Articles by Abril Gijsbers in JoVE
Fluorescensanisotropi som ett verktyg för att studera protein-proteininteraktioner Abril Gijsbers1, Takuya Nishigaki2, Nuria Sánchez-Puig1 1Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, 2Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México Proteininteraktioner är kärnan i en cell funktion. Kalorimetriska och spektroskopiska tekniker används ofta för att karakterisera dem. Här beskriver vi fluorescensanisotropi som ett verktyg för att studera interaktionen mellan proteinet muterat i Shwachman-Diamond syndrom (SBD) och förlängningsfaktor liknande ett GTPas (EFL1).
Other articles by Abril Gijsbers on PubMed
Guanine Nucleotide Exchange in the Ribosomal GTPase EFL1 is Modulated by the Protein Mutated in the Shwachman-Diamond Syndrome Biochemical and Biophysical Research Communications. Aug, 2013 | Pubmed ID: 23831625 Ribosome biogenesis in eukaryotes is a complex process that requires the participation of several accessory proteins that are not part of the mature particle. Efl1 is a yeast GTPase required for the cytoplasmic maturation of the 60S ribosomal subunit. Together with Sdo1, the yeast ortholog of the protein mutated in the Shwachman-Diamond Syndrome (SBDS), Efl1 releases the anti-association factor Tif6 from the surface of the 60S subunit allowing the assembly of mature ribosomes. We characterized the structural content and folding stability of the Saccharomyces cerevisiae and human EFL1 GTPases, as well as their enzymatic properties alone and in the presence of Sdo1 and SBDS, respectively. The human and S. cerevisiae EFL1 GTPases are composed of a mixture of α-helices and β-sheets. Despite being orthologs, the yeast protein elicited a non-two state thermal unfolding behavior while the human EFL1 was highly resistant to thermal denaturation. Steady-state kinetic analyses indicated slow GTP hydrolysis for both EFL1 GTPases, with kcat values of 0.4 and 0.3min(-1) and Km for GTP of 110 and 180μM respectively. In the presence of the effector proteins, their kcat values remained unaltered while the Km decreased twofold suggesting that Sdo1 and SBDS act as nucleotide exchange factors.
Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein The Journal of Biological Chemistry. Jul, 2015 | Pubmed ID: 25991726 Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1.