A random approach to stabilize a membrane transport protein for crystallization studies / Un enfoque aleatorio para estabilizar un transportador de membrana para estudios de cristalización

• Autores: Arturo Rodríguez Banqueri
• Directores de la Tesis: Manuel Palacín Prieto (dir. tes.), José Luis Vázquez Ibar (dir. tes.)
• Lectura: En la Universitat de Barcelona ( España ) en 2013
• Idioma: inglés
• Tribunal Calificador de la Tesis: Marc Le Maire (presid.), Alejandro Perálvarez Marín (secret.), Jordi Benach Andreu (voc.)
• Materias:
  • Química
    • Bioquímica
  • Ciencias de la vida
    • Genética
      • Ingeniería genética
  • Ciencias médicas
    • Ciencias clínicas
      • Microbiología clínica
  • Ciencias tecnológicas
    • Procesos tecnológicos
      • Cristalización
• Enlaces
  • Tesis en acceso abierto en:  TDX  Dipòsit Digital de la UB 
• Resumen

  • X-ray crystallography is, now at days, one of the most powerful techniques to study proteins at the atomic level. Unfortunately, obtaining high quality crystals of membrane proteins for x-ray diffraction is a difficult task due to the hydrophobic nature of these proteins. The low stability in solution of these proteins and their tendency to form aggregates are the biggest problems during crystallization studies. One of the most common strategies to overcome these problems consists on working with functional mutants of these proteins. It has been reported that single point mutations of key residues (normally within transmembrane segments) leads to a remarkable increase in the stability of some membrane proteins after detergent solubilization and extraction from the membrane. In addition, a single mutation can stabilize a specific conformer of the protein, decreasing its heterogeneity in solution. Despite this, predicting what mutations are going to improve the stabilization of a protein is virtually impossible. The main purpose of this thesis is to build up a medium-high throughput experimental protocol with the objective to generate and characterize random mutants of a membrane protein with more stability in detergent-solubilized solution and, therefore with a better probability to crystallize. The combination of random mutagenesis with rapid and sensitive screening protocols of protein expression and stability seems to be the best approach for this goal. The use of the green fluorescent protein (GFP) as reporter has enormously facilitated the studies of expression, purification and stability of a membrane protein. Also, with the aim of minimizing undesired effects of full-length GFP, we optimized an assay based on a split GFP to build and characterized the random mutants library. Specifically we focus on SteT, a Bacillus subtillis transporter that exchanges L-threonine by L-serine. SteT is an excellent prokaryotic model (30% of amino acid identity) of the mammalian L-amino acid transporter (LAT) family. Genetic mutations of some LATs are the direct cause of two types of aminoaciduries. Moreover, a member of this family, LAT1, is overexpressed in tumor cells, although the physiological role of this is still unknown. Unfortunately, SteT wild type solubility and stability in detergent solutions is very low and completely incompatible with crystallization tests. Our results suggest that random mutagenesis combined with the GFP split assay, appears to be an excellent strategy to build robustness in membrane proteins for structural studies. So far, using this strategy we found a mutant of SteT that currently is undergoing for crystallization screenings to study the structure and mechanism of mammalian LATs.