Sunday, March 6, 2011

Message (Crammed) in a Bottle

To sell a bottle of Champagne as "bubbly" a vendor must ensure the wine has absorbed between five and six atmospheres of carbon dioxide. To anyone who has ever popped the cork on champagne you know there is enough force there to do some serious damage to an innocent bystander in the event of poor aim.

Viruses spend part of their existence as a kind of bottle (capsid) with a message inside (DNA or RNA). The message inside carries the code or blueprint to make more viruses, more messages, more bottles. This message isn't short. If it were written out as As Ts Gs an Cs on regular 8 X 11" printer paper it would take up between 100-200 pages, depending on the specific virus we're talking about. Imagine trying to jam 100 leaves of paper into a wine bottle! The analogy isn't prefect as the viral message looks more like a rope than sheets of paper. Regardless, the message has to be crammed into an extremely tight space. The bottle's volume in this case being 0.065 cubic millimicrons. When the packing is done the DNA or RNA has been condensed from a free floating strand 6,ooo times like a tightly coiled garden hose at immense density. The pressure inside that tiny bottle reaches nearly 60 atmospheres. 10 times the pressure in the champagne bottle!

This raises many questions. How does a virus manage to pull or suck that long piece of rope inside its shell? Especially at the end when the pressure is at its peak? <--- The illustrated capsid cutaway shows the immense density with which the rope is crammed. How does such a tiny bottle not break under such intense pressure? Where's the cork and how does it stay put? And who or what pops the cork?

Dr. Carlos Bustamante of the University of California, Berkeley is answering these questions in incredible detail using single-molecule biophysical techniques. His lab is able to "grab the cat by the tail" and measure individual molecules directly. They specialize in DNA translocases. These are molecular machines or machine-like entities as he calls them.  Tiny "motors" that convert chemical energy into physical energy. When your eyes move to read these words the muscles are using the same chemical reaction's to pull your eyeballs from side to side as a virsus uses to pull that rope inside the bottle. This reaction is the hydrolysis of Adenosine triphosphate, the famed ATP, the energy "currency" of the cell. 

From Morais, M.C., Koti, J.S., Bowman, V.D., Reyes-Aldrete, E., Anderson, D.L., and Rossmann, M.G. 2008. Structure 16:1267–1274. © 2008, from Elsevier.
The viral machine is displayed in panel B. The capsid is pictured in gray while the motor sits at the "gate" and pulls the DNA inside. 

The motor is made of a five piece ring of small RNA molecules (pink) and five ATPases (lavender). In this particular single molecule study they are using two polystyrene beads to tether two ends of a long piece of DNA (the set up is pictured in panel A below). The strand is held in what is called an optical trap - those red sideways vase looking things. I blogged about how optical tweezers work before here's the link if you want to read about how this works. The goal in this study was to capture the capsid on one bead (coated with capsid protein antibodies) and the other end of the DNA  strand is bound to the other bead by a biotin-strepdavidin bridge. Not only did the researchers accomplish this feat but they were able to tug on either side of the system to measure how much force the motor was exerting on the DNA strand as it was sucking it into the capsid.
From Moffitt, J.R., Chemla, Y.R., Aathavan, K., Grimes, S., Jardine, P.J., Anderson, D., and Bustamante, C. 2009. Nature 457:446–1450. © 2009 Nature Publishing Group, a division of Macmillan Publishers Limited.

They discovered that with zero tension the capsid is packed with DNA at the rate of 100-120 base pairs per second. These studies also build a model for how they think the motor is working.  As said before, the motor requires a chemical reaction (hydrolysis of ATP) to work.  In this controlled environment the researches can set the concentration of ATP. When and only when all five site on the motor are loaded with their ATP molecules can the motor pull the strand.  Like a revolver laying with the cylinder open and someone pouring bullets on top of it, the "gun" can only fire after it is fully loaded by the random addition of bullets. When the concentration of ATP is low the rate of firing is less frequent, higher  - more frequent.

The simplicity of this model as understood by observing single molecules, for me, made years of Michaelis-Menten kinetics (the dry math that drives people away from studying biochemistry) come to life in a visual context.  I could see in my mind's eye exactly how concentration affects the rate of a reaction.

Not only were they able to define this requirement but they also show that only 4 of the five ATPs are used per "firing" and that each single ATP molecule hydrolyzing (thus power stroking the motor) corresponds to a pull of 2.5 base pairs.  And, that the motor moves the DNA inside the capsid at 10 base pair intervals (2.5 base pairs x 4 ATPs = 10 base pairs).

This basically means that a lot of energy is required from the surroundings for a virus to be able to package itself.

But when it's finished how does the capsid not explode from all that pressure? This has to do with the structure of the bottle.  The capsid is made of self-assembling capsid proteins that arrange themselves by interlocking joints. Like the ancient cultures of Bolivia who constructed their temples with interlocking rocks to withstand centuries of earthquakes these puzzle-boxes withstand 60 atmospheres of pressure by doing the same. 

The refinement of these models displays the best in what scientific inquiry has to offer in the twenty first century while still reminding us how new evidence can overturn old assumptions. As the trend toward studying single bio-molecules as they do the voodoo they do so well continues, our understanding of nature is refined to unprecedented clarity. 

These results were presented by Dr. Bustamante at the Inaugural Robert W. and A-Young M. Woody Lecture at Colorado State University last Thursday.  When a single lecture can breath new life into a graduate student in the throes of dissertating it says something about the power of good science and good scientific communication. 

Yu, J., Moffitt, J., Hetherington, C., Bustamante, C., & Oster, G. (2010). Mechanochemistry of a Viral DNA Packaging Motor Journal of Molecular Biology, 400 (2), 186-203 DOI: 10.1016/j.jmb.2010.05.002

Moffitt, J., Chemla, Y., Aathavan, K., Grimes, S., Jardine, P., Anderson, D., & Bustamante, C. (2009). Intersubunit coordination in a homomeric ring ATPase Nature, 457 (7228), 446-450 DOI: 10.1038/nature07637
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