Use the Force, Luke!

A couple nice force spectroscopy papers this week. The first comes out of our lab, by Matt Larson, studying the mechanism by which RNA hairpins cause termination in RNA polymerase. The common theory is that the U-rich RNA in the active site forms a "greasy" tract which is easily yanked out by the forming RNA hairpin. Matt finds evidence supporting this hypothesis, but finds that the mechanics of similar hairpin terminators (t500 and tR2, for instance) effect termination in mechanistically different fashions, which is quite surprising. The paper is worth a read, if for no other reason than to understand the real power of using single molecule force experiments to study mechanisms which are difficult to probe using traditional biochemical approaches.

There's also an interesting looking review in ChemPhysChem (what a bad name for a journal, by the way) looking at the use of AFM to learn about the structure of membrane proteins by yanking them out of the membrane. I think that finding ways to apply force spectroscopy to membrane proteins is going to be huge, but I think that just yanking them out of the membrane, while interesting from an energetics standpoint, is kind of crude. It's a tough problem, though, to figure out how to apply forces to membrane proteins without yanking them out of the membrane. Some of my graduate work was on ion channels, so I have some familiarity with the field, and I've spent some hours dreaming about it, but it's a tough nut to crack. Essentially, you want to be able to cleanly attach handles to membrane-bound objects in an in vitro setting, in such a way that you can still modulate them (by voltage, ligand, or mechanical strain, for instance) and then use your handle to probe and apply forces without pulling the whole thing out of the membrane. I've dreamed of using unilammelar vesicles, or lipid bilayers painted onto electrode patterned coverslips, but the complexity seems overwhelming. I'm still dreaming, though.

Also, taking the "Use the Force" metaphor a different direction, a shout out to my homey Greg Snyder who writes in to say, "I know this isn't single-molecule biophysics, but it is cool optics." White holes, black holes, fiber optics...next thing you know we'll be writing about Transdimensional Monte Carlo Hyperdrives.

Trans-dimensional Monte Carlo hyperdrive

• Shimon Weiss' lab has a nice paper out about a new laminar-flow mixer for protein folding studies. It's not particularly fast by fast mixer standards (15 - 20 ms), but it has the nice property of being constructed from off-the-shelf parts, using co-axial capillaries which have been pulled with a standard capillary puller. They show that it can be used for doing smFRET studies of protein folding, and identify a folding intermediate. (I did my undergrad thesis on microfab laminar flow mixers, in Bob Austin's lab at Princeton.)
• Joe Lakowicz has a paper out on coating metallic nanoparticles with fluorescently labeled proteins in order to create fluorescent probes with novel brightness and lifetime properties. This seems to have some nice advantages: the short lifetime allows the particles to be distinguished from cell autofluorescence by fluorescence lifetime imaging (FLIM). Unfortunately, the content is overshadowed by the fact that it appears to have gotten into press without ever having been edited by a native English speaker, and is just one linguistic gem after another. For instance, "Most MEF [metal enhanced-fluorescence] results are reported on the metal films, but to our knowledge, there are still some publications from us and other laboratories that describe the occurrence of MEF on single metal particles."
• Yet Another Paper in Biophys J on how to do a better job analyzing your noisy single particle tracking data. This isn't really notable, except for the fact that it apparently uses something called "trans-dimensional Sequential Monte Carlo". I have no idea what this is, but it sounds freaking awesome. Combine that with some bistromathics and an infinite improbability drive, and you'd have yourself a Nobel prize.
  

Where do they find the time?

This is how they do it (over at UMass)...


And apparently Ron Vale's mustache has been at the center of a political controversy:




Hat tip to Polly Fordyce for showing us some of this stuff when she was down here stealing our microspheres.

The ribo-BOMB

The long-awaited Bustamante ribosome paper is now out. But, at first glance, this paper doesn't have data on tethered ribosomes. Instead, it has tethered RNA, and tracks the sequential opening of a hairpin by the ribosome. This is still pretty neat, but I'm still breathless to see data about tethered ribosomes. None of the conclusions here appear to be revolutionary (the ribosome moves a codon at a time!!!1!!!1!!), but I'm not a ribo-expert, and there's some interesting looking stuff about the distribution of rate constants during the pausing.

UPDATE: Biocurious has posted a longer review of the paper here.

Bay Area Single Molecule Club?

Steve's recent visit to Harvard to deliver the Loeb lecture was apparently a rousing success, and I even made a new Facebook friend through somebody who had heard his lecture and found me through our lab web page. Steve came back and reported that Boston has a Single Molecule Discussion Club, and it looks like these guys have a lot of fun. I think it would be great to do thisout here as well. We could get folks together from Stanford, UCSF, Berkeley, and UC Santa Cruz (and anybody else who wants to come, of course) to have monthly talks, meetings, drink, eat, and schmooze. If you're interested, you can leave a comment, or e-mail me at:

If there's enough interest, maybe we can start to set something up. I'll probably try to send some e-mails around to the various labs (Vale, Bustamante, etc) and see if I can drum up interest. Suggestions are welcome!

Quantify Versus Quantitate: A Quantifactogorial Study

I had forgotten about this for a while, but here's a short paper I wrote back in 2004 demonstrating one of the key differences between physicists and biologists. The numbers are a bit dated (especially the Google search numbers) but the trends are still the same: physicists say "quantify", and biologists say "quantitate." From the conclusions:

As these results clearly demonstrate, the word “quantiate” is much more highly correlated with biology than with physics, and vice versa for “quantify.” Since, as everybody knows, physicists are smarter than biologists[2], this means that using the word “quantitate” is stupid.

[2] V. Adrian Parsegian. Harness the hubris: Useful things physicists could do in biology. Physics Today, pages 23–27, 1997. See counterpoint column by R.H. Austin p 27, e.g., “Having lived with biologists and biochemists for a number of years, I know damn well that many of them can’t reason their way out of a paper bag, and that they really need the analytical and experimental gifts of good physicists...”.

UPDATE: Forgot to add the link to the paper! Fixed.