor functions, although at low levels. It is likely that the observed defects resulted mainly from a loss of the cluster of hydrophobic residues, since replacement of Asp 403 or Arg 402 with alanine generated a mutant with less severe or no defects compared with those of the deletion mutant.

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Limited glutaraldehyde modification of tryptic myosin subfragment 1, which mainly consists of 26-, 50-, and 20-kilodalton (kDa) peptides, resulted in the selective cross-linking of the 20- and 50-kDa peptides. The cross-linking pattern was altered by nucleotides, depending on the base structure. Neither the reactive thiols on the 20-kDa peptide nor the reactive lysyl residue on the 26-kDa peptide was modified with the reagent, regardless of the presence or absence of nucleotide. Glutaraldehyde treatment of the protein resulted in marked increases in its Mg2+-ATPase activity and affinity for actin. High ATPase activity and actin affinity were not produced if the treatment was conducted in the presence of ATP. These ATPase and actin binding properties of the protein derivatives are explained by assuming that glutaraldehyde "freezes" the existing interactions between the 20- and 50-kDa peptides in the activated and nonactivated conformational states, respectively. Taking into account the previous reports that the ATPase site resides between the 26- and 50-kDa peptides, and the 50-kDa peptide binds either ATP or actin, the present results suggest that the 50-kDa peptide acts as a communicating apparatus between the ATPase and actin binding sites of myosin. A simple model for the intersite communication is also proposed.