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Foresight Update 7 (page 4)

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Table of Contents - Foresight Update 7

Recent Progress: Steps Toward Nanotechnology

by Russell Mills

Of the various research paths leading toward nanotechnology, protein engineering seems to be carrying the most traffic--a situation stemming from the ability of protein engineers to draw from a pre-existing treasure trove of protein designs: Earth's multi-billion-year accumulation of molecular tinkering. The sheer volume of exciting results causes this subject to dominate my column; I expect that it will for some time also dominate the "toolkit" of molecules and techniques that researchers develop for working with atoms.

Protein engineering itself follows several parallel tracks defined by the strategies used in designing and building new protein molecules. Let us look at three of these strategies: site-directed mutagenesis, transfer of structural cassettes, and induction of antibodies.

Site-directed mutagenesis

This widely used technique is based on the alteration of existing protein chains through addition, deletion, or substitution of amino acids at particular points along the chain. While in principle capable of producing any conceivable protein, this method's usefulness is circumscribed by our limited ability to make rational choices of sites and alterations. Modifying a protein to achieve a given structure or function may involve changes at dozens of sites. Experience can provide a rough guide to the changes needed, but pinning down the details requires the calculation of interactions between thousands of atoms--an ability still well beyond current computational techniques.

The work of Roger Bone and colleagues at UC illustrates the use of site-directed mutagenesis to explore protein function. The enzyme alpha-lytic protease is a protein that selectively cuts other protein chains having certain sequences of amino acids in accessible positions. Bone's group substituted amino acids at either of two locations in the enzyme's active site (the pocket in the enzyme's surface where substrate molecules are bound and transformed). A mutation at one location removed a bump in the active site, causing the enzyme to select larger substrate molecules for binding and modification. A mutation at the other location enlarged the active site by the same amount, but gave it a different geometry; the enzyme's overall activity declined drastically. Both mutations made the enzyme less rigid, broadening the class of substrates to which it could bind. [Nature 339:191-195,18May89]

Table of Contents - Foresight Update 7

Transfer of structural cassettes

This approach to protein engineering consists of transferring segments of one protein to another protein--cutting and pasting chains of amino acids, as it were. The rationale is that evolution has already optimized the structure of such segments for the functions and local environments in which they occur. The protein engineer still must pay attention to how the components join together--for example, rejecting candidate chains whose overall geometry would disrupt adjacent parts of the target molecule--but is spared the task of understanding and calculating every molecular detail.

Thomas Hynes and his colleagues at Yale and Stanford recently used this method to make a hybrid between two unrelated proteins. A chain of 5 amino acids from one protein was replaced by a 6-amino acid chain from the other, yielding a fully functional protein with somewhat reduced stability. In choosing the proteins and the transferred segments, the researchers ignored the amino acid sequences; they strove for similarity in the angles at which the segments fused to adjacent protein chains. [Nature 339:73-76,4May89]

Structural cassette transfer is a major shortcut in the development of the nanotechnological toolkit. Treating proteins as modular devices whose parts can be selectively interchanged is one way of accessing evolution's ancient but extensive engineering experience.

Table of Contents - Foresight Update 7

Induction of antibodies

Antibodies are proteins produced by mammalian immune systems in response to molecules that the immune system classifies as foreign. A given antibody binds tightly to a specific pattern of atoms (called an "antigen"); we say that it recognizes that antigen.

When a mammal (typically, a rabbit) is injected with a substance that provokes an immune reaction, the resulting antibody molecules can be separated from the blood and tested for reactivity with molecules resembling the original antigen. Often some of the antibodies are able to recognize molecules chemically distinct from the antigen but resembling it in shape or charge distribution. This has made it possible to induce the production of antibodies that catalyze chemical reactions, much as enzymes do. The trick is to choose an antigen resembling a chemical "transition state"--i.e., a transitory configuration of molecules in mid-reaction, as the system passes over the energy barrier separating the reactant configuration from the product configuration. By binding the transition state, the resultant "catalytic antibodies" make it somewhat less unstable, lowering its energy and thus lowering the energy barrier that hinders the progression of the chemical reaction.

In a new extension of this strategy, K. M. Shokat and others in Berkeley and Zürich have developed a degree of control over the microstructure of an antibody's binding site. Although it would be possible (in principle at least) to achieve the same results through site-directed mutagenesis or even by synthesizing the relevant parts of the antibody and "pasting" them into another antibody (as structural cassettes), the method used by Shokat's group is indirect and elegant: they synthesized an antigen that mimicked not the transition state of a chemical reaction but the complementary shape and charge distribution they hoped to realize in the binding site of an antibody. Using this antigen, they induced the production of antibodies with desired characteristics built into their binding sites. [Nature 338:269-271,16Mar89]

The procedure may be made clearer with this analogy: a mechanic (protein engineer) who wants a new kind of wrench (the catalytic antibody) makes a wax model of the wrench's jaws (the shape and charge distribution of the binding site) and gives it to a foundry (the rabbit). The foundry builds a plaster mold (the complementary shape and charge distribution of the binding site) around the wax model, melts out the wax, uses the mold to cast a metal wrench head (the actual binding site), and attaches the wrench head to a standard handle (the rest of the antibody). The resulting wrench (catalytic antibody) can now be used by the mechanic to carry out mechanical tasks (catalyze certain chemical reactions).

Like the structural cassette strategy, this method draws upon an ancient engineering legacy--that which produced the antibody factories of the immune system. By delegating much of the molecular engineering and fabrication work to these agencies, researchers stay within the practical limits of computational and experimental complexity.

Table of Contents - Foresight Update 7

Protein engineering in perspective

The discussion above portrays protein engineering as having begun to assemble a few very simple tools. It is mainly preoccupied with making very small changes in existing protein structures to test the relationship between structure and function, or to develop variations on useful functions already found in native proteins.

There are exceptions to this picture. The work of de Grado's group at Du Pont, discussed in previous issues of Update, belongs to yet another branch of protein engineering: de novo protein design, in which moderate-sized proteins are designed and built from scratch. These workers are confronted by the same basic problem plaguing all molecular engineering today: that of computing the structure and dynamics of large collections of atoms. A future generation of computers should make such computations feasible; meanwhile, protein designers simplify the problem: using short chains of amino acids (for which useful calculations can be done) they build larger structures from multiple copies of these chains by arranging them in simple patterns.

Table of Contents - Foresight Update 7

Short subjects

Molecular motors appeal to the mechanic in all of us, and evolution has managed to come up with several kinds--some drive rotary devices (like flagella), others drive muscle contraction, while still others haul loads along fibers (called "microtubules") inside cells. The last category includes dynein, which generates movement away from the growing end of microtubules, and kinesin, which moves in the opposite direction. Jonathan Scholey in Denver and his colleagues have now shown that kinesin consists of a pair of globular "heads" about 10 nm in diameter, a 45 nm stalk, and a fan-shaped "tail" about 20 nm long. The heads apparently are motor domains that bind to microtubules and generate force and motion. They contain a distinct ATP-binding site for intake of energy. The tail probably binds to cell organelles, which are then hauled along the microtubules to their destinations. The researchers speculate that the two heads take turns attaching and detaching as they track along a microtubule so that at least one head is holding on at any given time. [Nature 338:291-292,23Mar89]. (Chapter 4 of Engines of Creation describes an array of assemblers backed up by conveyors carrying reactive molecules to the assembly area. Rows of molecular conveyors--an engaging idea, but one with no prospect of fulfillment for several decades, right? ... Wrong. Prototypes exist already inside our every cell! Modifying microtubules and kinesin motors for use in other contexts should prove far easier than designing a whole new conveyor system from scratch.)

Some fascinating work by Seth Stern and colleagues (UC and Univ. Wis.) elucidates ribosome assembly and function. Ribosomes--those intracellular particles that read the genetic code and use the information to assemble proteins--are the archetype of all molecular assemblers. We already use them to produce proteins specified by recombinant DNA; when we learn to redesign them we should be able to manufacture almost any polymeric material with atomic precision. Ribosomes consist of three RNA molecules, and dozens of different proteins whose role has been obscure. Stern's group measured the accessibility and reactivity of all 1500+ nucleotides making up one of the three RNA molecules, doing this at each stage as this RNA folded into a functional molecule. The work revealed a folding process controlled and stabilized by the ribosomal proteins. The researchers constructed a 3-dimensional map of this RNA molecule, and found sites on the ribosomal surface for the binding of antibiotics and of molecules that assist in protein synthesis. The most significant findings, however, involved the accuracy with which ribosomes read the genetic code. The error-rate is affected by the conformation of the RNA chain, which in turn is modulated by external factors, such as the binding of streptomycin. Intriguingly, the authors mention a mutation that causes ribosomes to read hyper-accurately, and suggest that "translational accuracy is somehow held in balance at a low level of misreading." [Science 244:783-790,19May89]

Protein motions--vibrations, rotations, folding, unfolding, and other motions--unfortunately appear to be crucial for the function of biological macromolecules. The time-scale of these motions ranges from about 100 femtoseconds to more than a second. The motions treatable by computer models lie between 100 femtoseconds and about 300 picoseconds, but experimental verification has been lacking. Now Hans Frauenfelder of the Univ. of Illinois suggests that recent measurements of myoglobin dynamics not only permit comparisons to be made between theory and experiment, but also hint at a simple underlying unity between some protein motions and the dynamics of glassy materials. He believes that progress in protein dynamics will be rapid. (With 9 more orders of magnitude to cover, we'll be in trouble if it isn't!) [Nature 338:623-624,20Apr89]

Chemists Nadrian Seeman and colleagues at New York University say they aim to build three-dimensional structures out of DNA segments, then hook proteins or other catalytic molecules to the resulting framework. [Science News, 136:126,19Aug89]

[Webmaster's note: See Update 23 for a report on the award of the Feynman Prize in Nanotechnology to Dr. Seeman in 1995.]

Materials fabrication has reached nanometer dimensions (in one dimension) in experiments at Simon Fraser University in Burnaby, British Columbia. A paper by Anthony Arrott and others describes the use of molecular-beam epitaxy to lay down alternating layers of metals, each only a few atoms thick. The resulting materials exhibit such properties as magnetic fields of unprecedented strength and magnetic moments that can be switched from one direction to another by an electric current. [IEEE Spectrum, April 1989:12]

Dr. Mills has a degree in biophysics and assists in the production of Update.

Table of Contents - Foresight Update 7

Foresight thanks Dave Kilbridge for converting Update 7 to html for this web page.

From Foresight Update 7, originally published 15 December 1989.
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