Foresight Update 7 (page 4)
A publication of the Foresight Institute
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.
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
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
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
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
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
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
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
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,
Dr. Mills has a degree in biophysics and assists in the production of
Table of Contents - Foresight
Foresight thanks Dave Kilbridge for converting Update 7 to html for this
From Foresight Update 7, originally published 15
On to next page
of Foresight Publications | About the
Foresight Institute | Foresight Institute
Home Page |
Foresight materials on the Web are ©1986-1997 Foresight Institute.
All rights reserved.
Last updated 31March97. The URL of this document is: http://www.foresight.org/Updates/Update07/Update07.4.html
Send requests for information about Foresight Institute activities and membership
Send comments and questions about material on this web site and reports
of errors to email@example.com.