Peptide phosphorylation coupling

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

Another aspect of stimulus-secretion coupling in the mast cell in which specific peptides and compound 48/80 have been studied is the phosphorylation of specific protein bands in response to stimulation [211-214], In these experiments mast cells were pre-labelled with 32P and stimulated, and... 

While these observations clearly point to protein phosphorylation in the mast cell as an important aspect of stimulus-response coupling, how this may fit into the overall picture is unclear. It would be of interest to know, for example, whether activators of protein kinase C (for example, 1,2-diacylglycerol) will promote a similar pattern of phosphorylation or whether inhibitors of G protein involvement (for example, Pertussis toxin) alter the pattern of phosphorylation in response to peptide or immunologic stimulation. 

The rationale behind the synthesis of phosphohomoserine-containing peptides [Hse(P)] is that this phosphorylated residue is inert to base-mediated (3-elimination and thereby may provide an interesting surrogate for the study of Ser(P)-based processes. In initial work, Boc-Hse[PO(OPh)2]-OH[21l was prepared by a simple four-step procedure but was found to be unsuitable for use in Boc-based solution synthesis due to extensive amine-mediated intramolecular dephosphorylation of the Hse[PO(OPh)2] residue during mixed anhydride coupling of its N-terminus. This side reaction was subsequently minimized through the use of Fmoc-Hse[PO(OPh)2]-OH in a Fmoc-based solid-phase approach in which the use of a large... 

The possibilities of N-(dialkylphosphoryl)amino acids for the prebiotic syntheses of peptides and polynucleotides have been studied in a series of papers [24,116-122], However, it must be emphasized that the phosphoryl group does not behave as an amino-activating group, the hydrolysis of which would be coupled to peptide bond formation. Actually, further peptide elongation requires the subsequent hydrolysis of the N-terminal phosphoryl group of the ligated product. In the presence of an amino acid ester, dipeptide esters 16 with an unreacted N-phosphoryl protection are formed, support-... 

Bn groups and Cbz protection, under which condition the amino Boc protection is stable. GPI 74 is the first fully phosphorylated synthetic GPI that was feasible for future coupling with peptides or proteins. 

Kinase substrates can become resistant to the actions of proteases due to their phosphorylations. Thus, the fluorescence quench assays (described in Chapter 2 covering protease assays) can be used to measure kinase activity. The assays can be viewed as coupled because they require a second enzyme to convert a product or substrate into a detectable signal. With kinase assays, the formation of phosphopeptide inhibits the protease action on the peptide and the signal remains quenched and therefore decreased (Rodems et al., 2002). Inhibiting the kinase results in increases in protease sensitivity and in signal. 

Fig. 3.3 The structure of the N-terminal SH3 domain of Grb2 bound to a proline-rich Sos peptide has been determined by NMR.29.30 The structure of the Gbr2 N-terminal SH3 domain, compiexed with a 10-residue peptide, comprising residues 1134-1144 (VPPPVPPRRR-NHz) of Sos, is shown. The prolyl residues, P2, P3, P6, and P7, which interact with the SH3 domain of Grb2 are marked. (The ribbon model was reproduced with permission of the authors and J. Mol. Biol, from data in ref. 30, available In databanks.) A variation of this scheme is the recognition of a proline-rich sequence (APTMPPPLPP) in the GAP protein for Rho by the SH3-domain of the cytosolic c-Abi tyrosine kinase. i This interaction couples the Rho/GAP tightly to this cytosolic tyrosine kinase and brings the momomeric G protein, Rho, under the control of phosphorylation by the kinase.

Recently, a small molecule fluorophore phosphosensor technology referred as Pro-Q Diamond dye has been developed to detect and quantitate phosphorylated amino acids within peptides and proteins in microarrays. ° In addition to binding assays, fluorescence detection methods have also been developed for functional assays. For example, microarrays of quenched fluorescent substrates can be used to detect protease or esterase activities in the analytes. In this method, quenched fluorescent substrates are prepared by coupling the peptide substrate to coumarin, a fluorescent dye. These peptide substrates are then spotted onto the solid support... 

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