DataDungeon Intro

Just the facts, Ma'am:
Why we think what we think
Genetic data: Who's zoomin' who?

he "front end" of our approach to dissecting the inner workings of the myosin motor is to figure out 1) which parts of the motor contribute to which functions, and 2) how defective parts< can be restored to function by altering parts elsewhere in the machine. Unfortunately, analogies to engines and other macromachines break down at the level of proteins--while it does consist of definable "parts", all the parts are "strung together" as part of only protein chain, which is twisted into the whale shape. One useful way of thinking about the myosin motor is that it has several preferred "shapes". These "shapes" almost certainly involve different relative positions of the "parts" (protein structural elements called beta sheets and alpha helices), very few of which change their structures. The motor cycle occurs because the different interactions of the motor (with ATP, ADP+Pi, ADP, actin...) cause it to favor different shapes. The role of our genetic approach is to deduce these shapes and determine why myosin prefers each in its turn by finding out which amino acids are essential for which shapes, and how defective motors (presumably ones that favor a state too much or are reluctant to enter a state) can be tweaked back into "normal" behavior. Following is a graphical representation of what we have learned. As with our actual research, it's "not done yet", but represents a look at the patterns that are emerging in how different parts of the myosin structure influence each other and the state of myosin.

The arrows indicate that the mutant at the tail of the arrow can be suppressed (restored to function) by the mutant at the arrowhead; dual arrows indicate that either mutant alone lacks function (in some cases where single arrowheads are shown, we simply don't know if the relationship is symmetric). Boxed residues behave as a group genetically (i.e. all suppress or are suppressed by the same other mutations) and biochemically (see below). Thick arrows coming to or going from a box means most or all of the contents of the box show the indicated behavior; thinner arrows mean that only the mutant indicated is known to behave in the way indicated. The data are arranged in two columns, tho this represents a model of the relationships that we are only now testing. It is possible that boxes or mutants in column I are not similar to one another, and should be "swung" to the right to make a third column. These ideas are discussed again in the hypotheses section (or will be soon!).

ATPase data

ne way of measuring the properties of myosin is to look at the rate it burns through ATP, its fuel. An ideal myosin would never use an ATP molecule in the absence of actin, since it is only when binding and moving actin that it is performing useful work. Similarly, an ideal myosin might have a very high rate of actin-induced ATP use, IF this use was caused by successful running of the motor cycle. Thus we measure both the "basal" ATPase (this can be thought of as drooling by the whale, or idling by your car engine--something that happens, but isn't doing any good) and the "actin-activated" ATPase, or the rate at which ATP is consumed when actin is present. This data is represented not as the absolute rate, but the fold increase in ATPase induced by actin addition. Finally, we measure ATPase in the presence of calcium. Normally, the ATP molecule is accompanied by a magnesium ion. If we substitute calcium for magnesium, we're giving it a "funny looking" fuel packet. Wild type myosin actually burns this fuel rapidly in the absence of actin; we have examined the ability of many of our mutants to do the same.

The data shown here display the relative amount of ATPase activity (=ATP usage per unit time) for various mutants. All mutants are shown relative to wild-type myosin, whose value is indicated by dashed lines. Yellow = Basal ATPase, Orange = Actin-activation of ATPase, Red = Ca2+ ATPase. Note how the G680V mutant shows small basal ATPase while its suppressors show enhanced basal ATPase. On the other hand, G680V shows excellent actin-activation, while the suppressors fare poorly by this measure

Actin Binding/Release Data

nother way of "checking up" on the performance of a myosin motor is to check its response to actin under a variety of conditions. The wild-type motor binds strongly to actin in the absence of ATP (the so-called rigor state, as in rigor mortis), but interacts with it only transiently in the presence of ATP (mostly, myosin is hanging out playing with its food, as it were--hydrolyzing ATP and putting it back together again and again and... But every now and again, it bumps into actin in a fruitful way, and performs its job--gripping the actin tightly and moving it. This short-lived event represents much of the time a normal myosin motor is tightly associated with actin). We can also "force" the motor to freeze at selected points--for example, we can examine the "state of mind" of a motor in the ATP-bound state by feeding it ATPgammaS, an ATP analog which is very difficult for myosin to break and use. Similarly, by providing the motor with ADP only, we can see how it behaves when ADP is bound. Finally, we can to some extent investigate the STRENGTH of its grip on actin by seeing what happens to the system in the presence of lots of salt, which weakens many molecular interactions. The data shown below are taken by mixing actin, myosin, and whatever other substances we wish to study, quickly recovering the actin and observing how much myosin was stuck to it at the time we recovered it.
Release of mutant S1 from actin by ATP, ADP....
Bars depict how much more myosin is released from actin by a given treatment than is released by buffers alone. Wild-type is shown in the left half of each data group for direct comparison. Amounts listed next to treatments are in uM (concentration units). ATPgS is an analog of ATP that is very difficult for myosin to break, so it functions as a stable form of ATP. To simplify the presentation, only differences between wild type and mutant are shown. In cases where there is a minus sign (-) next to the wild type data, the mutant values are less than or equal to 0.

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States of the
Working Motor

One Bad

Dissecting a
mutant's Defects

2 Wrong
= Right

Fixing mutants
the hard way




Digging up
NavTable Comments Help!

Bruce Patterson