Protein and peptide prenylation is an essential biological process involved in many signal transduction pathways. Hence, it plays a critical role in establishing many major human ailments, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), malaria, and Ras-related cancers. Yeast mating pheromone a-factor is a small dodecameric peptide that undergoes prenylation and subsequent processing in a manner identical to larger proteins. Due to its small size in addition to its well-characterized behavior in yeast, a-factor is an attractive model system to study the prenylation pathway. Traditionally, chemical synthesis and characterization of a-factor have been challenging, which has limited its use in prenylation studies. In this chapter, a robust method for the synthesis of a-factor is presented along with a description of the characterization of the peptide using MALDI and NMR. Finally, complete assignments of resonances from the isoprenoid moiety and a-factor from COSY, TOCSY, HSQC, and long-range HMBC NMR spectra are presented. This methodology should be useful for the synthesis and characterization of other mature prenylated peptides and proteins.
|Original language||English (US)|
|Title of host publication||Methods in Enzymology|
|Editors||A. Joshua Wand|
|Publisher||Academic Press Inc.|
|Number of pages||32|
|State||Published - Jan 1 2019|
|Name||Methods in Enzymology|
Bibliographical noteFunding Information:
Mass spectrometry analysis was performed at The University of Minnesota Department of Chemistry Mass Spectrometry Laboratory (MSL), supported by the Office of the Vice President of Research, College of Science and Engineering, and the Department of Chemistry at the University of Minnesota, as well as The National Science Foundation (NSF, Award CHE-1336940). The content of this chapter is the sole responsibility of the authors and does not represent endorsement by the MSL or NSF. This work was supported in part by the National Institutes of Health (RF1AG056976, GM084152, and GM106082) and by the National Science Foundation (CHE-1308655). NMR analysis was performed at Minnesota NMR center. Funding for NMR instrumentation was provided by the Office of the Vice President for Research, the Medical School, the College of Biological Science, NIH, NSF, and the Minnesota Medical Foundation.
© 2019 Elsevier Inc.
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