February 13, 2007 | David F. Coppedge

Cells Perform Nanomagic

The cell is quicker than the eye of our best scientific instruments.  Biochemists and biophysicists are nearing closer to watching cellular magic tricks in real time but aren’t quite there yet.  They know it’s just a trick of the eye, but it sure is baffling how cellular machines pull off their most amazing feats.  Think, but don’t blink:

  1. Knot Wizardry:  Proteins needing a fold go into a private dressing room (05/05/2003).  The most glamorous and well-equipped room, the GroEL-GroES chaperone, helps the star emerge just right.  How it does this is as puzzling as watching a magician untie a Gordian knot under a kerchief.  There are thousands of wrong ways a protein could fold; how does the chaperone always perform the trick correctly?  Some of the bonds between domains (disulfide bridges) are a long way apart.  What brings them together, and what keeps the wrong bridges from forming?
        Some scientists at Howard Hughes Medical Institute, writing in PNAS,1 cheated and built the chaperone with one door open so they could peek inside.  They still couldn’t figure it out completely.  Something in the chaperone creates conditions that favor the correct “native” fold, but also fix the mistakes before the prima donna protein emerges.  Somehow they do this without any ATP energy cost.  “We conclude that folding in the GroEL-GroES cavity can favor the formation of a native-like topology, here involving the proper apposition of the two domains of TG [trypsinogen, the enzyme in the experiment]; but it also involves an ATP-independent conformational ‘editing’ of locally incorrect structures produced during the dwell time in the cis cavity.”
  2. Speed Solve:  Maybe you’ve watched a blindfolded man solve a Rubik’s cube in seconds and wondered how it was done.  You can imagine the bewilderment of German and Swiss scientists watching a protein fold in far less time.  Protein chains of hundreds of amino acids have to explore a vast space of possible folds yet arrive at the one correct fold, often in fractions of a second.  These scientists, writing in PNAS,2 used lasers to try to figure out in slo-mo how this happens.
        As with a Rubik’s cube, there are billions of ways a protein could fold incorrectly.  Parts of a nascent protein chain form loops in the process of solving the puzzle.  “Exponential kinetics observed on the 10 to 100-ns time scale [ns=nanosecond, a billionth of a second] are caused by diffusional processes involving large-scale motions that allow the polypeptide chain to explore the complete conformational space,” they said.  “The presence of local energy minima [e.g., loops] reduces the conformational space and accelerates the conformational search for energetically favorable local intrachain contacts.”  To catch these loops, they had to look fast.  “Complex kinetics of loop formation were observed on the 50- to 500-ps [picosecond] time scale,” they noted.  A picosecond is a trillionth of a second.  Good thing they had lasers that could flash up to a femtosecond (quadrillionth of a second), or it would all be a blur.
  3. Levitation:  With a feat better than defying gravity, “Cytochrome c oxidase catalyzes most of the biological oxygen consumption on Earth, a process responsible for energy supply in aerobic organisms,” wrote a Finnish team also publishing in PNAS.3  To do this trick, the enzyme has to go against the force.
        Scientists like to talk in dispassionate language, but they called this enzyme “remarkable,” so they must have liked the magic act.  “This remarkable membrane-bound enzyme also converts free energy from O2 reduction to an electrochemical proton gradient by functioning as a redox-linked proton pump,” they remarked about the remarkable.  The way this pump works has “remained elusive,” even though most of the structure has been known.  With special spectroscopic and electrometric techniques, they were able to observe the trick in real time.  Abracadabra led to eureka: “The observed kinetics establish the long-sought reaction sequence of the proton pump mechanism and describe some of its thermodynamic properties.”  OK, tell us.  What’s the secret?

    The 10-microsecond electron transfer to heme [iron complex] a raises the pKa of a “pump site,” which is loaded by a proton from the inside of the membrane in 150 microseconds.  This loading increases the redox potentials of both hemes a and a3, which allows electron equilibration between them at the same rate.  Then, in 0.8 ms, another proton is transferred from the inside to the heme a3/CuB center, and the electron is transferred to CuB.  Finally, in 2.6 ms, the preloaded proton is released from the pump site to the opposite side of the membrane.

    So, there.  Now you know the trick.  Uh, how’s that again?  Actually, they only figured out part of the trick; “some important details remain unsolved,” they confessed, “e.g., the identity of the proton-accepting pump site above the hemes.”  Their diagram of the enzyme looks for all the world like magician’s tightly-cupped hands, with the active site secreted within.  Maybe this could be dubbed sleight-of-enzyme.

In the introduction to this last paper, the authors described how the enzyme is essential to all life.  It is a key player in the transfer of electrons and protons that feed the ATP synthase motors that produce ATP – the universal energy currency for all living things.  Water is produced in the process that generates oxygen (in plants) and consumes it (in animals).  These reactions would not occur without the machinery to drive them against the physical forces of diffusion.
    The scientists are converging on a mechanical description of how the pumping action works.  “Each of the four electron transfer steps in the catalytic cycle of CcO [cytochrome c oxidase] constitutes one cycle of the proton pump, which is likely to occur by essentially the same mechanism each time,“ they said.  “Here, we report on the internal electron transfer and charge translocation kinetics of one such cycle, which is set forth by fast photoinjection of a single electron into the oxidized enzyme.”

1Eun Sun Park, Wayne A. Fenton, and Arthur L. Horwich, “Disulfide formation as a probe of folding in GroEL-GroES reveals correct formation of long-range bonds and editing of incorrect short-range ones,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0610989104, published online before print February 5, 2007.
2Fierz, Satzger et al, “Loop formation in unfolded polypeptide chains on the picoseconds to microseconds time scale,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0611087104, published online before print February 6, 2007.
3Belevich et al, “Exploring the proton pump mechanism of cytochrome c oxidase in real time,” Proceedings of the National Academy of Sciences USA, 10.1073/pnas.0608794104, published online before print February 9, 2007.

We may not be able to tell how it’s done, but we all know that a stage magic trick is just an illusion.  But a good trick doesn’t just happen, either.  It takes a lot of intelligent design to put on a good show.  Split-second timing, carefully engineered props, trained assistants, planning, and precise manipulation are all required.  If and when we figure out all the cell’s tricks, it should produce even more awe than a childish belief in magic.  It should produce a deeper respect for the planning and execution of a well-designed show – and a hearty round of applause.
    Need we say how disappointing it was for Nature to submit this Stupid Evolution Quote of the Week about the same time as this last paper appeared: “The invention of oxygenic photosynthesis was a small step for a bacterium, but a giant leap for biology and geochemistry.  So when and how did cells first learn to split water to make oxygen gas?” (John F. Allen and William Martin, “Evolutionary biology: Out of thin air,”  Nature 445, 610-612, 8 February 2007).  Shamelessly, they continued on and on: “Biologists agree that cyanobacteria invented the art of making oxygen, but when and how this came about remain uncertain.”
    It appears that some childish scientists still believe in magic.  We hope the growing brightness of design emerging from cell biology will not cause too much pain as it shatters their illusions.  If they maintain their illusions in spite of the evidence, though – well, willful blindness is its own punishment.

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