Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Šresolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the ß-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

Caracterización molecular con microsatélites amplificados al azar (RAMs) de Inchi (Caryodendron orinocense K.) / Molecular characterization with random amplified microsatellites (RAMs) of Inchi (Caryodendron orinocense K.)

El Inchi o Cacay (Caryodendron orinocense Karsten) es una de las especies más promisorias de la Amazonía y la Orinoquia colombiana. El principal producto del Cacay son sus almendras, de las que se extrae un aceite con aplicaciones cosméticas, fitoterapéuticas y alimenticias, además presenta un alto contenido de antioxidantes como los Omega 3, 6 y 9 y Vitaminas como la A y E. No existen estudios sobre la caracterización molecular de este recurso fitogenético, por lo cual el objetivo de esta investigación fue caracterizar la diversidad genética usando marcadores Microsatélites Amplificados al Azar (RAMs). El análisis de similitud al 0.50 formó cuatro grupos de acuerdo al sitio geográfico, siendo los materiales procedentes de Putumayo, Cacayal 19, Pauna y Castilla los de menor similitud. Los valores de heterocigosidad estimada fueron de 0.16 y 0.28 para los cebadores CGA y GT, respectivamente. El porcentaje de loci polimórfico varió entre 55% para el cebador CGA y el 90% para el GT. El valor de Fst promedio para los 27 materiales estudiados fue de 0.35, mostrando que la dinámica espacio-temporal de los materiales de Caryodendron tienden hacia una diferenciación genética, propio de sus procesos evolutivos e incidencia de la domesticación.

The Inchi or Cacay (Caryodendron orinocense Karsten) is one of the most promising species of the Amazon and Orinoco Colombian. The main product of Cacay are its almonds, from extracted oil cosmetic, phytotherapeutic and food applications, also has a high content of antioxidants such as Omega 3, 6 and 9 and vitamins like A and E. There are no studies on the molecular characterization of this plant genetic resource; therefore the objective of this research was to characterize the genetic diversity using Random Amplified Microsatellite markers (RAMs). The similarity analysis to 0.50 formed four groups according to geographical location, being materials from Putumayo, Cacayal 19, Pauna and Castilla lowest similarity. Estimated heterozygosity values ​were 0.16 and 0.28 for the primers CGA and GT, respectively. The percentage of polymorphic loci ranged from 55% for the primer CGA and 90% for the GT. The average Fst value for the 27 materials studied was 0.35, showing the space-temporal dynamics of materials Caryodendron tend toward genetic differentiation, due to their own evolutionary processes and domestication incidence.

Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments.

Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three-dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although noncomplementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts.