AMER. ZOOL., 29:593-603 (1989)
The Search for a Biological Energy Transducer1
MANUEL F. MORALES
Cardiovascular Research Institute, University of California, San Francisco, California 94143
SYNOPSIS. Muscle can be thought of as an engine—an assembly of devices capable of
performing useful work at the expense of degrading fuel. Among the devices the most
distinctive is the transducer, wherein energy of one form (chemical) is converted into
energy of another form (mechanical). The author contends that until recently muscle
research has examined the auxiliary devices, and that only currently is the transducer
under study.
THE SEARCH FOR A
BIOLOGICAL TRANSDUCER
An engine is an assembly of devices that
performs useful work at the expense of fuel
degradation. One of the devices is always
a transducer, the particular gadget that converts one form of energy into another, e.g.,
chemical to mechanical; besides the transducer there are numerous associated
devices that are essential for engine operation. Since the engines that we encounter
in everyday life—dynamos, electric motors,
rockets, automobile engines—are manmade, there is little difficulty in figuring
out how they work from reading their
schematics. In an automobile engine, for
example, the transducer is the arrangement in which the rapidly expanding gases
generated by a chemical reaction displace
a piston against resisting external forces.
Free energy, in the form of gasoline and
oxygen, is thereby converted into mechanical work. Of course the transducer operating alone would be useless. On the input
side there is needed a carburetor to mix
the fuel ingredients, a spark to catalyze
(accelerate) the oxidation, etc.; on the output side there is needed a crankshaft to
summate the forces from several cylinders,
a differential gear to change rotation direction, etc. Essential as these associated
devices are, the engine element that is truly
distinctive—different in principle in an
automobile engine and in an electric
motor—is the transducer.
1
From the Symposium on Science as a Way of Knowing—Cell and Molecular Biology presented at the Annual
Meeting of the American Society of Zoologists, 2730 December 1988, at San Francisco, California.
In biology it has long been recognized
that cells have engines. Well-fed human
beings perform true force x distance work
every day, and, in freshman biology classes,
glucose-fed frog muscles do the same. More
subtle, but equally obvious, are indications
that cells create and maintain non-equilibrium ion gradients—situations which, if
permitted to dissipate will spontaneously
perform charge x potential difference work.
So certainly biological engines exist. But
biological engines are studied in a different
context; in the absence of schematics, we
spend most of our time figuring out how
they work. Nevertheless, understanding an
engine has to mean the same thing in biology that it does in engineering—recognizing the various devices, their purposes,
identifying and describing the movements,
and, especially, knowing the physical principle of the transducer.
This essay is a brief history of how
researchers have gone about understanding the major biological engine called muscle. We will see that for one reason or
another research has "closed in from both
ends," that it has been successful in understanding the associated devices but is only
now beginning to identify and study the
transducer.
In biological engines both the chemical
fuel and the controlling catalysts are
exquisitely suited to their functions, and
our story will begin with them.
It is now classical knowledge that the
exotic ingredient in biological engine fuel
is adenosine triphosphate, or " A T P " (the
other ingredient is water). The products
of the fuelling reaction are adenosine
diphosphate ("ADP") and ortho-phosphate ("Pi"). ATP, ADP, and P; are all
593
594
M. F. MORALES
translational velocities of the reactants,
hence the energies spent at collisions) a
small region of the mixture enough for
reaction to occur; the energy thus liberated in turn heats neighboring parts, and
so on. A catalyst of a different sort, platinum dust, binds both H2 and O2, thus
bringing them close enough for reaction,
avoiding the waste of time of finding one
ATP + H 2 O - ADP + P^
another in the gas phase.
Biological catalysts are almost always the
can somehow be "coupled" to, or "geared"
into, the engine, the engine should per- specialized surfaces of large (protein) molform some fraction of the work equivalent ecules; they employ the principles that we
of 8 kcal per mole of fuel used. Experience have just illustrated as well as others, and
with man-made machines suggests that due do so with extraordinary specificity and
to fuel leaks, turbulence, friction, etc., the efficiency. Proteins with this ability are
fraction is likely to be 0.3 or 0.4 in good known as "enzymes," and the special class
engines. In fact, in muscle, the fraction is that catalyzes ATP hydrolysis is often called
shockingly close to 1.0, indicating that "ATPases." In muscle the catalytic propmuscle may be an engine worth emulating erty is one of several properties built into
a giant molecule, so we may think of a catin man-made designs.
Typically, uncoupled fuelling reactions alytic "patch" or "site" that is at some defresult in large free energy losses, as we have inite location on the giant molecule. There
just said above; however, the magnitudes is a general feature of enzymatic catalysis
of such losses are unrelated to the rates at that is of special importance for muscle
which the free energy is liberated, i.e., to ATPase: The enzymatic process always
the rates of the fuelling reactions. If done begins with the successive binding of the
with care, gasoline + O 2 (automobile), H2 reactants to the enzymatic site, and it always
+ O 2 (rocket), and ATP + H2O (muscle) ends with the dissociation (not necessarily
can all be mixed without explosive result; simultaneously) of the products from the
specifically, ATP dissolved in water is sta- enzymatic site. Normally, the process is
ble for weeks. The properties of having a indefinitely repetitive, since new reactants
large free energy release, but a very slow can bind as soon as old products are disintrinsic reaction rate, are very advanta- sociated. So far as the enzymatic site is congeous in fuels, since they allow the reaction cerned the process is therefore cyclical.
to be controlled with appropriate catalysts. Between the initial bindings and the final
A catalyst is an agent that greatly accel- dissociations the process invariably proerates the reaction rate without affecting ceeds in several discrete steps or "transithe net free energy change that accom- tions," i.e., the ATP or its chemical descenpanies the reaction. While the fuel is being dants bind first in one state, then the
stored, catalyst is withheld, but at the complex converts into another state, then
moment of use catalyst is introduced. The that into a third state, and so on. In such
fuelling reaction—the exchange of co-va- a sequence of events the bound pieces are
lent bonds between fuel molecules—often called "enzymatic intermediates," and they
requires as an initial step that the mole- can be detected by studying a very fast
cules increase their energy (e.g., to sur- succession of aliquots just after the reacmount long range repulsions) or reduce tants are mixed. A very important consetheir entropy (e.g., in assuming an advan- quence of this stepwise procedure is that
tageous orientation), i.e., that they acquire although the overall free energy yield is
an extra "free energy of activation." Basi- large, the free energy is actually liberated
cally, the catalysts reduce this requisite. For in a succession of relatively small packets.
example, a spark traversing a gasoline + If, as in this case, the energy-yielding reacO 2 mixture "heats" (i.e., increases the tion is "driving" another process, the other
rather highly charged, and interact with
common ions in muscle fluid (e.g., 10~7 M
H + , 10~s M Mg2+) in a way that affects the
free energy yield, but we can safely assume
that the free energy of ATP + H2O exceeds
that of ADP + P, by some 8 kcal/mole
(Podolsky and Morales, 1956). This means
that if the fuelling reaction,
595
BIOLOGICAL ENERGY TRANSDUCER
process will also be advanced in short steps.
Such an arrangement minimizes the chemical equivalent of "leaks, turbulence, and
friction," allowing each state to come into
approximate equilibrium with the system;
it is one of the reasons that the "efficiency"
factor approaches unity. Experimental
analysis has shown (Trentham et al, 1976)
that the ATPase of muscle proceeds
through at least 7 states, which will be enumerated later.
Besides the fuel and the catalyst there
are other important associated devices in
the muscle engine; these were deduced
when the relation between two unrelated
research efforts was finally perceived. An
early, very inspired effort (by Soviet and
by Hungarian scientists Engelhardt and
Ljubimova, 1939; Szent-Gyorgyi, 1947)
isolated two protein constituents, actin, and
myosin, showed one of them (myosin) to bear
the "catalytic patches" for ATPase, showed
that "threads" could be extruded from an
actin-myosin mixture, and—most spectacularly—showed that threads shorten or lift
weights when presented with ATP + H 2 O.
In the same era emerged a puzzle which
could not be easily explained: For the most
part, myosin and actin combined with high
affinity. If the complex was presented with
ATP (plus H2O + Mg2+) the result depended on ambient conditions (salt, pH, temp.,
etc.); under some conditions it contracted,
and under other conditions it dissociated.
What caused this ambiguity? Twelve years
later a second effort by two British teams
(Huxley and Niedergerke, 1954; Huxley
and Hanson, 1954) began, this time in ultracytology. It showed the "sarcomere" (the
structural building block of muscle fibers,
hence of muscle) to contain a double array
of filaments (Fig. 1). In sarcomere (hence
in muscle) shortening, the two end arrays
"telescoped" into the central array, i.e., end
filaments slid past central filaments. The
relationship between these two types of
research efforts began to be illuminated
when differential extraction showed the
central filaments to consist largely of
myosin, and the end filaments to consist
largely of actin.
After the classic studies, progress
depended on an interplay between chem-
W
myosin
Jr
actin 0
FIG. 1. Schematic diagram illustrating that when the
cytological unit (sarcomere) of a striated muscle fiber
shortens (bottom descending) actin filaments translate past myosin filaments, and the overlap (number
of myosins with access to actin) increases. The common speculation is that the S-l moieties of the myosins
are "impellers" whose movements cause the relative
translation of the filaments.
istry and cytology. For example, the assembly of the two kinds of filaments (from
monomeric actin and monomeric myosin),
and the consequent symmetry of the sarcomere, became known. Finally, it was
shown (Mueller and Perry, 1962) that the
myosin segments (so called "S-l" moieties,
or arms, of the giant Y-shaped myosin molecules) that radiate out from the central
filaments toward the adjacent end filaments, (a) had a strong affinity for actin,
and (b) bore the ATPase patches or sites.
Together, these various findings suggested
that the force responsible for the relative
sliding of the filaments (hence the contraction of muscles) resulted from some
interaction between the S-ls and the adjacent actins, in the presence of ATP (Huxley, 1957). The earlier effort had identified
actin, myosin, and ATP as the essential
596
M. F. MORALES
participants in generating force. Ultracytology showed that it was specifically the
S-l moieties of myosin that participated,
and that the filaments were associated
devices for summating and transmitting the
force contributions of acto-(S-l) pairs
(rather like the crankshafts of automobile
engines). These results ushered in "the
modern era."
It is possible to study, as a function of
muscle length (i.e., of sarcomere length),
(a) the static ("isometric") tension developed by an excited muscle, and (b) the
increment (upon excitation) in the rate of
fuel (ATP + H2O) consumption. Examination of these functions reveals that both
are linearly proportional to filament "overlap" (Gordons al., 1966; Ward et al, 1965),
i.e., to the number of S-ls that have actins
opposite to them. Once again it appears
that an S-l plus an adjacent actin constitute
a unit engine. But there is more. An S-l is
only 15 nm long, whereas a sarcomere can
shorten by 1,500 nm. The unitary engine
concept and the long traverse can be easily
reconciled by assuming that the unitary
engine operates repetitively throughout the
traverse, as an oar does throughout a long
canoe trip. If S-l functions as an impeller,
what is the nature of its work cycle? Speculatively one might expect an oar-impeller
to engage, rotate, disengage, rotate back
(repeat endlessly). The "water" in this case
is actin, and the "paddle" is S-l. During
the modern era, several investigations have
sought to check whether S-l in action
actually behaves in this way. A molecular
mechanical investigation (see Appendix) of
myosin revealed that S-l attaches to the
"stem" of the molecule by a universal
swivel, as an oar might contact an oar lock
(Mendelson etal., 1973); this finding makes
the postulated rotations quite plausible.
Another investigation, however, uncovered a surprise. The torque that turns S-l
originates at the (S-l)-actin interface, not
at the swivel (Nihei et al, 1974), i.e., the
torque does not originate at the oar lock,
but at the paddle-water interface! Meanwhile there began to build up evidence that
would eventually link up ATPase chemistry with structural-mechanical observations. As we remarked above, ATPase
activity consists of a succession of cyclical
chemical transformations. Cycles of
ATPase are completed at nearly 102 Hz
(100 "paddle strokes" per second), so it is
not easy to study structure during any one
state lasting a few milliseconds. Reference
to Figure 2 shows, however, that some states
can be held indefinitely. For example, the
"empty" state, in which the ATPase patch
is unoccupied, can obviously be held simply
by never providing ATP.
For simplicity, we have not mentioned
until now that muscle has an off-on
"switch." In the "off' position the transition between state M-ATP and state MADP-P, is blocked, so, if ATP has been
given with the switch off, all the patches
bear ATP*. Low levels of Ca2+ throw the
switch "on," so that another easy-to-hold
state is obtained by adding ATP but withholding Ca2. Physiologists call the empty
state "rigor," and the ATP-but-no-Ca2+
state "relaxation." A seminal EM and X-ray
diffraction study of a muscle fiber was made
in rigor and in relaxation, and it was concluded that in the 2 states the S-Is appeared
to have different spatial (orientational) attitudes (Reedy et al, 1965). Ironically, neither EM nor X-ray diffraction seems to have
produced clear results since that time
(1966), but this old observation strongly
encouraged the idea that S-l moieties execute oar-like, impulsive movements. Moreover, they suggested that if experimental
tricks could induce all the oars to assume
the same attitude even as they pull, "snapshots" of the swinging oar might, in effect,
be obtained.
Soon after the X-ray/EM result, experiments were initiated that sought to assess
the spatial attitude of S-ls by a different
physical effect (Aronson and Morales,
1969). Many relatively simple (in comparison with entire proteins) molecules absorb
light of characteristic wavelength; having
acquired energy by this means, some of these
absorbers later emit light of characteristic
(longer) wavelength; the emission is known
as fluorescence. It is interesting that both
absorption and emission are directional, i.e.,
these molecules behave as though embedded in them were receiving and sending
antennas. This being so, the likelihood that
BIOLOGICAL ENERGY TRANSDUCER
a molecule will absorb so-called "linearly
polarized" light, or that it will emit light
that is "polarized" in a specified direction,
depends sensitively on how the molecules'
own antennas are disposed in space. Turning the situation around, one can experimentally measure absorbance or fluorescence as a function of the polarization plane
of the light employed, and thereby deduce
the orientation of the molecules' antennas
(see Appendix). Knowing this, one can rigidly attach a simple molecule of this type
to an S-1 myosin arm (actually to many,
many similar S-ls) and deduce its orientation by appropriate optical experiments
on, say, a whole muscle fiber. This has come
to be known as the "spectroscopic probe"
method. In recent times it has been highly
perfected, both in theory and in practice
(Burghardt and Ajtai, 1988).
Spectroscopic methods began to detect
different static orientations of the myosin
arms (S-1 moieties) under different chemical conditions, and by 1972 it was conjectured that orientation of S-1 relative to the
actin filament (or fiber) axis was tightly correlated with the nature of the ligand bound
to the ATPase patch (Botts el al., 1972).
Recent, improved methods have borne out
this conjecture. For example, when "nothing" (i.e., the rigor state), and ADP are
compared, the S-1 attitudes are clearly different (Burghardt et al, 1983). In this and
similar experiments (Borejdo et al., 1979)
the ligand is chosen to be a chemically
immutable intermediate in ATPase, or an
immutable analog of an intermediate. The
reasoning applied is that if a series of statically-bound intermediates, results in a
succession of static attitudes, then, in normal ATPase, when intermediates spontaneously transform one into the next, the
S-1 attitudes will correspondingly change
one into the next—in other words, the S-ls
will move in a mechanical cycle. While this
is a very plausible interpretation, it would
be even more convincing to observe that
whenever a fiber is consuming ATP and
generating tension its S-1 moieties are in
repetitive rotational motion. An obvious
first experiment might be to study S-1 orientation during steady ATPase activity and
tension development. The result of such
597
FIG. 2. Seven states of the myosin arm (S-1). The
inner ring gives the names of the molecules that successively occupy the catalytic patch of S-1 (designated
as M). The outer ring indicates diagrammatically the
(S-l)-actin binding relation when the catalytic patch
is so occupied. The crosshatched ellipse is S-1 and the
open circle is actin.
an experiment is unexciting; a seemingly
static orientation (different, however, from
those observed with immutable ligands) is
observed. From independent measurements (Goldman, 1988) it is known that the
principal bound ligand in the steady state
is ADP-Pi**, i.e., states V and VI of Figure
2. To go beyond this result requires a little
reflection (that does not always precede
experimentation). The fundamental difficulty is that without any synchronizing
influence the S-ls, being simple parts of
molecules, exist in great numbers and
behave "stochastically," i.e., it is a matter of
chance when a particular S-1 begins, executes, or completes a particular cycle.
Under such circumstances it is clear that
at any instant there will be as many S-ls
rotating in one sense as in the opposite; at
best an observer will then record a steady
"average" orientation. This circumstance
will thus appear indistinguishable from the
circumstance in which truly nothing is
moving. Various ways of overcoming this
informational impasse have been tried,
some rational and some not. Our approach
was to study fluctuations. Suppose that the
598
M. F. MORALES
S-ls are really moving but do not seem to
be (for the reasons just explained). If we
could contrive to observe only one S-l and
neglect all others, we would of course detect
cyclical motion on making serial observations of spatial attitudes; however, present
technology is not sensitive enough to detect
just a single molecule. What we can do is
observe a limited number of molecules (say
106) but yet enough to be detectable. If the
conditions are right, then at any instant
there may be unequal numbers rotating in
one direction than in the other. This
inequality will vary eratically from one sense
to the other. The result will be that the
average S-l orientation will no longer be
steadily one value, but it will fluctuate
around an average; a suitable plot of angle
vs. time will be a shaky horizontal trace.
The existence of authentic fluctuations, not
due to instrumental artifacts, is qualitative
evidence that in fact the S-ls are moving,
but it is possible to go further. By mathematical analysis it is possible to infer the
"power spectrum" of the observed fluctuations. This power spectrum reveals the
frequencies that constitute the cyclic S-l
movements. To understand what this
means an analogy is helpful. A musical passage, no matter how complicated, can be
reproduced by summing the sounds made
by a battery of n, tuning forks of frequency
f,, n2 tuning forks of frequency f2, and so
on, but we have to allow for the n's to be
positive or negative. From analyzing the
passage mathematically we can obtain a plot
of n2 against f. This information does not
allow us to assemble tuning forks in the
right proportion to reproduce the passage
(because we know only n2, not n). These
frequencies can be related to other physical knowledge. In our work (Borejdo et ai,
1979) we found that when (and only then)
a fiber developed tension the measured
angle of S-l orientation fluctuated in time.
Analysis of these fluctuations revealed constituent frequencies similar to the (independently-known) frequency with which
ATP molecules are hydrolyzed in the same
fiber. This result is not as dramatic as a
movie of S-ls wagging to-and-fro, but for
some time it will probably remain the best
evidence that S-ls rotate cyclically in active
fibers, as one would expect if these S-ls
served as mechanical impellers of adjacent
actin filaments.
A second deduction concerns the energetics and kinetics of the system. The free
energy to be made available for performing external work depends solely on the
free energy change accompanying the net
ATP hydrolysis, whether the hydrolysis is
catalyzed or not. The change ("AG") is a
large negative number, characteristic of
fuelling reactions. Since the reaction is
enzyme-catalyzed, it is conducted in steps,
each with its individual free energy change,
and the sum of these changes is AG. The
correlation between mechanics and chemistry asserts that completion of any individual chemical step requires a corresponding geometric change in the relation of S-l
to actin. In solution these changes are
essentially unopposed, so AG is uselessly
dissipated. But in the fiber the associated
devices (the S-ls and actins are all lined up
infilaments)cause external forces to oppose
movement; if displacement occurs against
these forces, work is performed. Clearly,
the mechanical changes that accompany the
chemical ones are responsible for there
being energy transduction.
A final result of correlation between
chemical and mechanical states has to be
noted, although, strictly speaking, it is
unrelated to energy transduction. For the
sliding force to be generated there must
be a rotation of S-l while it is actin-attached,
but in order for the engine to achieve continuous relative displacement of filaments,
the S-l and actin must detach, and the S-l
must rotate back to engage the next actin
in line. The engine is well-designed to
accomplish these recuperative tasks. Reference to Figure 1 shows that when the
chemical patch is in certain states (binding
of intact ATP) the resulting S-l has very
little affinity for actin. Thus the time interval in which the catalytic patch is bound to
ATP, and is consequently unattached, is
the interval during which random rotation
(induced by temperature) of S-l around its
swivel allows it to search for the next actin.
The existence of states of the cycle that
call for thrust, and of other states that call
for dissociation, is, incidentally, the solu-
BIOLOGICAL ENERGY TRANSDUCER
tion of the puzzle of the 1950s. If we formulate our correlation a little more generally—for every chemical state of the
catalytic patch there is a particular (S-l)actin affinity, and a particular (S-l)-actin
geometrical relation—then we recognize
that the "program" of ATP degradation
provides—through the correlation—a
complete "program" for the work cycle.
The modern era, as recounted in the
foregoing pages, has left us with a muchimproved idea of how the muscle engine
operates. The relative translation of actin
and myosin filaments (that is clearly related
to muscle contraction) is occasioned by the
summated activities of tiny impellers which
are swivel-attached to the myosin filaments. Each impeller (an S-l moiety of a
myosin molecule) independently executes
a work cycle in which it: (1) binds to the
adjacent actin, (2) is turned by a force originating at its interface with actin, (3)
detaches from actin and finds the next actin
in line that has slid into place, (4) binds
again, and the cycle is repeated. It appears
that each mechanical state in this work cycle
is correlated with the (essentially) simultaneous chemical stage in the catalytic degradation of ATP + H 2 O, a degradation
that is proceeding at a separate location on
board S-l. The correlation between chemical and mechanical states expresses, in
effect, (free) energy transduction.
Most basic research on muscle today consists in "mopping up" (prettying up?) the
picture above. Should we be satisified with
this state of knowledge? To decide this
question the automobile analogy may help.
Let us say that we know everything essential about the chemistry of burning gasoline; we realize that each burning (in a cylinder) results from repeated sparks. We
have realized that the attitude of the piston
arm (driving the crankshaft) changes cyclically, and we have established a good correlation between chemical states following
the explosion and attitudes of the piston
arm. That is what we know. What we have
left out is that gases produced in the explosion expand and push the piston. Since we
do not know the transducer design we cannot apply the gas laws relating chemical
stage to pressure, this into force, and force
599
into turning torque. Our state of knowledge about the muscle engine resembles
this parody about the automobile engine.
We have a "correlation" between chemical
state and mechanical state, but we do not
know its physical basis because we have not
yet identified the transducer; without knowing
the mechanism of transduction in atomic
detail we cannot apply the physical laws
that would rationalize our observations. It
is safe to say that the transducer will be
sought out in the future, probably by a
massive posse; for now, however, the
searchers are few, and their knowledge is
sparse and shaky.
In exploring a scientific (or engineering)
puzzle it is often useful to cast it in the
simplest, most familiar light, so as to take
advantage of previous experience with similar problems. The structure and operation
of the muscle engine has (naturally) been
primarily a concern of physiologists who
have sought to know how a cell achieves
movement. For such investigators it is more
interesting to find out how the engine as a
whole—associated devices and everything—works rather than to delve into
abstract questions about the structure and
function of the protein machinery.
Accordingly, physiologists focus on how the
S-l impeller moves relative to the fixed
actin filament, because that is the essential
event in sliding the filaments and so on.
That may, however, prove to be a stultifying "Ptolemaic" viewpoint. With equal
justification we can imagine S-l to be fixed
in space and study how an actin monomer
moves relative to this S-l (a "Copernican"
viewpoint). Nothing is lost in describing
the impulsion that causes interfilament
sliding, since during the impeller cycle it
is the relational angles between S-l and
actin that change. The advantage of this
trivial change in viewpoint is that if we
adopt the Copernican viewpoint we can
speak of a protein particle with two patches
or "sites," one specialized to catalyze ATP
hydrolysis (N-site), and one specialized to
interact with actin (A-site) (Morales and
Botts, 1979). Our correlation asserts that
the A-site "knows" the state of affairs at
the N-site, and acts accordingly. Statements analogous to this have now been
600
M. F. MORALES
with, there are two ways of looking at this
phenomenon. We can imagine that the distortions impressed on the N-site by ATPbinding are mechanically communicated
to the distant A-site, there changing primarily angular relations among relatively
flat surfaces constituting the site. Such a
picture suggests that the communicating
structure may be special in disposition, and
certainly that it be traceable (Botts et al.,
1984). A different view (Shriver and Sykes,
1981) would be to imagine that there is a
pre-existing, possibly temperature-dependent, equilibrium between (S-l)-actin
related by one value of an angle and (S-l)actin related by another value of the angle.
FIG. 3. Angles (5, <j>, \p) by which we can describe theThe binding of ATP and its derivatives
orientation of one rigid body (cylinder) relative to might then "bias" the equilibrium by influanother (block); such a set of angles must be used to
encing a "joint," which may not have an
describe the orientation of actin relative to S-l.
obvious mechanical linkage to the A-site.
In this case the trace would go from N-site
made about many proteins, e.g., hemoglo- to the joint, rather than from N-site to
bin, and there is some information con- A-site. There is probably insufficient inforcerning the mechanism responsible for mation about the internal structure of S-l
transmission of information through their to decide between these viewpoints; what
structure. While no great generalizations information there is lends itself to the simhave emerged, the structural specialization pler first view (Morales, 1988; Fig. 4). Very
of protein regions to bind ATP (and its recent work (Lu et al., 1986; Rajasekharan,
derivatives) is roughly understood, and it 1987) suggests that as a result of ATPis also understood that such "ligands" are binding the "strings" connecting the two
engulfed by the specialized local protein sites translate relative to one another, and
region (N-site). Protein-protein binding is this translational movement may influence
well understood. To the extent that anti- the binding orientations at the A-site.
body-antigen binding can be a model, how- These details of what is happening inside
ever, we may expect the A-site of S-l to of S-l are of course very sparse, but they
consist of several contact areas, formed are already enough to make us feel that we
from rather flat protein regions that are are dealing here with a very interesting
remote from each other (Amit etal., 1986). device (see Appendix).
Undoubtedly both electrostatic attractions
The distortions caused in a protein strucand the "hydrophobic effect" contribute ture as a ligand settles into it are, of course,
to the stability of protein-protein com- exceedingly complicated, but in principle
plexes. Local distortions of both proteins they are completely predictable from well
occur, but there is no evidence of global established physical theory. The same is
changes in the shape of either. Nothing true for the "chemical" transformations
found in S-l or actin so far departs from that convert ATP into its successive
the foregoing observations on other pro- descendants, and for the protein distorteins. We have to continue to think that tions that the descendants in their turn
what changes the most as ATP degrades cause. On the theory that these N-site disare the angles that relate two quasi-rigid tortions are communicated "mechanibodies (Fig. 3).
cally" to distort the A-site, and hence to
We must now consider the crucial ques- change the (S-l)-actin orientations, the
tion of intersite communication. To begin communication and the change in actin
BIOLOGICAL ENERGY TRANSDUCER
Actin Binding
601
ATP Binding
Actin
Interface - . _ '
FIG. 4. Mechanical model suggesting what may happen inside of S-l when ATP binds to the ATPase site.
A continuous belt passes over pulleys A and B. The belt situation before adding ATP is indicated by full
lines, and that afterward by dotted lines. The numbers are residue numbers of certain cysteines (Cs) in the
polypeptide chain constituting S-l. Addition of ATP or ADP is known to change the proximities between
the thiols of two parallel strands. Initially, 697 is opposite to 540, and 707 is opposite to 522 (as indicated by
4 full circles [•]), while 526 and 510 are remote (indicated by open circles [O]). Upon adding ATP or ADP
(location indicated by dot), 697 moves closer to 707, 526 comes to lie opposite to 697, and 510 comes to lie
opposite to 707. Clearly these changes could result from simple movement of the belt over the pulleys. In
such a movement pulley A would turn counterclockwise through a definite angle; to accommodate the
approximation of 697 to 707 (which does not move) elasticity (indicated as a spring) must exist in the system.
The distance from the ATP location to pulley A is in excess of 4 nm. Pulley A has a tangent piece attached
to it, so if A turns the inclination of the piece changes. The piece represents the S-l surface to which actin
attaches. In this sense the model shows that changes resulting from ADP or ATP binding could, at a distance,
affect the angular relation between S-l and actin.
orientation are also in principle predictable, however monstrous may be the computational demands. But the phenomena
at the N-site are those that we conventionally call the liberation of chemical free
energy, and those at the A-site are what
we call the performance of mechanical
work. Therefore we have been describing
a transducer inside of S-l. Exploration of
this transducer will unquestionably take
time, much of it awaiting the development
of methods, as well as of advances made on
simpler systems. And, incomplete as is this
final phase of studying a biological engine,
it is satisfying to know that at long last we
have located the most distinctive element
in the engine ("expanding gases pushing
the piston"), and have started to figure out
how it works.
ACKNOWLEDGMENTS
The author gratefully acknowledges the
editorial advice of Ms. P. Morales and Prof.
L. Peller, as well as research support de-
rived from an AHA Career Investigatorship and NHLBI HL-16683.
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APPENDIX
The author belongs to a group investigating the
molecular basis of contractility. Among the most
effective techniques employed by this group are those
that use various features of the fluorescence phenomenon.
Fluorescence can be detected with great sensitivity,
so its value as a tracer is well known and appreciated,
but there are also other modes in which it has great
utility, particularly when the fluorescing molecules
("fluorophores") have been attached at unique sites
in myosin or actin and serve there as "reporters." It
is mentioned in the text that the effectiveness with
which a fluorophore can be excited by polarized light,
or with which it emits along a prescribed direction,
depends on how the fluorophore is oriented in space.
Therefore one can devise experiments capitalizing on
this feature to ascertain the unknown orientation of
a fluorophore (and of the protein to which it is
attached). This has been the basis for ascertaining the
orientation of S-l moieties in intact muscle fibers.
Fluorescence intensity can be measured with great
rapidity (as well as sensitivity), thus making it possible
to take serial "snapshots" of an object rotating in
space and computing its rotational velocity. By this
technique it was found that S-l could rotate much
faster than could myosin as a whole, thus suggesting
that S-l is swivel-attached to myosin. If a population
of fluorophores is illuminated by a flash of light
(receives a volley of quanta) a subset of these molecules will be "excited" (contain more energy than is
"normal"), and sooner or later they will lose the excess
energy and regain their normalcy. One of the ways
they can do this is by emitting quanta, i.e., by fluorescing. But there are other ways too, e.g., by collision or, if they have access to water, by twisting water
molecules with their own electric fields. The rate at
which the fluorescence of the population disappears
will of course be greater if these other mechanisms
of loss are operative, or lesser if the fluorophore is
safely hidden in an oily pocket of a protein. So, the
"excited state lifetime" is a good sensor of the reporter's environment, and has often been used to detect
structural perturbations, especially in myosin. Finally,
in the most recent research, fluorophores have been
used in a procedure called "proximity mapping,"
BIOLOGICAL ENERGY TRANSDUCER
whereby the structural organization of actin and S-1
have been studied on a nm scale. This application
depends on the phenomenon of fluorescence resonance energy transfer ("FRET"). The energy emitted
by a fluorophore under specified excitation can be
measured. If now a second fluorophore (chosen so
that it is able to absorb the emission of the first) is
placed near the first, it may turn out that emitted
energy of the first fluorophore is now reduced. The
difference can be used to calculate the fraction of
603
energy that has been transferred to the second fluorophore. This fraction transferred depends (among
other things) on the inverse sixth power of the distance
between fluorophores. Therefore if fluorophores A
and B can be attached to two points on actomyosin,
distance AB can be measured: if A and C, and B and
C, can be attached, then AC and BC can be measured,
and so on. In this way a lattice of chemically-defined
points has been built up. It is the basis for the real
geometric relations underlying the schema in Figure 4.