453 Fluorine: A new element in protein design
Benjamin C. Buer and E. Neil G. Marsh
Fluorocarbons are quintessentially man-made molecules, fluorine being all but absent from biology. Perfluorinated molecules exhibit novel physicochemical properties that include extreme chemical inertness, thermal stability and an unusual propensity for phase segregation. This review describes recent studies in which proteins have been designed that incorporate highly fluorinated analogs of hydrophobic amino acids with the aim of creating proteins with novel chemical and biological properties. Fluorination appears to be a general and effective strategy to enhance the stability of proteins, both soluble and membrane bound, against chemical and thermal denaturation, whilst retaining structure and biological activity.
589 Identification and removal of nitroxide spin label contaminant: Impact on PRE studies of α-helical membrane proteins in detergent
Brett M. Kroncke and Linda Columbus
NMR paramagnetic relaxation enhancement (PRE) provides long-range distance constraints (∼15-25 Å) that can be critical to determining protein structures when the number of restraints is limited. However, several challenges currently limit the use of NMR PRE on helical membrane proteins. One challenge is the nonspecific association of the nitroxide spin label with the protein-detergent complex resulting in spurious PRE derived distance restraints. The effect of the nitroxide spin label contaminant is evaluated and quantified and a robust method for the removal of the contaminant is provided to advance the application of PRE restraints to membrane protein NMR structure determination.
463 The mysterious RAMP proteins and their roles in small RNA-based immunity
Ruiying Wang and Hong Li
The repeat associated mysterious proteins (RAMPs) function in small RNA-based immunity in prokaryotes against invading nucleic acids. RAMPs arrange two RNA-recognition motifs in a defined spatial orientation that bind a remarkable range of RNA substrates. RAMPs expand the repertoire of RNA-binding proteins and their structures provide novel principles of RNA-protein interactions.
511 Induced heterodimerization and purification of two target proteins by a synthetic coiled-coil tag
Jesus Fernandez-Rodriguez and Thomas C. Marlovits
Protein-protein interactions are essential for life, but many are transient or too weak to be studied with individually purified components. We have introduced a previously described de novo designed coiled-coil, named E3/K3, as a fusion tag for the heterodimerization and purification of two weakly or non-interacting proteins. It shows high association specificity with a 1:1 ratio and allows the recovery of the target proteins with a high degree of purity. Additionally, the coiled-coil interaction is resistant to reagents commonly used in protein purification, such as high salt washes and detergent treatment. The E3/K3 tag system is a very promising tool for a general method of protein-dimerization and co-purification.
