Detour Ahead

For proteins with long half-lives, an siRNA phenotype can take days to emerge. Quicker inactivation is possible using small-molecule regulators, but this option is available for only a few proteins. An alternative approach is to fuse a small molecule binding domain to the protein of interest. For instance, rapamycin can trigger heterodimerization when one partner has an FKBP (FK506 binding protein) domain and the other an FRB (FKBP and rapamycin binding) domain, a property previously used to trap nuclear proteins in the cytoplasm. Seeking to generalize this method, Robinson et al. combined rapamycin-induced heterodimerization with mitochondrial outer membrane sequestration. The new system, described in Developmental Cell, involves an FRB domain fused to a mitochondrial targeting signal, while the factor to be sequestered is fused with FKBP. When rapamycin is added, the targeted protein heterodimerizes with the mitochondrial anchor and is blocked from normal function. Clearly, this strategy doesn't affect wild-type protein, so the authors also used siRNA knockdown of the target, being careful that the recombinant target-FKBP fusion contained no sequences complementary to the siRNA. The authors' test system involved adding FKBP to vesicular transport adaptor protein (AP) subunits. The alacrity of the response to rapamycin is astonishing: live-cell imaging showed rerouting after just 3 s. The phenotype of AP-2 sequestration 10 min after addition of rapamycin mimicked that seen 4 days into a standard RNAi experiment. In addition, rerouting gave clearer-cut insights into vesicular transport than siRNA, probably because this is a highly dynamic process and secondary effects can arise during gradual depletion. The authors contend their method—which they call “knocksideways” to evoke the quick blow to the cell's status quo—can be applied to virtually any cytosolic protein and they are investigating the feasibility of knocksideways mice.

Robinson et al. 2010. Rapid inactivation of proteins by rapamycin-induced rerouting to mitochondria. Dev Cell 18(2): 324–331.

Rapamycin-induced protein rerouting to the mitochondrial outer membrane. (Click to enlarge)

Get a Grip

In vitro selection of nucleic acids has produced aptamers to an assortment of small molecules and proteins. However, the resulting affinities are often in the micro-molar range, which is insufficient for biosensors. Writing in Nucleic Acids Research, Miyachi et al. propose finding higher-affinity aptamers by replacing the method for separating bound from unbound nucleic acids during selection. In conventional systematic evolution of ligands by exponential enrichment (SELEX), affinity chromatography distinguishes tight binders from nonspecifically-interacting molecules, with stringency set by factors such as wash conditions. Now, Miyachi et al. show how atomic force microscopy (AFM) can be used to select and assess aptamers. The authors prepared a library of biotinylated single-stranded DNA and dipped a streptavidin-coated AFM cantilever into the mixture. Meanwhile, they modified a gold chip with the aptamer target molecule—in this case, thrombin. As the DNA-coated AFM probe approaches the gold chip, some library members will interact with thrombin. When the cantilever draws away from the thrombin-coated chip, most ligand-binding DNAs will release thrombin. If there is an aptamer whose affinity for thrombin approaches the strength of the biotin-streptavidin attraction, however, the DNA itself could detach from the cantilever. These high-affinity thrombin binders are then eluted from the chip and used as input for a next cycle. After three rounds of AFM-SELEX (compared with eight or more rounds for conventional in vitro selection), the affinity force of the best binder was 206 pN, as opposed to 65 pN for a previously described thrombin aptamer, a difference corresponding to a 1000-fold improved dissociation constant. The affinity force of the AFM-selected aptamer also compares favorably with an anti-thrombin antibody, suggesting that AFM SELEX may suit biosensing applications.

Miyachi et al. 2010. Selection of DNA aptamers using atomic force microscopy. Nucleic Acids Res 38(4):324–331.

Linked N

N-linked glycosylation is a post-translational modification associated with cell adhesion and immune response. Characterizing these events would be simplified if homogeneous preparations of glycosylated proteins could be made. Although Campylobacter jejuni is capable of N-linked glycosylation, the attached glycan differs from eukaryotic N-glycans. In Nature Chemical Biology, Schwarz et al. describe engineering the prokaryotic glycosylation machinery to complement an in vitro enzymatic method to produce proteins with authentic eukaryotic N-glycans. First, the authors modified a C. jejuni glycosyltransferase operon to accommodate a eukaryotic N-acetylglu-cosamine (GlcNAc)-asparagine linkage. Reconstituted in Escherichia coli, this modified pathway generates a N-glycoprotein in which five to six N-acetylgalactosamine(GalNac) residues are attached to the asparagine-bound GlcNac for in vitro remodeling steps, including enzymatic treatment to excise all GlcNAc. Subsequent in vitro remodeling steps include enzymatic excision of the GalNAc residues and transglycosylation to add the desired eukaryotic N-glycan. This procedure requires adding a periplasmic targeting sequence and adapting the residues of the glycosylation site to a bacterial consensus sequence, cloning steps that represent additional time and effort, and may also interfere with protein function. Another limitation is that glycosylation efficiency varies, but despite these challenges, this study shows a promising pathway to new functional studies and biomedical applications of glycoproteins.



Schwarz et al. 2010. A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation. Nat Chem Biol 6:264–266.