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Foresight Update 30 (page 3)

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Table of Contents - Foresight Update 30

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 (delta)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.

Table of Contents - Foresight Update 30

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.

Table of Contents - Foresight Update 30


Special thanks this issue go to Ka-Ping Yee and Terry Stanley for their work on Web Enhancement: Ping for coding the Backlink Mediator, and Terry for programming the LinkMap. 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, 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

Table of Contents - Foresight Update 30

From Foresight Update 30, originally published 1 September 1997.
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