ABSTRACT
The centromere is epigenetically marked by a histone H3 variant-CENP-A. The budding yeast CENP-A called Cse4, consists of an unusually long N-terminus that is known to be involved in kinetochore assembly. Its disordered chaperone, Scm3 is responsible for the centromeric deposition of Cse4 as well as in the maintenance of a segregation-competent kinetochore. In this study, we show that the Cse4 N-terminus is intrinsically disordered and interacts with Scm3 at multiple sites, and the complex does not gain any substantial structure. Additionally, the complex forms a synergistic association with an essential inner kinetochore component (Ctf19-Mcm21-Okp1-Ame1), and a model has been suggested to this effect. Thus, our study provides mechanistic insights into the Cse4 N-terminus-chaperone interaction and also illustrates how intrinsically disordered proteins mediate assembly of complex multiprotein networks, in general.
Subject(s)
Chromosomal Proteins, Non-Histone , DNA-Binding Proteins , Kinetochores , Protein Binding , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Kinetochores/metabolism , Kinetochores/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Saccharomyces cerevisiae/metabolism , Molecular Chaperones/metabolism , Molecular Chaperones/chemistry , Models, Molecular , Intrinsically Disordered Proteins/metabolism , Intrinsically Disordered Proteins/chemistry , Centromere Protein A/metabolism , Centromere Protein A/chemistry , Binding Sites , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/chemistry , Cytoskeletal Proteins , Microtubule-Associated ProteinsABSTRACT
A well-characterized and fully validated ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometric (UHPLC-ESI-MS/MS) method was developed to reliably analyze combination of perindopril arginine and amlodipine besylate in bulk and tablet formulations. The chromatographic separation was achieved on a Waters ACQUITY UPLC® BEH C18 column with 1.7 µm particle packing which enabled the higher peak capacity, greater resolution, increased sensitivity, and higher speed of analysis using a volatile mobile phase ideally being at least 2 pH units below and above the perindopril arginine and amlodipine besylate pKa, respectively. Mass spectrometric detection was performed using electrospray ion source in positive ion polarity to profile the abundances of perindopril arginine and amlodipine besylate, using the transitions m/z 369 â m/z 172, and m/z 409 â m/z 238 for perindopril arginine and amlodipine besylate, respectively. Calibration curve was constructed over the range 0.25 - 500 ng/mL and 1.0 - 100 ng/mL for perindopril arginine and amlodipine besylate, respectively. The method was precise and accurate, and provided recovery rates > 80% for both compounds. Furthermore, the intra- and inter-assay precision in terms of % RSD was in between 0.1 - 3.7 for both perindopril arginine and amlodipine besylate. A specific, accurate, and precise UHPLC-MS/MS method for the determination of perindopril arginine and amlodipine besylate in bulk and tablet formulation.
