josh@cs.rutgers.edu (04/20/91)
+---------------------------------------------------------------------+ | The following material is reprinted *with permission* from the | | Foresight Update No 11, 4/15/91. | | Copyright (c) 1991 The Foresight Institute. All rights reserved. | +---------------------------------------------------------------------+ Recent Progress : Steps Toward Nanotechnology by Russell Mills The molecular-scale devices that evolution has made available to us form an impressive list. They include: * Energy converters -- motors (converters from electrochemical to mechanical energy); antennas (electromagnetic to electrochemical); mechanotransducers (mechanical to electrochemical); transfer chains for breaking energy packets into smaller pieces. * Information processors -- binding sites for recognizing shapes and charge patterns; codes and encoders for storing information, code-readers for retrieving it, proofreaders for correcting it, and translators for converting between codes; promoters and inhibitors for switching molecular processes on and off. * Constrainers -- structural elements for maintaining shapes of large molecules, organelles, and cells; membranes to prevent mixing; tracks to control molecular traffic. Here we see analogs of most basic devices upon which industrial technology is based. A more detailed list would include familiar items such as hinges, propellers, lids, plugs, tubes, ratchets, etc. It is anyone's guess whether the easiest approach to developing nanotechnology will be to design and build entirely new molecular devices, or to modify existing devices in a series of small steps by means of genetic and protein engineering. But it is certainly possible to imagine constructing molecular workshops by combining and modifying the molecular devices we have discovered but did not design. Let us keep this in mind as we look at some recent studies of several such devices. MOTORS Several kinds of motors have been found in living cells. Dynein and kinesin are motor molecules that transport objects within cells by hauling them along guiding fibers called "microtubules," using adenosine triphosphate (ATP) as an source of chemical energy. The motor molecule responsible for muscle contraction -- myosin -- works in a similar manner by pulling on actin fibers. Although dynein and kinesin motors are too small to see by light microscopy, the objects they transport can be seen and photographed. In a recent study [A. Ashkin, et al., Nature 348:348-352,22Nov90], researchers attached kinesin motors to silica beads, then used optical tweezers (a trap generated by a laser beam) to place the beads against a microtubule. Often the motors would attach and begin moving their load along this track. Beads having few motors moved only a short distance before coming loose, suggesting that individual motors go through cycles of attachment, movement, and detachment. During in vivo operation each load is presumably bound to the track by several motors, reducing the chance of derailment. In another study [Steven M. Block, et al., Nature 348:348-352,22Nov90], mitochondria were observed being transported by (presumed) dynein motors along microtubules inside the giant amoeba Reticulomyxa. Optical tweezers were used to halt and hold these loads momentarily; when laser power was reduced, the motors would overcome the trapping force and escape. The force generated by a single motor was determined to be 2.6x10(exp-7) dynes. Perhaps the most significant aspect of this work is the use of optical tweezers to manipulate single molecules by means of the larger objects to which they are attached. A similar technique might be of use in constructing a complex molecular machine. Components would be temporarily fastened to "handles" much larger than themselves; an operator using optical tweezers could then move each such unit to an assembly area where the unit would be allowed to bump its way randomly into a binding site; the handle could be removed chemically. Or perhaps optical tweezers could be used to transfer components from various storage depots to molecular motors running along tracks leading to an assembly area; the motors would haul the components into place. Such schemes involve molecular design and construction beyond current abilities, but they seem simpler than schemes requiring automation of the entire process -- parts acquisition, transport, and assembly. A flagellar motor is considerably more complex than kinesin, dynein, or actin motors. Found in many species of bacteria, including E. coli, this motor is embedded in the cell membrane; it drives a helical filament (or "flagellum") that projects into the surrounding medium, and can be switched between clockwise and counterclockwise rotation. An excellent review article by David F. Blair [Seminars in Cell Biology, 1:75-85,1990] surveys what is known about the structure, genetics and dynamics of the bacterial flagellar motor. He puts forward a low resolution model of the motor's structure and a plausible explanation of torque generation. According to the model, the motor and its supporting structure have eight-fold symmetry and consist of somewhat more than a hundred protein molecules of about a dozen different types. The helical flagellum connects to a rod via a flexible segment at its base. The rod passes through a bushing in the outer bacterial membrane and flares out to become a rotor element embedded in the inner membrane. The outer rim of the rotor holds approximately 1000 proton acceptors -- possibly carboxyl groups. The stator, located just inside the inner membrane, consists of eight proton channels spanning the inner membrane and an eight-pointed star-shaped structure bearing a cluster of negative charges at each tip. The rotor's rim passes within half a nanometer of the charge clusters. The energy source for the flagellar motor is a pH difference between the inside and outside of the cell. The proton (H+) density is high outside the cell and low inside. When a proton channel in the stator conducts a proton across the cell membrane, that proton is deposited in a negatively charged proton acceptor on the rotor rim, thus neutralizing the charge. The neutralized site can now move freely past the charge cluster on the stator, whereupon the proton passenger immediately jumps out into the low-H+ interior of the cell leaving the acceptor negatively charged again. The rotor has thus moved one notch ahead. The direction of movement is determined by the geometry -- if protons are deposited into sites on the clockwise side of the charge clusters then rotor will rotate counterclockwise. If the geometry changes so that the proton channel terminates on the counterclockwise side then the motor will run in reverse. Apparently there is a mechanism to accomplish this -- bacteria do reverse their motors, apparently. Some 35 genes are required for the assembly of a normal flagellar structure; many of the them have been cloned and some have been sequenced. About half of the genes code for proteins identifiable in the motor and flagellum; the other half may be involved in assembling, installing, and controlling the structure. Some of the assembly steps have already been deduced. The tools of site-specific mutagenesis are now being brought to bear to reveal the correspondence between structure and function. The bacterial flagellar motor has considerable appeal as a starting point for the engineering of complex molecular devices: * Genetic manipulation of bacteria is easier than that of eukaryotic cells. * E. coli has the best understood genome of any organism. * The output of individual motors can be easily monitored and measured. * The motor appears to be readily understandable in mechanical terms without reference to the intricacies of quantum chemistry. * The flagellar motor contains a ratchet, a screw, an ion channel, an axle, a bushing, and a rotary mechanism -- fundamental components that, with modifications, could be incorporated into other devices. MOLECULAR CHAPERONES When chemists recreate biological polypeptides outside the cells of origin -- either with protein synthesizers or by cloning and expressing genes in different organisms -- they often find that the resulting molecules donUt fold properly. The reason in many cases is that protein folding in the original organism was being assisted by molecular "chaperones." Chaperones are found in all living cells and in organelles such as mitochondria and plastids. They fall into several unrelated families. By definition, they are proteins that mediate the correct assembly of other polypeptides but are not components of the assembled structures. Some chaperones bind temporarily to specific regions of unfolded polypeptide chains, thereby preventing them from binding incorrectly to other parts of the chain until the latter are folded and out of reach. Others bind to already folded protein monomers, covering charges that might otherwise cause them to dimerize incorrectly. Chaperones are also involved in protein transport, DNA replication, masking of hormone receptors, and refolding of proteins damaged by heat. Some of the chaperones have been sequenced; their structure and mechanisms of action are currently being studied. A review by R. John Ellis [Science, 250:954-959,16Nov90] discusses the history and current status of chaperone research. Ellis suggests that the problem of obt [missing text in original -- JoSH] Molecular chaperones may offer shortcuts to nanotechnology. Their role, in essence, is to enable functional proteins to be made from amino acid sequences that otherwise would fold incorrectly. This translates into far greater freedom for protein designers who, in the future, will probably design several chaperones along with a target protein. HINGES AND PLUGS Recent work on T4 lysozyme (an enzyme that dissolves bacterial membranes) has shown that this molecule contains a hinge [Nature, 348:198-199,15Nov90]. It is thought that the molecule has two domains joined by this hinge -- like a pair of jaws. When the jaws are open, substrates can reach the enzymeUs active site; the jaws then close, creating conditions that favor reaction of the substrate. Work by Richard Aldrich et al. at Stanford has revealed that certain ion channels in nerve cells are opened and closed by a structure resembling a ball and chain [Science, 250:506-507, 26Oct90]. These ion channels, made up of protein molecules arranged around a central cavity, serve as pores connecting the inside and outside of nerve cells. By studying the consequences of altering amino acids in these proteins, Aldrich deduced that closure of the channel must be carried out by a ball formed from 19 amino acids, connected by an amino acid chain to the ion channel protein at the channel's cytoplasmic end. It remains to be seen how voltage changes cause this molecular plug to be inserted or removed. CHEMISTRY "Inclusion compounds" consist of a molecule with a large cavity and a small molecule confined within this cavity. A great variety of such compounds are possible. One group with interesting electronic properties is easily made by mixing metal iodides with alpha-cyclodextrin in water and then drying. A recent survey by E.A. Rietman [Mat. Res. Bull., 25:649-655,1990] discusses the structure and properties of these compounds. Alpha-cyclodextrin is a ring-shaped molecule that crystallizes in stacks -- like parallel stacks of donuts. When a metal iodide is present during crystallization, the iodine atoms take up residence in the channels formed by the holes, while the metal atoms occupy the spaces left between cyclodextrins in adjacent stacks. Most of these materials are poor conductors, but several can conduct in one dimension -- probably along the iodine chains. Future developments along these lines may lead to self-assembling conductors and semi-conductors with applications in molecular electronics. MOLECULAR MAINTENANCE As ordinary human endothelial cells grow old they become larger and lose the ability to divide. The lifespan of such cells is limited to about 70 cell divisions. Researchers at the American Red Cross have now developed a short DNA chain that appears to suppress this decline in the ability to proliferate. [Jeanette A.M. Maier, et al., in Science 249:1570-1574,28Sep90]. Earlier work had shown an accumulation in aging cells of a potent inhibitor of cell division, interleukin-1-alpha. If production of this molecule could be impeded, the investigators reasoned that the cells might retain the ability to divide. An "anti-sense" DNA molecule 18 bases long was therefore designed to inhibit the synthesis in vivo of interleukin-1-alpha. When cultures of human endothelial cells were exposed daily to this DNA they retained the ability to divide for about 140 doublings, and their appearance resembled that of young cells. Anti-sense DNA works by binding to specific sequences in messenger RNA molecules, thereby preventing these mRNAs from being translated into protein. Since all cells employ mRNA to convey information from genes to the devices that make proteins, anti-sense DNA holds promise as a molecular tool for controlling a broad range of diseases and other cellular processes. Anti-sense DNA can also bind to, and inactivate, the genes themselves; applications based on this ability are under active study in many laboratories. Dr. Mills directs a small research company in California. +---------------------------------------------------------------------+ | Copyright (c) 1991 The Foresight Institute. All rights reserved. | | The Foresight Institute is a non-profit organization: Donations | | are tax-deductible in the United States as permitted by law. | | To receive the Update and Background publications in paper form, | | send a donation of twenty-five dollars or more to: | | The Foresight Institute, Department U | | P.O. Box 61058 | | Palo Alto, CA 94306 USA | +---------------------------------------------------------------------+