Fragments, proteins that interact with

The blot overlay assay is a powerful method used to study protein-protein interactions and provides an especially useful means by which to identify potential protein ligands. In this approach, a radiolabeled protein probe is used to overlay protein samples immobilized on nitrocellulose. Because this assay allows detection of protein-protein interactions within the context of a complex mixture of proteins, this method is also useful to investigate the specificity of an interaction between two ligands. The blot overlay assay may also be used to define specific domains of a ligand involved in protein-protein interactions for example, proteolytic fragments of proteins or nested deletion fragments may be probed to determine which portion of a molecule contains the interactive site (Crawford ei al., 1992 Gilmore et al., 1992). In addition, the blot overlay procedure may be modified to probe cDNA libraries to identify bacterially expressed proteins that interact with labeled probes (Cicchetti et al., 1992 Blanar and Rutter, 1992). 

In the present case, each endpoint involves—in addition to the fully interacting solute—an intact side chain fragment without any interactions with its environment. This fragment is equivalent to a molecule in the gas phase (acetamide or acetate) and contributes an additional term to the overall free energy that is easily calculated from ideal gas statistical mechanics [18]. This contribution is similar but not identical at the two endpoints. However, the corresponding contributions are the same for the transfonnation in solution and in complex with the protein therefore, they cancel exactly when the upper and lower legs of the thermodynamic cycle are subtracted (Fig. 3a). 

The protein-DNA interactions have been analyzed in detail at high resolution in the complex between the 434 repressor fragment and the ORl containing 20mer DNA. A pseudo-twofold symmetry axis relates the halves of this complex. The symmetry is not exact since the nucleotide sequence of the DNA is slightly different in each half (see Table 8.2). However, the interactions between one protein subunit and one half of the DNA are very similar to those between the second subunit and the other half of the DNA since most of the bases that interact with the protein are identical in both halves. Details of the interaction are very similar to those in the complex with the palindromic synthetic 14mer of DNA shown in Figures 8.14 and 8.15. The base pairs at one end of the DNA, 1-14, 2-13, etc. are called base pairs 1, 2, etc. 

V NMR spectroscopy has revealed that the sub fragment 1 of the muscle protein myosin interacts with the various vanadates present at sufficiently high concentrations, preferentially so with tetravanadate " ] and decavanadate " ] (see the previous section). If the myosin subfragment 1 is irradiated with UV light in the presence of vanadates, cleavage occurs and vanadate is reduced to vanadyl, which is not active as an energy transmitter in photo-cleavage. ]... 

Figure 8.15 Sequence-specific protein-DNA interactions provide a general recognition signal for operator regions in 434 bacteriophage, (a) In this complex between 434 repressor fragment and a synthetic DNA there are two glutamine residues (28 and 29) at the beginning of the recognition helix in the helix-turn-helix motif that provide such interactions with the first three base pairs of the operator region.

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