Design, Synthesis and Application of New Heterobifunctional Photoaffinity Probe for the Studies of Protein-protein Interactions Involved in the Actin-linked Calcium-regulated System of Muscle Contraction [microform] PDF Download

Author: Pele Choi-sing Chong
Publisher: National Library of Canada
ISBN: 9780315160293
Category : Muscle contraction
Languages : en
Pages : 0

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Book Description
In the rabbit skeletal muscle system previous studies suggested that sulfhydryl (SH) groups might be in the proximity of the sites of interaction of the following protein complexes: troponin C - troponin I, troponin-tropomyosin, actin-actin, actin-tropomyosin, and actin-myosin. Therefore a bifunctional cross-linking reagent that could be specifically attached to these SH groups would be invaluable for the identification of the proteins in the vicinity of the labelled SH group and enable the determination at the molecular level of amino acid residues involved in the site of interactions. This information would aid us in understanding how the Ca 2+ -induced conformational changes in troponin C can be transmitted from troponin C to all other components of the regulatory complex. For this reason we have designed and synthesized a new heterobifunctional photoaffinity probe, N-(4-azidobenzoylglycyl)-S-(2-thiopyridyl)cysteine (AGTC) from cysteine via a coupling of the N-hydroxysuccinimide ester of 4-azidobenzoylglycine to S-(2-thiopyridyl)cysteine. The chemical stability and reactivity of AGTC have been characterized. AGTC is readily dissolved in an aqueous buffer at pH 7.5 and is stable at room temperature ranging in pH from 3 to 9. The disulfide bridge moiety of AGTC is stable to the conditions of photolysis used to activate the arylazido group for crosslinking. AGTC is readily incorporated (90%-100%) within 2 hr into such proteins as rabbit skeletal troponin C, tropomyosin, and actin through disulfide bridge formation. The degree of incorporation of AGTC into proteins can be monitored by spectrophotometric determination of the release of pyridine-2-thionine at 343 nm. AGTC has a cross-linking distance of 14 A. The aryl azide moiety is inert until photolysis, permitting the removal of the excess reagents from the modified proteins and control experiments to ensure the correct protein-protein interaction. Also the aryl azide is nonspecific, in that it does not require the presence of a particular reactive functional group at the binding site for cross-linking to occur. Demonstration of the general utility of AGTC to study protein-protein interactions has been carried out in three different protein complexes: the interactions involving troponin C and troponin I, tropomyosin and troponin, and the subunits of troponin complex. Troponin C was labelled specifically at cysteine 98 with radioactive AGTC to form AGC-TnC which was used to form a binary complex with S-carboxamidometnylated troponin I (CM-Tnl) in benign media. Photolysis of CM-TnI-AGC-TnC complex resulted in the formation of a 1:1 covalently cross-linked complex in 30% yield. The radiolabelled CM-TnI-AGC was isolated from the cross-linked complex by reduction of the disulfide bridge between AGC and TnC using DEAE-Sephadex chromatography in the presence of 8M urea and 1 mM EGTA. These results indicated that CM-Tnl was within 14 A of cysteine 98 of TnC. AGC-TM (AGTC was attached to cysteine 190 of TM via disulfide bond formation) was used to determine which component of rabbit skeletal troponin (CM-Tn) was in close proximity to cysteine 190 of TM. Photolysis of the CM-Tn-AGC-TM complex in the presence of Ca 2+ resulted in formation of a 1:1 covalently cross-linked complex in 7% yield. The radiolabelled troponin (CM-Tn-AGC) was isolated by hydroxylapatite chromatography. CM-Tn-AGC was further separated into its individual components on DEAE-Sephadex chromatography. Radioactive measurements and SDS-urea gel electrophoresis indicated that only troponin T (TnT) was radiolabelled. A limited (15 min) chymotryptic digest of CM-Tn-AGC resulted in the isolation of T2-AGC (residues 159-259 of TnT). More extended proteolysis of CM-Tn-AGC allowed the isolation of T2'-AGC (residues 159-227 of TnT). When the CM-Tn-AGC-TM complex was photolyzed in the absence of Ca 2 + compared to the presence of Ca 2+ there was 1.7 fold increase in the cross-linking yield. This result suggested that there was a Ca 2+ -sensitive conformational change in the binding region of TnT around cysteine 190 . A tightening of the complex about cysteine 190 in the absence of Ca 2+ could explain the decrease in reaction of the arylnitrene with solvent. Nevertheless, region 159-227 of TnT is in the vicinity of cysteine 190 of TM in both the presence and absence of Ca 2 + . The topographical relationship of the SH groups of rabbit skeletal troponin has been investigated to provide information for the specific labelling of the SH groups with AGTC for the purpose of monitoring the Ca 2+ -induced conformational changes in the troponin complex. The approach involved the reaction of 14 C-iodoacetamide with SH groups in native troponin and various binary complexes of troponin components in the presence and absence of Ca 2+ . The SH groups involved in interaction sites and those exposed were identified by peptide mapping using two dimensional paper electrophoresis and autoradiography. In the presence and absence of Ca 2+ cysteine 133 of Tnl in native troponin was exposed while cysteines 48 and 64 of Tnl and cysteine 98 of TnC were inaccessible to modification. Differential labelling of the Tnl-TnT complex showed that cysteine 133 of Tnl was again exposed and cysteines 48 and 64 were inaccessible. 14 C-S-carboxamidomethylation of the Tnl-TnC complex showed that all three cysteine residues of Tnl (48, 64, and 133) were accessible to modification in the absence of Ca 2+ while cysteine 48 and 133 were only partially accessible in the presence of Ca 2+ . Cysteine 98 of TnC was inaccessible to modification in both the presence and absence of Ca 2+ . These studies indicated that in native troponin, TnT was solely responsible for the inaccessibility of cysteines 48 and 64 of Tnl in absence of Ca 2+ , and 64 of Tnl in the presence of Ca 2+ . The inaccessibility of cysteine 48 of Tnl in native troponin in the presence of Ca 2+ may be due to a combined effect of conformational changes in Tnl induced by TnC upon Ca 2+ binding and the interaction with TnT. These studies have provided the information for selective and specific attachment of AGTC into Tnl for the study of Ca 2+ -induced conformational changes in the troponin complex itself or troponin in the thin filament. AGTC was attached to cysteines 48 and 64 of skeletal Tnl to determine which component of troponin was in close proximity to these cysteines. The reconstituted troponin complex (AGTC labelled CM-Tnl, TnT, and TnC) was photolyzed and separated using DEAE-Sephadex chromatography in the absence of reducing agent. Radioactive measurements indicated that 12% of the cross-linker reacted with solvent and 88% with proteins. The percentage radiolabel found in Tnl, Tnl-TnT, and Tnl-TnC complexes was 35%, 55%, and 10%, respectively. These results have indicated that both TnT and TnC are in the vicinity of one or both cysteines 48 and 64 of Tnl. Of the total radiolabel found in TnT, 33% and 23% was located in two CNBr fragments, CB4 (residues 176-230) and CB2 (residues 71-151). The most likely interpretation of the cross-linking results is that one of the interaction sites between Tnl and TnT is an ionic interaction involving the region around cysteines 48 and 64 of Tnl (residues 28-82) with the CB5 region of TnT (residues 135-185). Finally, combining the present photochemical cross-linking results and all other studies, a working model of the regulatory complex (TM-Tn) was constructed to aid us in future experimental design to expand our knowledge on how the Ca 2+ -induced conformational changes in TnC can be transmitted from TnC to all other components of the regulatory complex.