Physiological binding partners

Interactions of Cytochrome c with Physiological and Non-physiological Binding Partners... 

You can investigate protein-protein interactions (see also Sections 2.5 and 2.2.S.2). Example you have purified or expressed a protein X and you would like to know its function. Neither the sequence nor the purification provides any clues. It would be cool to know which proteins bind to protein X. Maybe you know their function, which would give you valuable pointers. Thus, you couple your protein X to a SELDI chip, if possible under different conditions. Then you wash off the unbound protein X and block unused groups. Now you incubate the chip holes with cell extract or wherever else you suspect binding partners to occur. Wash it off. Co-crystallize with matrix. Off with the chip into the M ALDl-TOF. After a few seconds you know whether a protein binds to protein X. Now this raises the question Is this protein the physiological binding partner To decide this, you need controls. In a control chip hole, you couple a protein that is similar to protein X in isoelectric point and MW. This hole should bind no proteins, or at least no other proteins. On the other hand, cell extracts or serums known not to contain binding partners for protein X should leave no trace in the mass spectrum. Furthermore, the protein should bind to X somewhat stoichiometrically. 

The Crk protein was first discovered as the transforming principle of the retroviruses CTIO and ASV-1. Abl tyrosine kinase is under discussion as a binding partner of the SH3 domain of Crk (Feller et al., 1994). Possible binding partners of the SH2 domain have been described but their physiological function is unclear. 

Further studies of ET between the protein and inorganic complexes have defined three sites for binding. Site I is positioned at the top left as pictured in Fig. 10 (17, 175), and includes residues Met-65, and Lys-89, -5, -86, -87 (92). This site is preferred by physiological protein partners (92). Site II includes Val-11, Ala-15, Thr-19, and Lys-7, -25, -27. Site III, to the left of the heme edge, may include Ile-81, Phe-82, Ala-83, and Lys-13,-72, -86 sites I and III have high affinities for anionic reactants. Cationic oxidants such as [Co(phen)3] interact mainly at site II, possibly a result of the presence of Glu-21 and fewer positively charged side chains in this region (16, 35). 

Example of application of solute quenching in protein studies One of the main aims in biophysical studies of the stracture and function of proteins is to identify the protein domains which are responsible for the interaction of the entire protein with physiologically relevant binding partners. Proteins usually contain several tryptophan residues, which may be distributed among the different protein domains. Since each of these tryptophan residues is located in a distinct environ-... 

Four crystal structures of Ni CODHs have been determined from the following organisms C. hydrogenoformans [87,88], Rhodospirillum (R.) rubrum [89] and the bifunctional CODH/ACS from M. thermoacetica [90,91]. In each case CODH has a very similar homodimeric quaternary structure with a diameter of about 100 A in the largest dimension and a total of five FeS clusters (Fig. 3A). An initially unexpected [4Fe - 4S] center, now called the D-cluster, is coordinated by the two subimits very close to the molecular surface. Each subunit also binds an additional [4Fe-4S] center, called the B-cluster, as well as the catalytic Ni-containing C-cluster. There is an electron transfer pathway between the physiological redox partner, the exposed D-cluster, then the B-cluster of one subunit and finally the C-cluster of the other subunit. The electron flow direction will depend on whether the enzyme reduces CO2 or oxidizes CO (Eq. 2). 

Since Eddowes and Hill first reported that 4,4 -bipyridyl (4,4 -bipy) modified gold and platinum electrodes, which facilitate a reversible (rapid) electrode reaction of cyt. c, numerous surface modifiers have been reported [60]. Among these surface modifiers, carboxylic acid-terminated alkanethiol SAM are most attractive for mimicking physiological ET partners with positively charged binding sites [61]. 

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