RPA-1 from Leishmania sp.: Recombinant Protein Expression and Purification, Molecular Modeling, and Molecular Dynamics Simulations Protocols

• Autor(a) principal: Fernandes, Carlos A. H. [UNESP]
• Data de Publicação: 2021
• Outros Autores: Morea, Edna G. O. [UNESP], Cano, Maria Isabel N. [UNESP]
• Tipo de documento: Capítulo de livro
• Idioma: eng
• Título da fonte: Repositório Institucional da UNESP
• Texto Completo: http://dx.doi.org/10.1007/978-1-0716-1290-3_10; http://hdl.handle.net/11449/206210
• topic: Leishmania; OB-fold; RPA; TEBP; Telomeres; Trypanosomatids

RPA is a conserved heterotrimeric complex and the major single-stranded DNA (ssDNA)-binding protein heterotrimeric complex, which in eukaryotes is formed by the RPA-1, RPA-2, and RPA-3 subunits. The main structural feature of RPA is the presence of the oligonucleotide/oligosaccharide-binding fold (OB-fold) domains, responsible for ssDNA binding and protein:protein interactions. Among the RPA subunits, RPA-1 bears three of the four OB folds involved with RPA-ssDNA binding, although in some organisms RPA-2 can also bind ssDNA. The OB-fold domains are also present in telomere end-binding proteins (TEBP), essential for chromosome end protection. RPA-1 from Leishmania sp., as well as RPA-1 from trypanosomatids, a group of early-divergent protozoa, shows some structural differences compared to higher eukaryote RPA-1. Also, RPA-1 from Leishmania sp., similar to TEBPs, may exert telomeric protective functions. Remarkably, different pieces of evidence have pointed out that trypanosomatids may not have OB fold-containing TEBPs. Moreover, recent data indicate that trypanosomatid RPA-1 may be considered a TEBP since it shares with TEBPs conserved functional and structural features. However, it is still unknown whether the RPA-1 protective telomeric role is exclusive to trypanosomatids or is also present in other primitive eukaryotes. Here, we describe a protocol to obtain highly purified and biologically active Leishmania amazonensis recombinant RPA-1, and to perform molecular modeling and molecular dynamics simulations methods which could be probably applied to functional and structural studies of homologous proteins in other primitive eukaryotes.