Cross-linking experiments

Pearson, K.M. et al. (2002) Intramolecular cross-linking experiments on cytochrome C and ribonuclease A using an isotope multiplet method. Rapid Comm. Mass Spectrom. 16, 149-159. 

The covalently bound hybrid was prepared in the presence of Ca2+. Cross-linking experiments in the absence of Ca2+ were also performed using CaM and PDE. However, we could not obtain the hybrid between CaM and PDE in the fraction pattern without Ca2+. This finding suggests that the assembling of the present molecules requires that CaM is associated with the specific CaM binding domain of PDE when the two biomolecules are linked with remaining as much cooperatively as possible. 

This method is currently very difficult to implement in the case of two proteins, although some of the cross-linking experiments mentioned in the next paragraph... 

Chemical cross-linking experiments between rhodopsin and Gat indicate that residue 240 in IC 3 of rhodopsin is near the N- and C-termini of Ga, as well as the a4//J6 loop (Fig. 5 Cai et at., 2001 Itoh et at., 2001). Five residues at the C-terminus of Ga were shown to specifically interact with four noncontiguous residues in IC 3 (Liu et at., 1995). Peptides corresponding to IC 3 also cross-link to the N-terminus of Ga and the C-terminus of G/J (Taylor et at., 1994, 1996). As mentioned previously, the binding site for the C-terminal peptide of Gat was mapped to the inner face of helix VI (Janz and Farrens, 2004), and both the Ga and Gy C-termini of Gt interact with IC 4 of rhodopsin (Ernst et at., 2000). These data provide important information about specific contacts between receptors and G proteins however, more constraints are needed to improve our current model of the receptor-G protein interface. 

B. Interhelical Cross-Linking Experiments and the Functional Relevance of the... 

In the MMO OB3b system, the values could be used to predict the concentration dependence of the MMOB enhancement on the rate of the multiple turnover reaction. The fit to the experimental data predicts that the maximum rate is attained when a stoichiometric ternary complex (based on active site concentration) is established. Excess MMOB is inhibitory, apparently due to the formation of inactive MMOB-MMOR and MMOB-MMOB complexes, or perhaps binding of MMOB in the MMOR binding site. Cross-linking experiments were used to demonstrate the formation of each of these inhibitory complexes. Component complexes also play a significant role during the single turnover reaction as described below. 

The observation that nucleocapsid assembly in the absence of nucleic acids was inhibited indicated that interaction of the coat protein with the RNA is an essential and early step in the assembly pathway. The availability of mutant coat proteins that retained nucleic acid-binding activity but could not assemble further provided an opportunity to identify a possible coat protein-nucleic acid assembly intermediate. Cross-linking experiments revealed the presence of a coat protein dimer that could be detected only in the presence of nucleic acid and for those types of mutant proteins that had retained nucleic acid-binding activity. The protein dimer itself could not assemble into cores but was incorporated into cores in the presence of wild-type protein. These and other results strongly suggested that the cross-linked dimer represents a genuine intermediate of nucleocapsid core assembly. 

The six subunits probably exist as a heterohexamer. Cross-linking experiments could test the model and help determine which subunits are adjacent to one another. 

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