[{"oa_version":"None","volume":707,"date_updated":"2025-10-22T06:40:54Z","title":"Generation of TIM chaperone substrate complexes","doi":"10.1016/bs.mie.2024.07.051","scopus_import":"1","OA_type":"closed access","author":[{"first_name":"Undina","last_name":"Guillerm","full_name":"Guillerm, Undina","id":"bb74f472-ae54-11eb-9835-bc9c22fb1183"},{"first_name":"Iva","full_name":"Sučec, Iva","last_name":"Sučec"},{"full_name":"Schanda, Paul","last_name":"Schanda","id":"7B541462-FAF6-11E9-A490-E8DFE5697425","first_name":"Paul","orcid":"0000-0002-9350-7606"}],"day":"13","language":[{"iso":"eng"}],"department":[{"_id":"PaSc"}],"status":"public","external_id":{"pmid":["39488384"]},"pmid":1,"publisher":"Elsevier","article_processing_charge":"No","corr_author":"1","date_published":"2024-09-13T00:00:00Z","_id":"18167","publication_status":"published","publication_identifier":{"issn":["0076-6879"]},"intvolume":"       707","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","page":"391-422","publication":"Methods in Enzymology","type":"book_chapter","date_created":"2024-10-01T10:58:27Z","abstract":[{"text":"Holdase chaperones are essential in the mitochondrial membrane-protein biogenesis as they stabilize preproteins and keep them in an import-competent state as they travel through the aqueous cytosol and intermembrane space. The small TIM chaperones of the mitochondrial intermembrane space function within a fine balance of client promiscuity and high affinity binding, while being also able to release their client proteins without significant energy barrier to the downstream insertases/translocases. The tendency of the preproteins to aggregate and the dynamic nature of the preprotein—chaperone complexes makes the preparation of these complexes challenging. Here we present two optimized methods for complex formation of highly hydrophobic precursor proteins and chaperones: a pull-down approach and an in-vitro translation strategy. In the former, attaching the client protein to an affinity resin keeps the individual client protein copies apart from each other and decreases the client self-aggregation probability, thereby favouring complex formation. In the latter approach, a purified chaperone, added to the cell-free protein synthesis, captures the nascent precursor protein. The choice of method will depend on the desired client-chaperone complex amount, or the need for specific labeling scheme.","lang":"eng"}],"citation":{"ista":"Guillerm U, Sučec I, Schanda P. 2024.Generation of TIM chaperone substrate complexes. In: Methods in Enzymology. vol. 707, 391–422.","ama":"Guillerm U, Sučec I, Schanda P. Generation of TIM chaperone substrate complexes. In: <i>Methods in Enzymology</i>. Vol 707. Elsevier; 2024:391-422. doi:<a href=\"https://doi.org/10.1016/bs.mie.2024.07.051\">10.1016/bs.mie.2024.07.051</a>","mla":"Guillerm, Undina, et al. “Generation of TIM Chaperone Substrate Complexes.” <i>Methods in Enzymology</i>, vol. 707, Elsevier, 2024, pp. 391–422, doi:<a href=\"https://doi.org/10.1016/bs.mie.2024.07.051\">10.1016/bs.mie.2024.07.051</a>.","apa":"Guillerm, U., Sučec, I., &#38; Schanda, P. (2024). Generation of TIM chaperone substrate complexes. In <i>Methods in Enzymology</i> (Vol. 707, pp. 391–422). Elsevier. <a href=\"https://doi.org/10.1016/bs.mie.2024.07.051\">https://doi.org/10.1016/bs.mie.2024.07.051</a>","short":"U. Guillerm, I. Sučec, P. Schanda, in:, Methods in Enzymology, Elsevier, 2024, pp. 391–422.","chicago":"Guillerm, Undina, Iva Sučec, and Paul Schanda. “Generation of TIM Chaperone Substrate Complexes.” In <i>Methods in Enzymology</i>, 707:391–422. Elsevier, 2024. <a href=\"https://doi.org/10.1016/bs.mie.2024.07.051\">https://doi.org/10.1016/bs.mie.2024.07.051</a>.","ieee":"U. Guillerm, I. Sučec, and P. Schanda, “Generation of TIM chaperone substrate complexes,” in <i>Methods in Enzymology</i>, vol. 707, Elsevier, 2024, pp. 391–422."},"quality_controlled":"1","year":"2024","month":"09"},{"month":"10","abstract":[{"lang":"eng","text":"Brassicaceae are blessed with specialized metabolites called glucosinolates (GSLs), which along with their degradation products, are beneficial in agriculture and human health. To date, more than 130 GSL structures have been identified, mostly derived from the amino acid methionine. The biosynthesis of methionine-derived aliphatic GSLs starts with a side-chain elongation step involving a recursive three-step cyclic process that incorporates a new methylene group into the 2-oxo acid to form a series of elongated 2-oxo acids. Methylthioalkylmalate synthase (MAMS) catalyzes the first committed step in the side-chain elongation of methionine-derived GSLs. The substrate specificity of MAMS with different 2-oxo acids determines whether reaction products of a given cycle enter for an additional round of chain elongation or enter into core GSLs structure formation. Multiple MAMS encoding genes are present in the Brassicaceae species and are known to play a central role in shaping the diverse profile of aliphatic GSLs. We recently established a highly sensitive LC-MS/MS-based methodology that quantifies the MAMS activity by estimating the amount of the next intermediate of the pathway, the 2-malate derivatives. Overall, this chapter describes the protocol for the expression, purification, and steady-state kinetic analysis of the recombinant MAMS protein."}],"date_created":"2023-01-16T10:05:13Z","quality_controlled":"1","year":"2022","citation":{"chicago":"Kumar, Roshan, Michael Reichelt, and Naveen C. Bisht. “An LC-MS/MS Assay for Enzymatic Characterization of Methylthioalkylmalate Synthase (MAMS) Involved in Glucosinolate Biosynthesis.” In <i>Biochemical Pathways and Environmental Responses in Plants: Part A</i>, edited by Joseph Jez, 676:49–69. Elsevier, 2022. <a href=\"https://doi.org/10.1016/bs.mie.2022.07.019\">https://doi.org/10.1016/bs.mie.2022.07.019</a>.","ieee":"R. Kumar, M. Reichelt, and N. C. Bisht, “An LC-MS/MS Assay for Enzymatic Characterization of Methylthioalkylmalate Synthase (MAMS) Involved in Glucosinolate Biosynthesis,” in <i>Biochemical Pathways and Environmental Responses in Plants: Part A</i>, vol. 676, J. Jez, Ed. Elsevier, 2022, pp. 49–69.","ista":"Kumar R, Reichelt M, Bisht NC. 2022.An LC-MS/MS Assay for Enzymatic Characterization of Methylthioalkylmalate Synthase (MAMS) Involved in Glucosinolate Biosynthesis. In: Biochemical Pathways and Environmental Responses in Plants: Part A. Methods in Enzymology, vol. 676, 49–69.","ama":"Kumar R, Reichelt M, Bisht NC. An LC-MS/MS Assay for Enzymatic Characterization of Methylthioalkylmalate Synthase (MAMS) Involved in Glucosinolate Biosynthesis. In: Jez J, ed. <i>Biochemical Pathways and Environmental Responses in Plants: Part A</i>. Vol 676. Elsevier; 2022:49-69. doi:<a href=\"https://doi.org/10.1016/bs.mie.2022.07.019\">10.1016/bs.mie.2022.07.019</a>","short":"R. Kumar, M. Reichelt, N.C. Bisht, in:, J. Jez (Ed.), Biochemical Pathways and Environmental Responses in Plants: Part A, Elsevier, 2022, pp. 49–69.","mla":"Kumar, Roshan, et al. “An LC-MS/MS Assay for Enzymatic Characterization of Methylthioalkylmalate Synthase (MAMS) Involved in Glucosinolate Biosynthesis.” <i>Biochemical Pathways and Environmental Responses in Plants: Part A</i>, edited by Joseph Jez, vol. 676, Elsevier, 2022, pp. 49–69, doi:<a href=\"https://doi.org/10.1016/bs.mie.2022.07.019\">10.1016/bs.mie.2022.07.019</a>.","apa":"Kumar, R., Reichelt, M., &#38; Bisht, N. C. (2022). An LC-MS/MS Assay for Enzymatic Characterization of Methylthioalkylmalate Synthase (MAMS) Involved in Glucosinolate Biosynthesis. In J. Jez (Ed.), <i>Biochemical Pathways and Environmental Responses in Plants: Part A</i> (Vol. 676, pp. 49–69). Elsevier. <a href=\"https://doi.org/10.1016/bs.mie.2022.07.019\">https://doi.org/10.1016/bs.mie.2022.07.019</a>"},"page":"49-69","publication":"Biochemical Pathways and Environmental Responses in Plants: Part A","type":"book_chapter","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","editor":[{"first_name":"Joseph","full_name":"Jez, Joseph","last_name":"Jez"}],"intvolume":"       676","alternative_title":["Methods in Enzymology"],"publication_status":"published","publication_identifier":{"issn":["0076-6879"],"isbn":["9780323955614"]},"date_published":"2022-10-21T00:00:00Z","_id":"12294","publisher":"Elsevier","article_processing_charge":"No","pmid":1,"status":"public","external_id":{"pmid":["36280361"]},"language":[{"iso":"eng"}],"doi":"10.1016/bs.mie.2022.07.019","scopus_import":"1","day":"21","author":[{"last_name":"Kumar","full_name":"Kumar, Roshan","id":"50e95140-3016-11ec-9e1a-881b648a1367","orcid":"0000-0002-0346-9125","first_name":"Roshan"},{"full_name":"Reichelt, Michael","last_name":"Reichelt","first_name":"Michael"},{"last_name":"Bisht","full_name":"Bisht, Naveen C.","first_name":"Naveen C."}],"date_updated":"2025-07-03T14:02:58Z","title":"An LC-MS/MS Assay for Enzymatic Characterization of Methylthioalkylmalate Synthase (MAMS) Involved in Glucosinolate Biosynthesis","volume":676,"oa_version":"None"}]