Protein engineering for molecular electronics
Stephen G. Sligar and F. Raymond Salemme
University of Illinois at Urbana-Champaign, Urbana, Illinois and Sterling Winthrop
Pharmaceutical Research Division, Malvern, Pennsylvania, USA
Recombinant DNA technology allows the manipulation of the physical
properties of proteins that perform electron transport and photochemical
processes. Recent work is reviewed that has a potential impact on the
development of molecular electronic devices, within a general framework
outlining strategies for device fabrication. This review is also published in
Current Opinion in Structural Biology 1992, 2:587-592.
Current Opinion in Biotechnology 1992, 3:388-393
Introduction
This review is p r o m p t e d b y the recent enthusiasm
for the use of biological macromolecules as building
blocks for the construction of molecular electronic devices (MEDs). Molecular electronics is a term that has
a dual meaning. One meaning refers to electronic devices that use materials w h o s e unique properties result
from their molecular structure. In this regard, proteins
can be used in MEDs to sense, r e s p o n d to, or record
chemical, electrical, or physical stimuli. Examples include the use of the bacterial photopigment bacteriorhodopsin as a three-dimensional medium for the
storage and readout of optically e n c o d e d information
[1",2",3]. The second definition of molecular electronics embodies the concepts that individual molecules
are functional units responding to stimuli, and that
they have the potential to b e interconnected in order to replicate electronic circuits functionally. G o o d
examples of such molecular electronic devices do not
yet exist, but the principles necessary for their design
and assembly are beginning to emerge from a variety
of areas.
From the perspective of practical device design, there
are several levels at which the ability to modify protein
molecules using recombinant DNA technology could
have an impact on the fabrication of MEDs. These
include alteration of intrinsic optical and electronic
properties, increasing protein stability for operation in
non-biological environments, and modification of surface properties to facilitate d e n o v o design of mesoscale
molecular assemblies or molecular circuits. Current activities in each of these categories are described below.
Modification of protein electronic and physical
properties
Naturally occurring proteins incorporate a wide range
of physical properties that are of potential use in electronic devices. In most cases, the useful properties
derive from a prosthetic group such as a metal center, organic r e d o x cofactor, or chromophore. Although
naturally occurring redox and photoactive proteins exhibit great functional diversity, this is achieved through
a limited n u m b e r of prosthetic groups w h o s e properties are modulated through interaction with amino acid
side chains of the surrounding protein moiety. Attempts to engineer proteins in order to modify their
properties in useful ways have followed precedents
suggested b y the study of structure-function relationships in natural systems. In this respect, heme-containing proteins, which comprise a diverse set of molecules
with electron transfer, ligand binding, or catalytic function, are of particular interest. Alterations in the electronics or chemistry of the h e m e iron center frequently
produce large changes in optical spectroscopic or magnetic properties which are useful as indicators for state
assignment or device readout. Following pioneering
w o r k on cytochrome b5 [4], myoglobin [5-8], and cytochrome p450 [9], recent w o r k reporting site-directed
modifications of residues that are heme iron ligands, or
can otherwise influence h e m e spectral properties, include additional studies on cytochrome p450 [10] and
myoglobin [11], together with studies on cytochrome
c peroxidase [12,13] and iso-l-cytochrome c [14]. Although the study of the structure-property relationships that affect heme proteins remains an area of
active interest [15"], few systematic efforts have b e e n
Abbreviation
MED--molecular electronic device.
388
© Current Biology Ltd ISSN 0958-1669
Protein engineering for molecular electronics Sligar and Salemme 389
directed at producing unusually stable molecules that
could increase the reliability of state assignment. H o w ever, a serendipitous enhancement of protein stability
was reported for a site-directed mutant of iso-l-cytochrome c [14], in which an internal asparagine had
b e e n changed to a hydrophobic isoleucine residue.
This alteration resulted in the loss of an internal water molecule w h o s e location in the interior of the native
protein was postulated to b e energetically unfavorable.
Similar hydrophobic enhancements could direct further
efforts towards the important goal of stabilizing redox
proteins in non-aqueous environments.
A property central to the function of many MEDs is
the regulation of electron-transfer rate between proteins or b e t w e e n proteins and an external oxidoreductant. Physical aspects of the electron-transfer p r o cesses in proteins are n o w relatively well understood,
and suggest that electron-transfer rates d e p e n d primarily u p o n prosthetic group separation, difference in
oxidoreduction potential and molecular reorganization
energy [16",17]. This is consistent with recent site-directed modifications of the invariant Phe82 residue in
yeast iso-l-cytochrome c, suggesting that intermolecular electron transfer does not require the participation
of specific aromatic amino acids as electron wires between protein prosthetic groups [18,19]. Nevertheless,
translation of the physical requirements for efficient
electron transfer into a specific modification strategy
remains complicated, as s h o w n by the results obtained
b y Barker et al. for yeast iso-l-cytochrome c [20]. In
addition to heine-containing redox proteins the introduction or regeneration of copper-binding sites in proteins has b e e n investigated. In one study, both type I
and type II c o p p e r site properties could be obtained b y
addition of an appropriate external ligand to an azurin
mutant in which one of the native histidine c o p p e r
ligands had b e e n deleted b y site-directed mutagenesis [21]. In a second study, site-directed modifications
of cytochrome c were performed in order to introduce
a c o p p e r ligand site [22]. In both cases, the modified
proteins a p p e a r e d to lack the intrinsic stability of the
native molecule, but may point the w a y to successive
generations of molecules with useful functions.
Interaction specificity
Because rates of intermolecular transfer d e p e n d very
strongly on prosthetic group separation distance [16-,
17], reactions between reversibly binding electrontransfer proteins d e p e n d on interactions that ensure
the proper relative intermolecular orientation. Early
w o r k that m o d e l e d the interactions of cytochromes c
and b5 established the importance of solvent exclusion
and complementary electrostatic interactions in radox
protein interactions [23]. This precedent has since b e e n
generalized to m a n y other biological electron-transfer
interactions. With the advent of site-directed mutagenesis methods, investigation of intermolecular recognition interactions has intensified [24-29]. Most notably,
methods have recently b e e n developed that discriminate between the relative contributions of the principal components of intermolecular recognition: polar
interactions that result from hydrogen b o n d s and salt
bridges forming in the interaction domain, and non-polar van der Waals interactions that occur w h e n protein
surfaces dehydrate upon formation of the c o m p l e m e n tary complex [30"]. This m e t h o d o l o g y also facilitates
m a p p i n g the interaction domain between two associating macromolecules, a k e y step in the d e v e l o p m e n t
of generalized strategies to both study natural systems
and engineer alternative interactions a m o n g molecules
suitable for use in MEDs.
Bacteriorhodopsin in MED applications
Bacteriorhodopsin (molecular weight 26000 D) functions as a light-driven proton p u m p in the purple m e m brane of the salt-marsh micro-organism Halobacterium
halobium [1",2"]. Bacteriorhodopsin incorporates a retinal chromophore, which is covalently b o u n d as a protonated Schiff base in an all-trans conformation in the
resting state. U p o n absorbing a light photon, the retinal photoisomerizes and subsequently undergoes a
multistep photocycle that spans much of the visible
spectrum. The photocycle can b e stopped at specific
intermediates by low temperature trapping and recycled b y thermalization or application of a second light
pulse of the appropriate wavelength [1",2"]. Photoactivated retinal switching occurs with high quantum
yields at disparate wavelengths, which facilitate state
assignment, and produces b o t h changes in protein refractive index and a photoelectric potential in oriented
assemblies. These properties, together with the excellent stability of the protein w h e n immobilized in polym e r films or gels, form the basis for a variety of prototypes for data storage media, holographic memory,
a n d electro-optic applications [1",2",3,31,32"]. Three-dimensional photochromic memories have incorporated
bacteriorhodopsin that has b e e n oriented b y an external electric field and then immobilized in a solid
polyacrylamide matrix [2"]. This application exploits
the two-photon absorption cross section of bacteriorhodopsin and requires that a small v o l u m e of the
matrix be simultaneously illuminated by intersecting
laser beams for data storage.
Readout is achieved by reillumination, which produces
an electrical signal on the cuvette surface that depends
o n the state of the irradiated volume in the data storage
matrix. In this context, it is interesting to note recent
experiments demonstrating the maintenance of redox
protein properties w h e n immobilized in silicate glasses
'[33"], as well as preservation and control of proteolytic
e n z y m e activity in a photochromic azobenzene copolym e r [34]. Although most readily applied in biosensor
applications, both schemes could potentially be useful in three-dimensional optical m e m o r y applications
that utilize photochromic properties of incorporated
390
Protein engineering
proteins or that couple protein catalytic properties to
the photochromic matrix.
Current w o r k on bacteriorhodopsin with relevance to
molecular electronics involves finding additives, alternative pigments, or site-directed mutants of the protein
that will alter the relative stabilities of the photo-intermediates [35], enhance stabilization of the final M
intermediate at r o o m temperature [36], or alter other
aspects of photocycle-coupled proton translocation
[37,38] that might facilitate readout from information
storage applications. Consequently, investigations of
the Asp96 Asn modification [39], which alters coupling
of retinal photoisomerization to proton translocation,
continue to have substantial practical interest [40]. The
physical changes in the modified protein include an
increase in the lifetime of the intermediate from 10 ms
to 750 ms, together with improved diffraction efficiency
and photochromic sensitivity relative to the native protein. Considerable latitude for property improvement
exists using a combination of site-directed modifications at the c h r o m o p h o r e binding site, introduction
of alternative chromophores, or the engineering of
modified ion-binding sites, all of which can affect intermediate lifetimes and spectral properties [1",2"]. In this
regard, the recent structure determination of bacteriorhodopsin by electron diffraction methods [41"], coupled with rapidly advancing methods of computational
simulation [42"], may provide n e w insights into the engineering of bacteriorhodopsin for MED applications.
Oriented protein immobilization on surfaces
prosthetic groups in sensor or non-linear optical applications depends critically on the ability to control
prosthetic g r o u p orientation precisely relative to the
electrode or optical w a v e g u i d e surface. Site-specific introduction of anchoring points in a protein of k n o w n
three-dimensional structure provides a straightforward
solution to this problem. As an example, recent site-directed modifications of the heine protein cytochrome
b5 carried out at the University of Illinois introduced
cysteine residues at specified positions on the molecular surface. This allowed oriented coupling to a silane
substrate and produced immobilized molecular arrays
demonstrating a high level of heme prosthetic group
orientation as determined b y linear dichroism spectroscopy.
Methods to introduce two-dimensional periodicity on
surface molecular arrays d e p e n d either on engineering both intermolecular interactions [30"] and anchoring sites for surface immobilization, or on schemes that
erect molecular assemblies on a scaffold already possessing periodic order on the few tens of angstroms
scale. Scaffold possibilities include natural two-dimensional lattices [43], DNA lattices [51"], or the very highly
ordered synthetic lattices formed from streptavidin anchored to surfaces through biotinylated phospholipids
[52"]. As streptavidin is a tetrameric protein with four
biotin-binding sites arranged with approximate tetrahedral geometry, two biotin-binding sites per tetramer
remain f r e e in the two dimensional arrays and provide
a regular lattice for attaching additional molecules (Fig.
1).
Molecular electronics
Streptavidin
Bacteriorhodopsin is not unique in its ability to order
spontaneously in two-dimensional films that resemble
its native m e m b r a n e state and, indeed, other m e m brane protein systems have b e e n suggested for use or
have potential in a variety of MED or related biosensor
roles. Recent work includes studies of engineered bacterial transmembrane pores [43] and isolated bacterial
reaction centers immobilized on electrode surfaces [44].
In these cases, surface orientation of the protein complexes results from preexisting interactions between
the protein complexes or their m e m b r a n e environments. However, a n u m b e r of experiments have b e e n
carried out that involve tethering proteins to electrode
surfaces through polymeric 'wires' [45,46] or through
surface electrostatic interactions [47] in order to enhance electron-transfer efficiences. Related studies of
interest examine properties of soluble proteins conjugated to photo- or redox-active organic substituents
that enhance enzyme catalytic function in the absence
of natural cofactors or regeneration systems [48-50].
It is easy to envision hybrid systems that incorporate proteins with conjugated cofactors immobilized
on electrode surfaces. Although the forgoing studies
emphasize electron conduction b e t w e e n device components, the efficiency of m a n y devices incorporating
components
Biotinylated redox protein
Biotinylated DNA [~ giotinylated lipid
~
Lipid-anchored
redox protein
Hypothetical molecular electronics architecture
Fig. 1. Illustration of molecular electronics components and potential architectural features of self-assembling systems restricted to diffusion in two-dimensional lipid films.
Protein engineering for molecular electronics Sligar and Salemme
Strategies for protein patterning and circuit
fabrication
Although it a p p e a r s possible that quite complicated
molecular assemblies could b e erected o n two-dimensional protein lattices using a combination of chemical conjugation, site-directed introduction o f a n c h o r
points, and molecular fusions at the DNA level (rev i e w e d elsewhere in this issue), the materials described
thus far all c o m e u n d e r our first definition o f molecular electronics as bulk materials. Although these substrates could u n d o u b t e d l y b e patterned using lithogr a p h y m e t h o d s similar to t h o s e used in fabricating integrated circuits, optical diffraction w o u l d limit detail
to scales substantially larger than individual molecular
assemblies. Such devices w o u l d not realize the ultimate
in miniaturization of individually assignable devices
inherent in molecular electronics of the s e c o n d definition, w h e r e a s individual molecular assemblies w o u l d
lack the u n i q u e addressability and specific connectivity
b e t w e e n c o m p o n e n t s c o m m o n l y associated with electronic circuits. Nevertheless, it is interesting to consider
h o w it might be possible to construct true molecular
circuits, as well as the dual p r o b l e m at the molecular
level of h o w such devices could be addressed a n d
read. Atomic p r o b e m i c r o s c o p y could potentially b e
u s e d to manipulate and assemble molecules as a means
o f constructing devices, as well as a m e a n s of setting
molecular states or reading o u t information [53"]. The
limitations of manipulating only o n e or a few c o m p o n e n t s at a time, however, w o u l d s e e m ultimately
to defeat the objectives o f creating a molecular device. Alternatively, it m a y b e possible to 'condition'
a preexisting molecular lattice of uniformly interconnected molecules so that it is able to store or process
information as a neural n e t w o r k analog. Information
processing using a distributed a p p r o a c h requires each
m o l e c u l a r - a s s e m b l y to sense the state of its nearest neighbors a n d alter its configuration accordingly.
With phasing to an external clock, this s c h e m e has the
formal structure o f a cellular a u t o m a t o n operating with
individual m a c r o m o l e c u l e s as building blocks. In such
a highly cooperative device, it m a y n o t be necessary
to manipulate individual molecules directly, but utilize
intermolecular c o m m u n i c a t i o n s c o m m o n to m a n y cooperative multimeric proteins to p e r f o r m s o m e analog
c o m p u t i n g function. Fabrication possibilities also exist
using defect tessellation automata [54"], w h e r e very
complicated structures can be generated b y defect
introduction into a periodic lattice subject to a regular site r e p l a c e m e n t protocol. Although presently only
the subject of c o m p u t e r simulation, it seems possible
to e m b o d y the required properties in ligand-capture
a n d displacement schemes using symmetric protein
molecules with multiple b i n d i n g sites. Access and readout could potentially be achieved b y any of the methods outlined above. While these devices are presently
w h o l l y conceptual, and m a y ultimately be limited b y
molecular noise that will necessitate distributed or red u n d a n t processing to ensure reliability, it seems clear
that g e n u i n e a p p r o a c h e s exist to investigate the limits
o f this technology.
Conclusion
Practical application of e n g i n e e r e d proteins in molecular electronics and sensor materials applications are
clearly o n the near horizon. Protein molecules represent the ultimate miniaturization possible in individual
devices w h o s e state can potentially be controlled indep e n d e n t of its neighbors. This property, together with
the e m e r g e n c e of a k n o w l e d g e base allowing protein
properties and interaction specificity to be e n g i n e e r e d
with relative ease, will u n d o u b t e d l y lead to progressively m o r e sophisticated assemblies, w h o s e functional
limitations can only n o w be g u e s s e d at. Multidisciplinary p r o g r a m s such as those in the Soviet U n i o n that
p i o n e e r e d d e v e l o p m e n t o f b a c t e r i o r h o d o p s i n based
devices [1",3], or the Frontier Research P r o g r a m of the
Riken Institute in J a p a n [55"], represent f o c u s e d efforts
to realize the potential of this technology.
References and recommended reading
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review, have been highlighted as:
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Optoelectrical R e s p o n s e s o f B a c t e r i o r h o d o p s i n Films.
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This paper draws an interesting parallel between bacteriorhodopsin
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The redox proteins copper-zinc superoxide dismutase, cytochrome
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A breakthrough paper describing the bacteriorhodopsin three-dimensional structure at the level of detail required for systematic
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A state-of-the-art study of dynamical processes that are key to bacteriorhodopsin function.
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DNA lattices offer extraordinary potential for two- and three-dimensional scaffolding for MEDs, particularly when they additionally
incorporate internal or terminal biotinylation sites. These sites can
interconnect DNA lattices with avidin or streptavidin molecules to
produce addffional symmetric, multivalertt biotin attachment sites.
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DARSTSA, AHLERSM, MELLERPH, KUBALEKEW, BLAKENBURG
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Streptavidin monolayers are demonstrated to have extraordinary surface order which, together with their ability to irreversibly bind virtually anything that can be conjugated to biotin, suggest a central role
for the system in MED fabrication.
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EIGLER DM, SCHWEIZER EK: P o s i t i o n i n g Single A t o m s
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344:524-526.
An initial demonstration of the possibility for patterning surfaces by
manipulation of single atoms,
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A remarkable paper illustrating the temporal evolution of complex
interconnected lattices bearing a striking resemblance to integrated
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SaSABEH: P r o c e e d i n g s o f t h e F r o n t i e r R e s e a r c h F o r u m
o n Bioelectronic Materials. 1991, Nishina Memorial Hall,
RIKEN, Wako-Shi, Japan.
Creative efforts to capitalize on the use of proteins as biomolecular devices has come from extensive work by the Frontier Research
Program at the RIKEN Institute in Japan under the leadership of Dr
Hiroyuki Sasabe. This has included ordered assembly of such photoactive systems as nitrile hydratase, photosynthetic reaction centers
and bacteriorhodopsin, as well as nonqinear optical properties of
polymer protein systems.
SG Sligar, School of Chemical Sciences, University of Illinois at
Urbana-Champaign, 303 Roger Adams Laboratory, 1209 West California Street, Urbana, Illinois, IL 61801, USA.
PR Salemme, Sterling Winthrop Pharmaceutical Research Division,
9 Great Valley Parkway, Malvern, Pennsylvania, I1 61801, PA 19355,
USA.