MYOSIN II: MUSCLE MOTION
Myosin V is no doubt fascinating due to its iconic, long-legged walking motion. But the original myosin, and the one most linked to the motions of animals that so fascinated biologists from Aristotle onward, is myosin II. The molecular machine that makes muscles work, myosin II was the first molecular motor discovered—by Andrew Huxley, in 1969. Strictly speaking, in evolutionary history, myosin II existed before muscles. “Nonmuscle” myosin II in cells pulls on actin filaments, which are part of the cell’s skeleton, and thereby helps the cells change their shape. As is common in evolution, once multicellular organisms evolved, the actin-pulling machines were co-opted to move the whole organism—using muscles.
It is quite easy to see how nonmuscle myosin may have turned into muscle myosin: Muscles are made of bundled bundles of muscle fibers— each fiber a single cell. Each cell contains myofibrils, which are structures composed of actin filaments interlaced with fibrous bundles of myosin II (Figure 7.8). Muscle cells are cells with an exaggerated network of actin and myosin—otherwise, myosin does what it has always done, even before muscles existed: pull on actin.
The structure of muscle and the presence of a molecule called myosin was known long before 1969, when Huxley proposed the first model to explain how an army of molecular motors could produce macroscopic motion. At the time, it was already known that muscle contraction was due to the relative sliding of two different filaments against each other: actin filaments and myosin filaments. But what generated the force that made the two filaments move relative to each other? Using electron microscopy of muscle fibers, Huxley suggested that there were distinct cross-bridges between the two filaments and that these bridges generated force and motion by tilting. The crossbridges turned out to be myosin heads, and thus muscle motion was now attributed to the concerted efforts of legions of myosin molecules pulling on actin filaments.
FIGURE 7.8. The structure of muscle.
It is worth considering the scales involved in the various components of muscular movement. Muscles are macroscopic objects with a typical dimension of centimeters (or inches). As we zoom in, we first find that muscles are made of large strands, each of them a bundle of smaller strands, the muscle fibers. A single muscle fiber may be centimeters in length, but is only about 50 micrometers (a millionth of a meter or a 25,000th of an inch) in diameter. A muscle fiber is a very long cell. Inside the cells are bundles of actin and myosin. Each myosin head, the active force-generating part of the whole assembly, is only 30–40 nanometers in size. Every gram of muscle contains about 1017 (100 million billion) myosin molecules.
The process by which myosin II generates motion is still not completely understood, but substantial progress has been made by structural (X-ray, electron microscopy), biochemical, fluorescence, and laser tweezer studies. The cycles of attachment and detachment, as well as ATP binding, hydrolysis, and release, are essentially the same for myosin II and myosin V. However, myosin II does not seem to be processive. It does not walk along actin filaments for long distances, as myosin V does. On the other hand, myosin II can produce faster motion than myosin V can.
As mentioned in Chapter 6, some people believe that myosin II is a weakly coupled motor, while others believe in a distinct, fixed-distance power stroke. Although myosin II was the first molecular motor discovered, it remains one of the most enigmatic. For example, as in myosin V, the role of ATP hydrolysis does not seem to be settled: In dozens of papers, most authors seem to reduce the function of ATP hydrolysis to an “increase in affinity to actin,” while a few researchers assume that ATP hydrolysis is associated with some prepower stroke. Moreover, as in myosin V, the ATP hydrolysis proceeds in several steps: splitting off a phosphate to create ADP-P, and only then releasing the phosphate, which results in a bound ADP. These steps happen too fast for scientists to clearly link them to mechanical motions of the myosin. Solving this problem will require the development of faster fluorescence techniques. The hydrolysis of ATP releases a fair amount of energy, so it is difficult to imagine that it would simply serve to make the myosin head more attractive for actin. Perhaps it plays a role in biasing diffusion, for example, by internally changing the strain in the molecule.
How, then, is muscle motion controlled? One explanation is that some mechanism controls the supply of ATP to muscle. If we want to move, our bodies supply ATP; if not, they shut off the ATP supply. But this is not how it happens. As a matter of fact, in myosin II, the binding of ATP allows the myosin head to detach from actin. Therefore, a lack of ATP leads to rigor mortis: The myosin gets stuck on the actin and the muscles harden up. Instead, the motion of muscle is controlled by a molecular switch called tropomyosin. Tropomyosin binds to actin and blocks the site where myosin takes hold of the actin filament. When we think about lifting an arm, nerves send a signal to the muscle cells, which release calcium ions. The calcium ions bind to the tropomyosin and it releases the actin, thereby exposing a patch to which myosin II can bind.
THE MYOSIN SUPERFAMILY
Myosin, like kinesin, forms a large family with many functions (Figure 7.9). Muscle myosin II generates power in muscles, while nonmuscle myosin II helps cells change shape, move, and divide. Myosin V is a short-distance transporter in our cells. What about the other myosin molecules? Myosin I, unlike myosin II and V, is a single-headed motor used in cell motion. It attaches to the cell membrane on one end and to actin at the other. Then it pushes actin filaments around as they are polymerized (generated). In this way, it shapes the internal skeleton of the cell and provides tension between the skeleton and the cell membrane.
FIGURE 7.9. An example for the family tree of a molecular motor: the family of myosins. Reproduced/adapted with permission from Tony Hodge, M. Jamie, and T. V. Cope, “A Myosin Family Tree,” Journal of Cell Science 113 (2000): 3353–3354.
A Myosin Family Tree
Tony Hodge and Jamie Cope
Abbreviations
Ac
Acanthamoeba castellanii
Acl
Acetabularia cliftonii
Ai
Aequipecten irradians (scallop)
At
Arabidopsis thaliana (thale cress)
Bm
Brugia malayi
Bt
Bos taurus (cow)
Cc
Chara corallina
Ce
Caenorhabditis elegans
Cr
Chlamydomonas reinhardtii
Dd
Dictyostelium discoidium
Dm
Drosophila melanogaster
En
Emiricella nidulans (Aspergillus)
Eh
Entamoeba histolytica
Gg
Gallus gallus (chicken)
Ha
Helianthus annus (sunflower)
Hs
Homo sapiens (human)
Lp
Limulus polyphemus (horseshoe crab)
Ma
Mesocricetus auratus (hamster)
Mm
Mus musculus (mouse)
Oc
Oryctolagus cuniculus (rabbit)
Ov
Onchocerca volvulus (a nematode)
Pf
Plasmodium falciparum
Pg
Pyricularia grisea (rice blast fungus)
Rc
Rana catesbeiana (bullfrog)
Rn
Rattus norvegicus (rat)
Sc
Saccharomyces cerevisiae (yeast)
Sm
Schistosoma mansoni
Ss
Sus scrofa domestica (domestic pig)
Tg
Toxoplasma gondii
Tt
Tetrahymena thermophila
Xl
Xenopus laevis
Zm
Zea mays
Adren
Bovine Adrenal (myosin I)
Bb
Brush Border Myosin I
CaA
Cardiac alpha (myosin I
I)
CaB
Cardiac beta (myosin II)
csm
Chitin synthase-myosin
FSk
Fast Skeletal (myosin II) = striated
FSkE
Embryonic Fast Skeletal (myosin II)
HMWMI
High Molecular Weight Myosin I
neur
Neuronal (myosin II)
nm
Non-muscle (myosin II)
PDZ
Human myosin with a PDZ domain.
Peri
Perinatal (myosin II)
sm
Smooth muscle (myosin II)
Myosin VI is a motor protein like myosin V, but it moves in the opposite direction of myosin V. While myosin V moves lipid-encased cargo to the outside of the cell, myosin VI moves cargo to the inside. It is involved in endocytosis, which is the process through which the cell ingests molecules from the outside of the membrane. Myosin VII seems to be involved in the cells in our ears: Mutations in the myosin VII gene can lead to deafness. Myosin IX may play a role in sending signals to the cell that initiates rearrangement of its actin filament network. Clearly, considering the large number of motor proteins and the multiple functions they fulfill, more research will be needed to figure out what they all do.
The Recharging Station: The Machinery of Energy Transduction
Through hydrolysis, molecular motors turn ATP into ADP. Then they release ADP and the spent molecular fuel pellet floats away. What happens to it? Wherever possible, cells are keen on recycling. Why not recharge the ADP by reattaching a phosphate and turning it back into ATP? Indeed, this is what the cell does, and it happens in one of the cell’s most important organelles: mitochondria.
Mitochondria are the fuel recharging stations of cells (they are sometimes called the cell’s power stations). They use energy derived from food to recharge ADP. The details took decades to decipher; the process is surprisingly complicated and proceeds through many steps. Fortunately for our story, at the end of the process, there is a marvelous molecular machine—a machine that, like no other, illustrates how, at the nanoscale, different types of energy can be converted into each other with very high efficiency.
But before we talk about this amazing machine, let’s briefly look at how food turns into ATP-energy. As described in Chapter 1, scientists slowly came to the realization that the heat of the body was generated by a slow burning process. Experiments by Lavoisier, Liebig, Helmholtz, and others showed that the energy generated from food is roughly equivalent to the burning of food in air. But how do our bodies burn food? In our cells, a number of complicated, multistep biochemical reactions can turn various food molecules—sugars, proteins, fat—into standard, high-energy molecules used by the cell. In each biochemical step, some energy is removed in the form of ATP or other energy-carrying molecules, and the end product has correspondingly less energy than the original food molecules. Thus, breaking down the burning process into many steps makes burning a very slow and, consequently, very efficient process.
In one such burning process, glucose (a sugar) is turned into two molecules of pyruvate (a small organic molecule), while generating two ATP molecules. Prior to the availability of free oxygen on our planet (courtesy of photosynthesis), this reaction was the end of the road. You could get two ATP molecules out of one glucose molecule, and that was it. In an aerobic bacteria (bacteria that do not use oxygen), pyruvate is waste. But pyruvate still has energy to offer. In aerobic cells (like our own), pyruvate becomes feedstock for the next set of biochemical reactions. All in all, the two pyruvate molecules that result from the burning of one glucose molecule generate another thirty ATP molecules. Now, that’s efficiency!
The first step in digesting pyruvate is to break it down further, releasing CO2 (which we breathe out) and creating another intermediate product, an acetyl, which gets attached to a carrier called coenzyme A. This product, acetyl coenzyme A, then enters the next cycle, called the Krebs cycle after its discoverer, and after more CO2 is released, we are left with several energy-carrying molecules called NADH and FADH2. I’ll spare you what these acronyms stand for—what’s important is that NADH and FADH2 are electron-rich molecules, which means they can easily give up electrons.
When NADH gives up electrons to an enzyme embedded in the membrane of the mitochondrion, NADH turns into NAD+ (Figure 7.10). The split-off hydrogen ion (H+, the same thing as a hydrogen nucleus, which in hydrogen’s case, is a single proton) passes through the membrane-spanning enzyme to the other side of the membrane. The electron is passed through several carriers along a chain of more enzymes (called complexes I, III, and IV), which use the electron energy to pump more protons across the membrane. In the end, this electron transfer chain succeeds in pumping a lot of positively charged protons from one side of the membrane to the other. This movement of charges (protons) leads to the development of a voltage across the membrane. Thus, the purpose of the electron transfer chain is to recharge a biological battery. The first energy conversion is complete—the cell has turned chemical energy into electrical energy.
FIGURE 7.10. Chemical energy in the form of sugar is converted in mitochondria into energy in the form of ATP. This process takes many steps. First the energy is fixed in molecules called NADH and FADH2. These molecules are then processed by different molecular machines in the mitochondrial membrane; the machines use the energy to pump hydrogen ions (H+) from one side of the membrane to the other. NADH is processed by complex I, while FADH2 is processed by complex II (not shown). The excess of hydrogen ions on one side of the membrane is then used to drive the ATP synthase, which recharges ADP to ATP.
The idea of storing energy as an excess of protons on one side of a membrane goes back to the Scottish biochemist Peter Mitchell, who in 1961 suggested this “chemi-osmotic” process as a key to understanding mitochondria. His ideas were subjected to much criticism. Most biochemists believed in a purely chemical process of recharging ADP. Many years were wasted searching for an enzyme that would chemically recharge ADP, but none was found. Finally, Mitchell’s ideas were accepted and he was awarded the 1978 Nobel Prize for Chemistry. In the late 1970s, however, the details of how it all worked were still quite sketchy. How did the stored electrical energy lead to the recharging of ADP molecules?
ATP SYNTHASE AND THE AMAZING SPINNING BATON
Wayne State University hosts the Ahmed Zewail Gold Medal Award and lecture (named after the Nobel Prize–winning Egyptian scientist who pioneered ultrafast measurements of chemical reactions). In 2010, the recipient of the award was another Nobel laureate, Sir John E. Walker, who deciphered the structure of ATP synthase, the molecular machine that constitutes the last step in the slow-burning process in our bodies. I thoroughly enjoyed Walker’s lecture, and during the question and answers, I asked about what was known about the evolution of these amazing machines. He raised an eyebrow and responded, in the laconic style of English intellectuals, “I usually only get questions like this in Texas,” alluding to the creationists’ use of the intricacies of our cells as proof of special creation. I laughed and assured him that I was genuinely interested in evolution and was not from Texas (my German accent should have been a giveaway).
Walker shared his Nobel Prize with UCLA biochemist Paul Boyer. Where Walker figured out the structure of ATP synthase, Boyer figured out how it worked. ATP synthase, as the name suggests, is a machine that makes ATP. The enzyme does not make it from scratch, but rather recycles ADP by attaching fresh phosphate groups. Attaching a phosphate to ADP and turning it into ATP requires energy, and ATP synthase takes this energy from the stored electrical energy provided by the electron transfer chain: The protons, laboriously moved to the outside of the mitochondrial membrane by the complexes of the transfer chain, are now pumped back—but not before giving up their electrical energy to the synthase machine.
In 1977, Boyer suggested that the flow of protons back through the membrane would drive a little rotary motor. Like the dial on a gumball machin
e, each turn of the motor would perform a different step of the ATP synthesis: binding an ADP and a phosphate, attaching the phosphate, releasing ATP. All of these steps would happen in the same enzymatic pocket. The rotation would somehow modify this pocket during each turn so that it would be most suitable for the particular reaction step it was assisting. Having three such multifunction pockets would allow three ATPs to be produced per full turn of the machine.
Boyer also suggested that the energy supplied from the protons moving across the membrane (the proton-motive force, as it was called) was not used to attach a phosphate to an ADP. This apparently happened readily in the pocket of the enzyme. Instead, energy was needed to release the newly formed ATP from the pocket. As Boyer and his group continued their research, using a special isotope of oxygen, 18O, to follow the movement of molecules, they discovered a curious phenomenon: When they ran the process in reverse, letting the synthase break down ATP, rather than synthesizing it, they found that removing the resulting ADP from the surrounding solution stopped the reaction. This made no sense. In chemical reactions, the removal of the product will only speed up a reaction, since it creates a large imbalance between reactant and products. How could the removal of the product, ADP, stop the reaction? One of Boyer’s students suggested that the still-unknown enzyme responsible for ATP synthesis (or breakdown, if run in reverse) only worked when somewhere in the enzyme, ADP was attached. This idea implied some kind of collaborative, allosteric interaction within the enzyme. Slowly, it became clear that the enzyme had three catalytic sites, which processed ADP in a sequential, coordinated way. The finding led Boyer to suggest that the ATP synthase went through a rotational cycle as it attached phosphates to ADP.
Life's Ratchet: How Molecular Machines Extract Order from Chaos Page 22