Structural and Biochemical Studies of Membrane Proteins CFTR and GLUT1 Yield New Insights into the Molecular Basis of Cystic Fibrosis and Biology of Glucose Transport

Integral membrane proteins (IMPs) assume critical roles in cell biology and are key targets for drug discovery. Given their involvement in a wide range of diseases, the structural and functional characterization of IMPs are of significant importance. However, this remains notoriously challenging due to the difficulties of stably purifying membrane-bound, hydrophobic proteins. Compounding this, many diseases are caused by IMP mutations that further decrease their stability. One such example is cystic fibrosis (CF), which is caused by misfolding or dysfunction of the epithelial cell chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Roughly 70% of CF patients world-wide harbor the ΔF508-CFTR mutation, which interrupts CFTR’s folding, maturation, trafficking and function. No existing treatment sufficiently addresses the consequences of ΔF508, and the substantial instability that results from this mutation limits our ability to study ΔF508-CFTR in search of better treatments. To that end, my colleagues at Sanofi generated homology models of full-length wild-type and ΔF508-CFTR +/- second-site suppressor mutations (SSSMs) V510D and R1070W, and performed molecular dynamics (MD) simulations for each model. Using information obtained from this analysis, I tested several hypotheses on the mechanism by which ΔF508 destabilizes full-length CFTR and how SSSMs suppress this effect. Leveraging studies of the purified NBD1 subdomain and of full-length CFTR in a cellular context, I confirmed the prediction of a key salt-bridge interaction between V510D and K564 important to second-site suppression. Furthermore, I identified a novel class of SSSMs that support a key prediction from these analyses: that helical unraveling of TM10, within CFTR’s second transmembrane domain, is an important contributor to ΔF508-induced instability. In addition, I developed a detergent-free CFTR purification method using styrene-maleic acid (SMA) copolymer to extract the channel directly from its cell membrane along with the surrounding lipid content. The resulting particles were stable, monodisperse discs containing a single molecule of highly-purified CFTR. With this material, I optimized grid preparation techniques and carried out cryo-EM structural analysis of WT-hCFTR which resulted in 2D particle class averages which were consistent with an ABC transporter shape characteristic of CFTR, and a preliminary 3D reconstruction. This result establishes a foundation for future characterization of ΔF508-CFTR in its native state. I have also applied this SMA-based purification method to the facilitated glucose transporter GLUT1 (SLC2A1). SLC2A1 mutations contribute to a rare and developmentally debilitating disease called GLUT1-deficiency syndrome. Using SMA, I successfully extracted GLUT1 in its native state. With the application of this method, I was able to purify endogenous GLUT1 from erythrocytes, in complex with several associated proteins as well as the surrounding lipids, in its monomeric, dimeric and tetrameric forms without the use of cross-linking or chimeric mutations. These results point to the potential for studying isolated IMPs without the use of destabilizing detergents and thereby offer a pathway to analysis of wild-type and mutant membrane protein structure, function and pharmacodynamics

Membrane protein nanoparticles: the shape of things to come

The use of styrene-maleic acid (SMA) for the purification of a wide range of membrane proteins (MPs) from both prokaryotic and eukaryotic sources has begun to make an impact in the field of MP biology. This method is growing in popularity as a means to purify and thoroughly investigate the structure and function of MPs and biological membranes. The amphiphilic SMA copolymer can effectively extract MPs directly from a native lipid bilayer to form discs approximately 10 nm in diameter. The resulting lipid particles, or styrene-maleic acid lipid particles (SMALPs), contain SMA, protein and membrane lipid. MPs purified in SMALPs are able to retain their native structure and, in many cases, functional activity, and growing evidence suggests that MPs purified using SMA have enhanced thermal stability compared with detergent-purified proteins. The SMALP method is versatile and is compatible with a wide range of cell types across taxonomic domains. It can readily be adapted to replace detergent in many protein purification methods, often with only minor changes made to the existing protocol. Moreover, biophysical analysis and structural determination may now be a possibility for many large, unstable MPs. Here, we review recent advances in the area of SMALP purification and how it is affecting the field of MP biology, critically assess recent progress made with this method, address some of the associated technical challenges which may remain unresolved and discuss opportunities for exploiting SMALPs to expand our understanding of structural and functional properties of MPs

Genética molecular del virus del síndrome reproductor y respiratorio porcino : aspectos evolutivos, diagnósticos e inmunógenos

El síndrome reproductor y respiratorio porcino esta producido por un virus arn, encuadrado en una reciente familia: arteriviridae. Se han desarrollado tres objetivos en este trabajo: 1/ el desarrollo y puesta a punto de una técnica rt-pcr para el diagnostico directo de la enfermedad; 2/ la expresión de la proteína codificada por la orf-5 vírica tanto en sistemas procariotas como eucariotas; 3/ el estudio de variabilidad existente entre cepas europeas del virus. Los resultados del primer objetivo indican que la técnica puesta a punto es altamente sensible y especifica, permitiendo la detección de aproximadamente 100 di50ct en muestras de campo. Con respecto al segundo objetivo, se ha logrado la caracterización de la antes mencionada proteína, así como la purificación parcial de ella. Por ultimo, los estudios de variabilidad demostraron una elevada semejanza entre las cepas analizadas, aunque si bien mayor entre las de un mismo paí