Fresh from a Nobel Prize win in Chemistry for his work with green fluorescent protein (GFP), Roger Tsien shows no sign of slowing down. One of the latest tools from his laboratory, dubbed ChIEF, is described in a recent issue of the Biophysical Journal. In this case, the goal is an engineered protein to facilitate photostimulation of neurons. Light-gated channelrhodopsins can be induced by bursts of blue light to propagate action potentials; this exogenous control of neuronal excitation has applications from mapping neuronal circuits to modulating neural activity. Chlamydomonas channelrhodopsin (ChR)2, which is a nonselective cation channel, is more widely used than ChR1, which shows bias for protons. However, ChR1 outperforms ChR2 in being less rapidly inactivated by ongoing light exposure. Using elements of ChR1 and ChR2 and site-directed mutagenesis, Lin et al. design a best-of-both-worlds chimera, ChIEF. This new variant shows nearly 3 times less inactivation than ChR2, making it more responsive to light pulses of 25 Hz or higher, the type of stimulus generally required to trigger action potential chains. Notably, the improved temporal precision does not come with the downsides of ChR1, as ChIEF conducts cations other than protons and so does not require acidic buffer conditions. Like the many flavors of fluorescent proteins Tsien's lab has pioneered, this engineered light-gated channel promises to become a tool that is widely and creatively applied.
Lin et al. 2009. Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys J 96:1803–1814.The Five-second Rule
Every kid knows the claim: momentary contact of food with the floor doesn't count. Skeptical adults aside, the five-second rule has some surprising support, at least in the field of epigenetics. The evidence appears in a recent PLoS ONE article from Schmiedeberg et al. Working with mutants of the methylated DNA-binding protein MeCP2, the authors observed dramatically different nuclear localization patterns between live-cell imaging of green fluorescence protein (GFP) fusion proteins and immuno-chemistry of fixed cells. In the latter, the staining was diffuse, suggesting defects in MeCP2's ability to bind methylated hetero-chromatic foci. Because the live-cell images showed the same punctate staining pattern as wild-type protein, however, the authors hypothesized that transient DNA-protein interactions were being missed by the formaldehyde fixation. Sure enough, closer investigation of binding dynamics by using fluorescence recovery after photobleaching (FRAP) showed that binding times under 5 s led to diffuse nuclear staining. The authors propose that protein-DNA cross-linking takes at least 5 s; if the binding time is less than that, only intramolecular crosslinks will occur, quickly inactivating the protein and trapping it in a diffuse staining pattern that erroneously suggests a complete absence of DNA-binding ability. Seeing the same pattern in ChIP, the authors warn that transient but biologically relevant protein-DNA interactions may be missed by common assays. Alternative procedures such as DamID (tagging DNA binding sites in vivo by fusing a putative DNA-binding protein to Dam DNA methyltransferase) may help investigators sort fact from fiction.
Schmiedeberg et al. 2009. A temporal threshold for formaldehyde crosslinking and fixation. PLoS ONE 4: e4636.Digesting the Evidence
Driven by every mouthful we eat and shaped by every macromolecule we require, our metabolic pathways form an extraordinarily complex system. A human cell may have over 3000 metabolites, each differing in size and properties and varying in concentration by several orders of magnitude. Understanding the underlying networks is the unenviable task of metabolomics, of which a primary aim is a quantitative description of the identities and levels of all relevant metabolites. This makes technique development critical, and a recent offering in Analytical Chemistry from Büscher et al. contributes to this goal by providing the first systematic comparison of pre–mass spectrometry (MS) separation strategies for quantitative metabolomics. The authors paired time-of-flight MS detection with six separation protocols, two each for the general approaches of gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). To keep the test manageable, the authors concentrated on central carbon metabolism. Of 75 metabolites or isomeric groups contained in a reference mixture of pure compounds, all but 3 could be detected by at least 1 method, but just 33 were detected by all 3 approaches (GC, LC, and CE), with the latter 2 showing the best coverage. To detect matrix effects, dilution series of the reference mixture were combined with a 13C-labeled yeast extract prior to analysis. In this test, CE using bared silica capillaries excelled, while all four GC and LC methods suffered from matrix effects. Because CE suffered the poorest reproducibility of the bunch, the authors recommend LC for most situations. Given the ubiquity of matrix effects, Büscher et al. point out the necessity of consistent use of internal controls. Though there are no easy shortcuts, the authors’ carefully collected evidence will be invaluable guidance for the flood of metabolomics studies to come.
Büscher et al. Cross-platform comparison of methods for quantitative metabolomics of primary metabolism. Anal Chem [Epub ahead of print, February 28, 2009, doi: 10.1021/ac8022857].