Foresight Update 30
Page 3
A publication of the Foresight Institute
Jeffrey Soreff's
Technical Progress column is continued from the previous page.
 Enzyme Design and Analysis
Enzymes are important to nanotechnology in several ways. They
are examples of atomically precise functional structures. They
may be useful as tools to construct the building blocks for more
rigid, polycyclic structures. The papers described below report
advances in the design and analysis of enzymes.
A.D.Mesecar, B.L.Stoddard, and D.E.Koshland Jr., writing in [Science
277:202-206 11Jul97--MEDLINE
Abstract] have experimentally demonstrated the
sensitivity of enzyme catalysis rates to small changes in the
alignment of the substrates. They studied isocitrate
dehydrogenase (IDH). This enzyme transfers hydrogen from
isocitrate to nicotinamide adenine dinucleotide phosphate (NADP),
performing one of the reactions in the Krebs cycle. The geometry
of the active site was determined by x-ray diffraction of the
Michaelis complex of IDH with its substrates and a metal ion
cofactor (Mg2+ in the normal case). Three crystal
structures were compared: the structure of a complex of the
normal substrates, the normal metal, and a mutant (Y160F) enzyme,
the structure of a complex of a modified substrate (nicotinamide
hypoxanthine dinucleotide phosphate NHDP), the normal metal, and
the normal enzyme, and the structure of a complex of the normal
substrates, Ca2+, and the normal enzyme. At the
reaction center, a hydride is transferred from the isocitrate to
the nicotinamide ring in all three of these cases. None of the
modifications studied changes the identity of the atoms which
participate directly in the reaction. The modifications do
alter the geometry of the reaction site.
In the experiment modifying NADP to NHDP, the "substitution
occurs at a position that is >12Å and 21 bond lengths away
from the hydride that is transferred and has no effect on the  G0 of the
reaction." The substitution does, however, shift the
positions of the substrate atoms in the complex, propagating to
the nicotinamide ring, where it "causes the distance of the
hydride donor-acceptor pair to increase by 1.55 Å (a covalent
carbon-carbon bond distance is 1.54 Å) and the angles of
approach to deviate between 10° to 20° from the more in-line
geometry observed for the NADP structures." The effect of
this shift in geometry is to reduce the catalysis rate by a
factor of 3.8 x 10-5.
In the experiment substituting Ca2+ for Mg2+
a cascade of geometric changes starts with the enlargement of the
coordination sphere from the normal 6 ligands (for Mg2+)
to 8 ligands (for Ca2+). The net effect at the
reaction center is "an adjustment of isocitrate, NADP, and
the side chains of the aspartate residues, thereby decreasing the
distance between the hydride donor-acceptor pair by 0.55 Å and
altering the attacking and dihedral angles." The effect of
this shift in geometry is to reduce the catalysis rate by a
factor of 2.5 x 10-3. The authors emphasize that the
Ca2+ data rule out a simple dependence of the
catalytic speed on the distance between the hydride donor and
acceptor. They write that: "The results provide evidence
that orbital overlap produced by optimal orientation of reacting
orbitals plays a major quantitative role in the catalytic power
of enzymes."
These experiments have a number of implications for
nanotechnology. First, they provide direct experimental evidence
for sharp geometrical control of reaction rates, even in the
unfavorable case of hydride transfer (with maximal tunneling
effects). This strengthens the claim that controlling the
approach of a reagent to a workpiece can permit selective
reaction at just one of a number of nearby chemically equivalent
sites. Second, this adds to the desirability of very stiff,
heavily crosslinked "diamondoid" structures for
controlling reaction state geometries. Third, these experiments
imply that purely x,y,z control of reactant positioning, as in
current STM and AFM piezoelectric actuators, is likely to be
insufficient for efficient mechanosynthesis. Control of
orientation via tiltable stages or similar mechanisms is likely
to be necessary to steer reagent orbitals towards desired
reactions.
Writing in [C&EN 35-36 30Jun97], S.Borman reports on
recent work from J.P.Caradonna's lab, which redesigned a
noncatalytic host protein, thioredoxin, to incorporate an active
iron center from superoxide dismutase (SOD). They were able to
demonstrate catalytic activity analogous to that of SOD (albeit
four orders of magnitude slower) in the hybrid structure. In a
separate modification, they were also able to introduce an Fe4S4
group into thioredoxin and to demonstrate the redox activity of
this group in the modified protein. Other groups have added metal
binding groups into proteins previously, "but this is the
first time metal-modified proteins have exhibited functional
activity such as catalysis." A program called Dezymer
(originally from H.W.Hellinga) was used to do the combinatorial
search to find feasible modifications to thioredoxin in order to
accommodate the SOD active site while preserving the folding of
the host protein. Caradonna said that they "are using
Dezymer as a tool to systematically investigate the effect of the
protein matrix on reactivity at metal centers, just as the effect
of ligand substitutions on the reactivity of small metal
complexes have been studied." From the perspective of
nanotechnology, our best current technologies for building
atomically precise 3D structures are currently biomolecules such
as proteins. This work helps to disentangle this geometrical
design work from the design of reactive tips, which are roughly
analogous to the metal centers in catalytic proteins.
Single Molecule Techniques
The techniques described in the sections above all rely on
some form of self-assembly to contruct structures. An alternative
strategy for constructing and analyzing atomically precise
structures is to modify one molecule at a time. These techniques
allow direct control of position with atomic precision. Some type
of replication technique needs to be used with this strategy in
order to produce large numbers of structures.
I. Amato, writing in [Science 276:1982-1985
27Jun97] presents a brief summary of the history of STM and AFM
microscopies. Most of the work that he touches on will be well
known to the readers of this column, but one group's work was
unfamiliar to me, at least. R. Dunn's group at the University of
Kansas has been monitoring cellular pores with an AFM probe.
Amato quotes Dunn as saying: "In the open state you see a
channel, but after triggering the pore [with calcium ions] you
see something like a piston stick up and block the central part
of the channel." This experiment demonstrates techniques
that should be useful in debugging nanoscale machinery. First, it
detected a nanometer scale mechanical event rather than
just a static image. Second, the mechanism for triggering the
event (Ca2+ addition) was independent of the AFM, so
modification of the AFM tip for more specialized imaging can be
attempted without interfering with the trigger mechanism. Third,
since the experiment used an AFM rather than an STM, it is
applicable to insulating samples as well as to conducting ones.
Writing in [Nature 387:688-691 12Jun97], F. Kulzer
et. al. "report light-induced reversible frequency jumps [of
the absorption frequencies of] single molecules of the aromatic
hydrocarbon terrylene embedded in a particular site of a
p-terphenyl host crystal at a temperature of around 2K."
When p-terphenyl crystals lightly doped with terrylene are
condensed from vapor, the terrylene can be inserted into four
different crystal sites, called X1-X4, with
different electronic dynamics. The authors found that
"although single molecules in X2 are very stable
with respect to changes in absorption frequency, we find
reversible, light-induced frequency jumps in site X1."
The authors examined isolated molecules by keeping the doping
sufficiently light that there were typically fewer than five
terrylene molecules in a 5-µm focal spot.
More specifically, when a terrylene molecule in an X1
site is illuminated at resonance with a laser field of 0.25 W cm-2
for 10-60 seconds, it jumps to a second state (denoted as XY)
where its absorption frequency is 843 GHz higher than in its
original state. Illumination at the new resonant frequency
switches the molecule back to its original spectral position. The
frequency jump is a small fraction (0.16%) of the absorption
frequency, but it is quite distinct, "more than 10,000 times
greater than the homogenous line width of terrylene in
p-terphenyl." It is also quite consistent from sample to
sample. The authors "have investigated ~50 molecules in
seven different crystals taken from four sublimation runs."
They have "always found the same reversible jumps over 843
± 2GHz, with a variation of at most 500 MHZ for molecules in the
same crystal." Illumination at XY's resonant frequency
doesn't always switch the molecule back to the X1
state. About 10% of the time, the molecule makes a transition to
a series of other states, although it can be returned to the X1
state by annealing at 40K. The states are sufficiently stable
that the authors followed the light-induced state transitions in
a "spectral diary" of one molecule over a period of 22
days.
The authors interpret these states as changes in the orientation
of phenyl groups in the local environment of the terrylene
molecule. They analyzed the vibronic structure of the
fluorescence from molecules in the various states and conclude
that "a central ring flip of the adjacent p-terphenyl
molecule...alters the dipolar host-guest coupling and shifts the
absorption frequency of the chromophore."
This work affects nanotechnology is several ways. First, as the
authors write: "Although many obstacles still exist, this
observation of reversible, reproducible frequency jumps might
pave the way towards the design of host-guest systems suitable
for optical switching and storage functions at the
single-molecule level." Second, this provides a new
technique for predictably reorienting single molecular
environments, possibly applicable where STM or AFM techniques are
not feasible. Third, together with a localization technique (such
as near-field optics or possibly Stark effect shifting of
resonant frequencies in STM fields), this technique may provide a
novel patterning mechanism for selectively reorienting individual
molecules.
Not all work that creates a nanoscale structure directly aids the
development of molecular manufacturing. Writing in [Science
276:1401-1404 30May97], M. Park et. al. describe a novel
method for constructing a pattern with a 30-40 nm pitch. Their
technique relies on phase separation in diblock copolymers. They
synthesized diblock copolymers from polystyrene-polybutadiene
(PS-PB) and also from polystyrene-polyisoprene (PS-PI). Two
different block length combinations were used, SB with 36
kilodalton PS blocks and 11 kilodalton PB blocks, and SI with 68
kilodalton PS blocks and 12 kilodalton PI blocks. The basic
ordering mechanism is evident even "in bulk, [where] the SB
36/11 microphase separates into a cylindrical morphology and
produces hexagonally ordered PB cylinders embedded in a PS
matrix; SI 68/12 adopts a spherical morphology and produces PI
spheres in a PS matrix with body-centered-cubic order." The
diameters of these structures is set by the chain lengths of the
blocks. In a 50 nm layer on Si3N4, SB 36/11
produces a sandwich structure, with a hexagonal array of PB
spheres in a PS matrix, bounded above and below by PB
"wetting layers", which coat the Si3N4
substrate and the air interface.
Since both PS and PB etch at similar rates under reactive ion
etching conditions, the authors needed to use special chemistry
to distinguish them. They used two techniques, which changed the
effective thickness of the PB in opposite ways. When they wished
to make the PB volume act like a smaller volume of polymer, they
ozonated the wafer. This "attacks the carbon-carbon double
bonds in the PB backbone" and converted the PB (but not the
PS) into fragments that can be removed with water. When they
wished to make the PB more resistant to etching than the PS, they
exposed it to OsO4, which "adds across the
carbon-carbon double bonds in the PB backbone," loading the
PB with etch-resistant osmium. When these two modified films are
exposed to CF4/O2 reactive ion etching,
they yield holes and dots respectively. The holes, for instance,
are approximately 15 nm deep, and have a period of 30 nm.
While this technique does generate nanometer scale patterns, and
it does build the patterns up from molecular patterning, it does
not appear to directly aid the development of molecular
manufacturing. At the molecular scale, the patterns of polymer
blocks are too imprecise. The blocks have approximately the
stated molecular weights, but they do not all have precisely the
same length (unlike specific proteins, for instance) nor can they
be expected to pack into regions with atomically precise
boundaries. At the other end of the length scale, the authors do
not appear to have attempted to synchronize their patterns with
the arbitrary long range patterns made possible by
photolithography. In fact, the patterns formed in this experiment
have "a polygrain structure that has an average grain size
of 10 by 10," so it loses coherence after about 300 nm. If
this work is extended in both of these directions, then it may
provide a mechanism for exploiting conventional photolithography
to order atomically precise structures, but it does not currently
do this.
Jeffrey Soreff is a researcher at IBM with an interest in
nanotechnology.
 Thanks
Special thanks this issue go to Ka-Ping Yee and Terry Stanley
for their work on Web Enhancement: Ping for coding the CritLink mediator, and Terry for
programming the CritMap. See the "Inside Foresight"
column in this issue for more details. Thanks also to our many
informal advisors on the project, who include Mark Miller (who
designed the backlink approach along with Ping), Norm Hardy, Marc
Stiegler, and Dean Tribble. Also of help have been Dave Forrest,
Wayne Gramlich, Ralph Merkle, and Russell Whitaker, and for PR
guidance, Lew Phelps and Ed Niehaus.
A discussion of hypertext publishing credits should also thank
the two main visionaries, Ted Nelson of Project Xanadu and Doug Engelbart,
now with Bootstrap Institute;
the primary corporate supporter, John Walker (now at www.fourmilab.ch), co-founder
of Autodesk which funded Xanadu; and Foresight's own Eric Drexler
for promoting the concept in
Engines of Creation and expanding it in the
paper "Hypertext
Publishing and the Evolution of Knowledge," both of
which are on the web in full text due to work by Russell
Whitaker.
For arranging for Foresight to have a London office, we
vigorously (or should we say, vigourously?) thank Chris Portman
and Philippe Van Nedervelde.
Continual thanks go to Gayle Pergamit, co-author of Unbounding the
Future, for frequent assistance in briefing the media
on nanotechnology and Foresight.
Ongoing thanks as well to the '97 nanotechnology conference team,
including volunteer co-chairs Ralph Merkle and Al Globus,
tutorial chair Deepak Srivastava, and planner Marcia Seidler.
For assistance to our summer policy intern, Franklin Van Ardoy,
we thank Prof. Pat Parker (U.S. Naval Postgraduate School),
Duncan Forbes, and Margaret Jordan.
For editorial contributions to this issue, thanks to Richard H.
Smith, II of Georgetown University and Chris Worth of Singapore.
For sending information, we thank Jon Alexandr, Richard Counihan,
Dave Forrest, Chris Fry, Richard Kluckhorn, Markus Krummenacker,
Anthony Napier, Chris Portman, Salvatore Santoli, Nadrian Seeman,
Richard Smith, Steve Vetter, and Russell Whitaker.
—Chris Peterson, Executive Director, Foresight Institute
From Foresight Update 30, originally
published 1 September 1997.
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