RESUMO
Dissecting the function of proteins' post-translational modifications (PTMs) is seriously hindered by the difficulty in obtaining the homogeneous protein with the PTMs of interest. Chemical protein synthesis offers a great potential to overcome this limitation. Here, a detailed protocol is introduced for chemical synthesis of HMGA1a protein with site-specific modifications via Ser/Thr ligation strategy, by which we can systematically study the function of the triple phosphorylation (3pSer) in the HMGA1a acidic tail. For complete details on the use and execution of this protocol, please refer to Wei et al. (2021).
Assuntos
Proteína HMGA1a , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes , Técnicas de Síntese em Fase Sólida/métodos , Proteína HMGA1a/síntese química , Proteína HMGA1a/química , Proteína HMGA1a/metabolismo , Fosforilação , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina/química , Serina/metabolismo , Treonina/química , Treonina/metabolismoRESUMO
High-mobility-group (HMG) proteins are a class of abundant non-histone nuclear proteins, among which HMGA1a is well-known for its association with transcription regulation as well as tumor formation and disease development. To study the functions of post-translational modifications, homogeneous HMGA1a protein with site-specific lysine acetylations (64/66/70/73) has been chemically synthesized. The full-length HMGA1a protein was assembled through two Ser/Thr ligations of three peptide fragments at Gly37-Thr37 and Thr75-Thr76 sites, respectively. Further inâ vitro studies with chemically synthesized proteins suggested that these acetylations did not significantly affect the CK2-catalyzed phosphorylation on the HMGA1a acidic tail.
Assuntos
Técnicas de Química Sintética/métodos , Proteína HMGA1a/síntese química , Lisina/química , Serina/química , Treonina/química , Acetilação , Sequência de Aminoácidos , Proteína HMGA1a/química , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-TraducionalRESUMO
The first chemical synthesis of nuclear protein HMGA1a via Ser/Thr ligation is reported. Notably, Hmb (2-hydroxy-4-methoxybenzyl) exhibits crucial improvement of both the difficult coupling during solid phase peptide synthesis and the poor ligation encountered in protein synthesis. These efforts led to preparation of HMGA1a analogs with well-defined phosphorylation and methylation patterns (9 synthetic proteins in total), thus overcoming the heterogeneous and combinatory problems inherent to protein post-translational modifications (PTMs), and facilitating the study of the regulatory roles of such PTMs.