Protein arrays: Concepts and subjects

Adv.

PROTEIN

ARRAYS:

KUNIAKI

CONCEPTS

Biophys.,

AND

Vol.

34, pp. 3-23

(1997)

SUBJECTS

NAGAYAMA

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo 153 and National Institute for Physiological Sciences, Okazaki, Aichi 444, Japan

Can biological materials be adapted to the materials used in our modern technologies? So far as tissues and organs are concerned, they do not seem to satisfy the requirements of the materials for various engineering utilizations. Biological materials have long been considered inadequate for engineering purposes, since they are prone to deterioration, are uncontrollable and expensive. But these difficulties are now gradually being overcome, through biotechnological achievements. Going down along the biological hierarchy to the level below cells, we can view a material world manageable with current technologies. The recent advent of protein engineering and supramolecular chemistry is providing a basic technique for a new type of technology where making and assembling proteins into an appropriate architecture is the central concern. In the governmental, ERATO Protein Array Project (199019954, we have been attempted to establish a technology for fabricating two-dimensional (2D) protein arrays with desired molecular orientation in the form of crystalline films. The basic strategy we took was to explore an interface able to connect two completely dissimilar worlds in its scale of basic components, protein molecules and macroscopic devices. To bridge this scale gap, we sought an interface in a stable thin liquid film on a substrate, flat and well defined on the nanometer scale.

4

K.

NAGAYAMA

The 2D space realized in the thin liquid film is a new working place for protein molecules which moved from their original living space, cell suspension. We found that protein molecules self assemble into the form of 2D crystals in this space, maintaining their original functions and structures and exhibiting a novel character as engineering materials.

I.

PROTEIN

ARRAY

AND

ITS

RELATIVES

The fate of proteins, originally written in DNA as genetic information, is expressed in the form of their interactions with protein counterparts and the surrounding environment, which finally leads to the self-assembling of constituent proteins to particular biological architectures. Though life is, for the time being, only one unique representation of many possible combinations and arrangements of protein molecules, there could be another form artificially constructed with equal biological complexity and, at the same time, useful in our technologies (la). Proteins, preserving their original functions, are the starting raw materials in our engineering; and complex assemblies designed for a special device, such as supramolecules and microcrystals with intelligent functions, are the end products. Our way of constructing large architectures with protein molecules is thus following the spirit of Nature’s creation as shown in Fig. 1 by putting special emphasis on 2D arrays. A protein array is generally defined as various forms of assembly:

Biosystem:

Proteins

Genetic I’------------‘-

Fig.

1.

Construction

+

Supramoiecules

+

Organelle

+

Cells

Control Seecific Interactions EkwironmentaVProcessing

of large

architectures

with

proteins

and Control

in biosystems

and

pro-

tein array systems. In either system, the genetic information is primarily transferred to the structure of protein molecules, particularly to their surface structures. Surfaces, then, characterize interprotein interactions and determine the fate of the protein assembly to supramolecules. Further organization depends on the control of the

environment

(adapted

from

Nagayama

(lb)).

PROTEIN

ARRAYS:

CONCEPTS

AND

SUBJECTS

5

globular, filamentous and layering of protein molecules. But here we restrict our interest to the 2D form of protein arrays using an analogy to the 2D patterns imprinted on the surface of a silicon wafer, reminiscent of LSI (large-scale integrated circuits). Nature’s way of manufacturing has to be remodeled to fit our goal by finding appropriate interfaces. The key we took in the ERATO project to open the door was to develop an interdisciplinary field able to connect molecular biology which has the know-how to play with proteins and surface sciences with skills to deal with materials. One special field was thought to satisfy our demand: electron microscope crystallography, where various methods of preparing 2D crystals of proteins have been developed over the last two decades for treatment of samples. Tiny crystals microscopically observable which develop on surfaces, mainly on water, do not have obvious industrial applications but the preparation methods of protein 2D crystals themselves can be incorporated into the fabrication of protein 2D arrays in general. To give a rational basis to our expectations, held prior to the start of the project on protein 2D arrays as an engineering entity, let me survey neighboring fields which have philosophies similar to ours in the way of utilizing small molecules and particles. Among the many which are related, I have selected the following fields: self-assembled monolayers in supramolecular chemistry, protein adsorption in surface chemistry, organic-inorganic complex in biomineralization, and nanostructural devices in nanochemistry and nanotechnology. Most of them have their own motivation for practical applications, contrary to our keen interest about the methodology involved in the fabrication of protein arrays themselves. 1. Self-ussembled Monolayer in Supramolecular Chemistry Initiated by the Nobel lecture by J. -M. Lehn (Z), the term supramolecular chemistry has rapidly disseminated among people who intend to develop more complex materials beyond conventional host-guest chemistry based on the lock and key principle. The term is flexible and has been utilized to infer molecular assemblies harnessing various kinds of non-bonded interactions (3, 4) or complex molecular forms such as inclusion compounds (5), macrocycles (6), monomolecular films and Langmuir-Blodgett films (7), and complex polymers (8). The common feature there observed is the establishment of suitable interfaces for recognition and assembly of molecules into an organized array with a given function (photoactive, electroactive, ionoactive, thermoactive, or chemoactive) (9). Among those, a self-assembled

6

K.

NAGAYAMA

monolayer of molecules or particles prepared on an appropriate surface has an internal connection to our protein array. Functionalized organic surfaces can be used to control the deposition of protein molecules from aqueous solutions. Self-assembled organic monolayer-s at the water-air interface or the solid substrate surface can homogeneously adsorb protein molecules, when the monolayer surface is functionalized with ligands which possessspecific interactions with the concerned proteins. Various affinity ligands, haptens (IO, II), metal chelating lipids (12-13 and biotinylated lipid (15, 16) have been introduced for the lipid monolayer. Lipid bilayers have also been used for the inclusion of proteins (I 7). A monolayer film with poly(benzyl-L-histidine) developed at the water surface is also utilized to introduce the electrostatic interaction for the adsorption (18). Due to flatness and inertness of the crystalline gold, its surface is widely employed for a reaction place for self-assembled monolayers. A biotinylated organic thiol monolayer bound to the gold surface was prepared on which to form a streptavidin array, and this was then utilized as the substrate secondarily adsorbing another species of proteins, for example, biotinylated immunoglobulin (19). The functionalized gold surface is now a standard device to study biological reactions with the surface plasmon resonance apparatus (19, 20). Lipid monolayers developed on glass were used in the pioneering work to prepare antibody sensors (21). 2. Protein Adsorption in Surface Chemistry Time-dependent deterioration (aging), if it occurs, is a serious problem in the application of protein arrays, as the original protein function can be severely damaged by this change. The structural and functional aspects of the adsorbed protein molecules with a gross and averaged resolution have been investigated by surface chemists as a phenomenon of protein adsorption. The degree of conformational change of adsorbed protein molecules varies from one protein to another. For example, a stable protein such as ribonuclease is known to be highly resistant to structural change upon adsorption compared with more hydrophobic and less stable proteins like serum albumin (22-24). There are few techniques which directly approach the problem of structural changes of adsorbed proteins. Using of infrared spectroscopy, time-dependent conformational changes of serum albumin adsorbed to germanium or polyurethane have been reported (25), while fibrinogen or prothrombin adsorbed onto a silica surface has resulted in no change in conformation (26). Surface force apparatus

PROTEIN

ARRAYS:

CONCEPTS

AND

SUBJECTS

7

studies have also shown no structural change for lysozyme on mica (27) but a great change for mucin on mica (28). The complexity in the structure and the charge distribution of proteins causes them to adsorb on various surfaces with a variety of orientation and organization. The orientational order is crucial in evaluating the varying degrees of conformational changes of adsorbed proteins reported to date. Arnebrant and Nylander suggested molecules adsorbed to hydrophobic or hydrophilic surfaces with different orientations of insulin (29). Advent of novel instrumentation, scanning tunneling microscopy (STM), or atomic force microscopy (AFM) seems appropriate to study this problem though it is still in the embryonic stage. Lysozyme monolayer array adsorbed from a solution onto a graphite surface was reported by Haggerty and Lenhoff using STM (30). They claimed a highly ordered molecular arrangement of oblique lattice. Individual images of protein molecules randomly adsorbed were also reported but their resolution seems slightly lower than required to clarify the molecular orientation (31-34). For biosensory use, the molecular orientation of immunoglobulin adsorbed to a solid support is of primary interest. Using a combination of adsorption isotherm and electron microscopy, the side-on or end-on adsorption was critically distinguished (35). The lateral diffusion (mobility) of adsorbed proteins at the surface is also important for the rearrangement and arraying of protein molecules after adsorption (36, 37).

3.

Organic-inorganic

Complex in Biomineralization

There are numerous fields inspired by the biological way of material processing by which units are self-assembled, self-constructed, and finally self-organized into highly ordered large architectures. One biomimetic field is simply borrowing the idea from biominerals such as microskeletons of diatoms, mesoporous aragonites, and bones in general. Biomineralization utilizes biomaterials (proteins, lipids, and polysaccharides) as a template for complex construction, in which minerals are guided to assemble or crystallize in a suitable form (38, 39). Parallel to the scheme shown in Fig. 1, the basic construction processes in biomineralization are supramolecularly preorganized, interfacially molecular-recognized (ternplating), and cellularly processed (38). From the viewpoint of protein array fabrication, supramolecular complexes of proteins and inorganic materials are of greater significance. The fixed size of the cavity observed inside a protein, ferritin, was utilized to synthesize nanoscale inorganic materials, manganese oxide or iron sulfide (40, 41). Ferritin arrays have always been one of the

8

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NAGAYAMA

target systems in our protein array project as described later. There had been no reports of ceramic films ternplating the protein 2D arrays, but ceramic films of silica, calcium carbonate, calcium phosphate, iron oxide, and alumina developed on surfaces functionalized with alkyl chains have recently been reported (42, 43). Inorganic particle arrays adsorbed to the lipid monolayer were also announced as described in the next section. 4. Nanostructures in Nano-chemistry and Technology As reviewed (44), three and a half decades have elapsed before the theoretical prediction by Feynmann to create a small world has finally come true owing to the invention of the atomic and molecular scanning tunneling microscopes (45), where nanostructures can be made by catching, moving, and aligning atoms with microscopic tweezers. This top-down approach, where macroscale tools are utilized to create nanoscale structures, however, has been criticized because of its enormous cost in assembling billions of atoms into a final form. The top-down approach must be compared with the conventional bottom-up approach, where nanoscale components are utilized to create structures. Chemistry is actually a field utilizing atoms and molecules and changing their forms by self-assembly, but Nature’s law inevitably sets a limitation on the product, the way of assembling atoms. For example we cannot make CHs with carbon and hydrogen atoms in place of CH4. On a scale higher than atoms or molecules, nanostructures with arbitrary forms can be made of matters by combining various kinds of tools in science and technology, as reviewed by Whitesides et al. (46). Nanostructures can be manufactured as follows: 1) produce material parts on the nano-scale; 2) process these parts into components at the nano-scale; 3) order molecular components into a structure and interconnect them; and 4) an interface system connecting with macroenvironment. Protein arrays can skip the first step by borrowing material from Nature. Steps 2) and 3) must be developed by us as shown in Fig. 1. The various self-assembly methods illustrated here are contributing to step 3). Ultrafine particles of which structures are defined on a nanometer scale, for example, fullerenes (47) or nanotubes (48), are materials developed in step 2). The technology of fine integrated circuits and related chemical approaches like microlithography (49) belong to steps 3) and 4). But the key issue to be targeted is always in the middle step 3), as emphasized in this article. Bioelectronics motivated by the molecular electronics (50) and

PROTEIN

ARRAYS:

CONCEPTS

AND

SUBJECTS

encouraged by a specific functional protein, bacteriorhodopsin, be the first example in a nanotechnological field (51).

II.

TWO-DIMENSIONAL

COLLOIDAL

9

must

ARRAYS

Uniform particles, whether atomic, molecular, or colloidal, can organize to form ordered solids when attractive and repulsive interparticle forces are properly balanced. Three-dimensional colloidal crystals have been attracting scientists for about 50 years (52-60) and still remain an active area highlighted by the fabrication of quantum dot superlattices (61). Here I restrict my survey to the 2D colloidal arrays, since they have many common features useful in the fabrication of protein arrays. From the days of Faraday (64, gold colloid has been one of the most popular materials for chemists. It is still used in the study of formation of ordered lattices (63, 64) but due to the polydisperse size distribution, its position has gradually been replaced by polystyrene (PS) latex particles (size: O.l-1Opm). The size uniformity of PS particles is extremely good and nowadays those commercially available are within an accuracy of three digits. Actually, we can trace back the study of latex film of ordered colloids to the success of the synthesis of highly monodisperse PS latexes about 50 years ago (53, 65, 66). After the removal of water from the PS suspension, small domains (about 10 pm) of 2D crystalline arrays were unexpectedly found together with many thin deposits (67-69). The preparation of well defined 2D arrays under a controlled condition was initiated by two groups, Goodwill et al. (70) and Pieranski (71), when they introduced PS particles (2-4 pm) to the air-water interface whereby they were coagulated. The particles adsorbed to the interface are mobile leading to the assembly of 2D arrays resulting from the balance of attractive and repulsive forces. Both amorphous and crystalline 2D arrays of PS particles were found and the particle density at the interface was proposed to be important in controlling the two forms (71). The assembling process of colloidal arrays has been investigated microscopically (72, 73), but details of the underlying mechanism await further studies (74). Small particles confined in a thin dispersion layer sandwiched by two pieces of glass were reported to be ordered in two-dimension (7476). They showed hexagonal or tetragonal thin colloidal crystals with forms of mono- to multi-layers depending on the thickness of the confining space. But arrays of hexagonally ordered PS particle monolayers have recently been obtained in our project in connection with the fabrication of protein arrays (77). Interestingly, the assembling

10

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NAGAYAMA

method we employed relies on a mechanism which works only in thin liquid films freely suspended in the air or developed on a supporting surface facing air (see Fig. 2B). After intensive study, two unique processes were found to be the cause of the particle self-assembly: particles, mobile in thin liquid films which have a thickness comparable to the particles, self-assemble and pack closely into crystalline arrays. The first step is driven by the convective flow of water, which compensates for the evaporation of the water at the growing array boundary and carries the input colloidal particles from the meniscus of thick liquid suspension to the array boundary (called convective assembly) (7843). Then, the particles are compressed to the array boundary in a closely packed state (usually hexagonal) by the combination of the flow impact (84,85) and the long range attractive force induced by surface tension of suspension solvent (86-95), which is analogous to the elevation of water in the glass capillary (called lateral capillary force). Continuous growth of a monolayer of particle arrays with crystalline order was obtained with a simple apparatus, where a wettable glass is vertically dipped into and withdrawn from a suspension of input particles (84, 85). This technology was applied to reproduce Nature’s art, the brilliant coloring of the Morpho-butterfly, by mimicking its surface texture (diffraction grating) with mono-particle crystalline films (96).

LIQUID (one

SURFACE surface)

\

LIQUID FILM (two surlaoes) B

A

Gas

Gas

I Fig.

Two-dimension!

Two-dimension? 2.

(A)

An

illustration

of the

growth

of protein

arrays

at the

water

surface

driven by the diffusion type of self-assembly and molecular recognition processes, which are physicochemical events inherent in the dispersion phase. (B) An illustration of a rapid growth of protein arrays in thin liquid film driven by the active selfassembling process of solute condensation such as evaporation, solvent suction, or lateral capillary al. (102)).

force

acting

among

protein

molecules

(adapted

from

Nagayama

et

PROTEIN

ARRAYS:

CONCEPTS

AND

11

SUBJECTS

The 2D condensation of colloids and the subsequent closely packed array formation can also be driven by the adsorption process. Solid surfaces immersed in the colloid dispersion were used for the layering deposition of the charged colloidal particles with electrochemical adsorption (97, 98). Two methods for particles to be twodimensionally developed on solid surfaces were reported. Spin coating widely used in industry to make polymer films was also employed to obtain monolayered latex films (99-101). Contrary to the amorphous 2D arrays observed for the spin coated colloidal films, the completely regulated ordered pattern of dielectric spheres like PS (- 1 pm) was obtained by the optical binding method (103, 104), which trapped particles into the laser-induced speckle pattern that acts as a kind of optical tweezers. The lipid monolayer developed at the air-water interface has also been utilized as the substratum for the particle adsorption (see Fig. 2A). This system widely utilized to prepare Langmuir-Blodgett films was intensively studied by Fendler and his coworkers in their work on fabrication of nanostructured materials (105). More than 30 papers have been published by his group during the last few years which dealt

TABLE Fabrication

I of Two-dimensional

Method

-

Evaporation

Colloid Supporting

Arrays surface

Material

Reference

67-69 97

Glass Polymer

Ps”

condensation Electrophoretic deposition

IT0 glass Silicon wafer

PS Gold

None”

PS

Self-assembly air-water

at interface

Lipid

PS

monolayer

colloid

98 63, 64

Semiconductors

70-73 105-113

Colloids, Colloids Self-assembly thin

liquid

in films

Spin-coating Optical a Polystyrene two pieces

binding

metal

oxides

None‘

PS

Glassd Fluorinated-oild

PS PS

Mercuryd Silicon waferd Soap film’

PS PS PS

74-76 77-81, 82 81 151 152

Silicon

PS

99-101

wafer

Glass

latex sphere. b Direct of glasses. d One surface

PS spreading. of liquid

’ Thin colloidal dispersion film facing the air. ’ Two

83-85,

96

103,104 sandwiched surfaces facing

between the air.

12

K. NAGAYAMA

with nanocrystalline particulate films made of semiconductors like CdS (l&5-208), PbS (109, 110) and metal oxides like TiOz (1 I I), Fez03 (112), and metals (113). This method has great versatility in its application to various colloidal particles but the quality of ordering of 2D arrays is not as excellent as we might wish compared with the liquid film method we developed. This seems to be due to the lack of a strong packing force at the water surface in the lipid monolayer method. The strong packing force is only found in the thin liquid film, as extensively discussed in the theory and experiments on lateral capillary forces (89, 91). Table I summarizes the fabrication of 2D colloidal arrays.

III.

TWO-DIMENSIONAL

PROTEIN

ARRAYS

Protein molecules are characterized by irregular but unique three-dimensional structures. This molecular nature adds one more dimension to the resultant assembly of protein arrays compared with colloidal arrays, namely the orientational order, though there are many fabrication parallels between them such as the ways of 2D condensation. The biggest concern in protein 2D arrays is thus the molecular orientation of individual proteins. In our Protein Array Project, the major utility of the crystalline array form lies in the control of its molecular orientation. Not only the nature-providing crystal form but also the artificially designed form of crystals should be studied in array engineering. Protein is a material which particularly accepts such a design owing to the large area at the surface and possible manipulation of interprotein interactions suitably modified through mutagenesis. Here, our five years of effort in developing protein arrays in this sense are summarized in three aspects of fabrication, characterization, and application.

1. A)

Fabrication 20 arrays in general

In biological systems we can find various examples of protein arrays, 2D crystals of proteins embedded into or onto the bilayer membrane (114). One example is the 2D crystals made of only proteins, the surface layer, observed on the bacterial cell wall (115-118), which serves as a life protector to various chemicals in cooperation with the membrane skeleton. Another is the ordered protein-lipid complex observed on bacterial plasma membrane, called purple membrane, which includes disks of hexagonal crystals of bacteriorhodopsin, whose structure has long been studied by electron microscope crystallography (119-122).

PROTEIN

ARRAYS:

TABLE

CONCEPTS

.4ND

SUBJECTS

Protein

Arrays

13

II

Fabrication -~

of Two-dimensional Supporting

Method Self-assembly

in

surface

None”

dispersion

Material

Reference” ___-___

Bacteriorhodopsin Membrane proteins

119 122 115 122

Bacterial Wafer-soluble Self-assembly liquid-solid

at interface

Lipid Mica Carbon Graphite

Self-assembly air-water

at interface

Self-assembly liquid-membrane

Lipid

monolayer/gold film

Liposome Supported

layers proteins

Streptavidin Viruses, catalase Alcohol oxidase,

ribosome

Lysozyme monolayer

Noneb at

surface

membrane

19 132-134 118, 135-137 138

Immunoglobulin

122, 123 139,140

Annexin V, ferritin Immunoglobulin

141, 142 143

Ferritin,

streptavidin

interface Self-assembly thin

liquid

in film

Mercury Glucose Silicon

solution wafer

None’ ’ Za situ

crystallization.

b Direct

Ferritin,

ATPase,

chaperonin

Ferritin, Ferritin

c-reactive

protein

151

Bacteriorhodopsin spreading.

’ Free

soap

144-148 131, 149, 150

film.

vesicle d The

first

152 or most

commonly

cited.

Various artificial supporting surfaces, replacing the biological membrane, such as air/water, air/graphite, air/metal, water/graphite, water/organic liquid, and air/lipid/water have been reported (122). Historically, the Langmuir film (lipid monolayer film developed on the air/water interface, see Fig. 2A) was the one first applied to a protein, ferritin (123), to obtain a crystalline film. To make the protein-substrate interaction more specific, ligand-anchored or charged lipids were also introduced and 2D crystals of proteins such as antibodies were prepared with improved quality (124-131). Due to the limitation of coherent length of the templating lipid monolayer (132), there is a size limit in 2D crystals obtained by adsorption to the monolayer surface. Supporting surfaces other than lipid monolayers have also been investigated. Those are mica (133, 134, carbon film (218, 135-137), graphite (30, 138), liposomes (141, 142), supported membrane (143), mercury (144-248), denatured protein films (149, ISO), and silicon wafers (151). Key elements conventionally used in the fabrication of 2D arrays are summarized in Table II. The initiation of 2D protein assemblies starts from the 2D condensation of the molecules on an’appropriate surface or interface. As

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mentioned above, adsorption condensation to the lipid monolayer at the air/water interface (Fig. 2A) or to the lipidated surface of solids has been employed by various groups, Both techniques originated in and grew from a traditional science, colloid and surface chemistry, backed up by the material assistance of organic chemistry. However, we used, another remedy to enhance the 2D condensation using an analogy with the successful fabrication of colloidal arrays described in the previous section (77), namely the 2D confinement of protein molecules in the thin liquid film (Fig. 2B). The most striking feature of our method compared with others such as the lipid monolayer film method (Fig. 2A) is the presence of two interfaces confining particles and the suspension liquid in between them (see Fig. 2B). A free soap film was also employed to obtain the stable liquid film. Combination of rapid freezing and trapping molecules in soap film (vitrified film method) brought about successful (252) 2D crystallization of relatively large supramolecules, a polyhedral cluster of bacteriorhodopsin (153). A thin liquid film (154) could be ideal for particle assemblage, since the movement of particles is twodimensionally confined and hence more easily handled than that in bulk solution or suspension where the Brownian motion is overwhelming. Thin liquid film systems developed during the project are summarized in the bottom lines of Table I I _ B)

Supramolecules

Large-sized protein molecules can be more tractable by the fabrication method developed here with liquid films, as thicker liquid films can be stably prepared without rapture. Several techniques for the conversion of protein molecules to large supramolecules have been developed as described below. Carbohydrate-glued protein supramolecules: A new face of supramolecular assembly using carbohydrate gluing (lectin-polysaccharide cognitive binding) was found in the architecture of a huge protein comprised of about 200 protein subunits, giant hemoglobin from the marine-worm annelid (155, 156). This newly found supramolecular architecture, was applied to the protein, ferritin, to obtain its multimeric supramolecules. Proteoliposome-like protein supramolecules: Liposomes were employed as templates to assemble many ferritin molecules through lockand-key interactions: avidin-biotin and lectin-polysaccharide (142). Virus-like protein vesicles were organized by tailoring the specific (lock-and-key) an d non-specific (electrostatic) interactions. Genetically and chemically fused protein supramolecules: Either co-

PROTEIN

ARRAYS:

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15

valently bonded dimer or trimer of ribonuclease H from Escherichia coli was synthesized by the gene engineering technique of genetic fusion (257). A covalent trimer of ribonuclease H was also produced by connecting three mutant monomers, which have extra cysteine on their surface, with newly synthesized trifunctionals (chemical fusion).

C)

20 arrays in thin liquidfilms

Since the hexagonal lattice is a natural form for the close packing of ball-shaped particles, proteins with symmetrical shape such as threefold or six-fold symmetry can naturally assemble in this form. This was actually observed for various protein samples tested: ferritin (145, 149, 158), H+-ATPase (144, I&3), c-reactive protein (131), chaperonin (146), LP ring of flagella motor (147), proteasome and streptavidin. Generally speaking, better quality in the crystal order is obtained for larger proteins with higher symmetry. Hexagonal, tetragonal, and oblique lattices of ferritin 2D crystals were prepared in thin liquid film developed on glucose solution with use of interprotein interactions modified with the mutagenesis of surface amino acids (158, 159). On the mercury surface due to the strong capillary force only the hexagonal lattice was observed, while hexagonal and tetragonal lattices were obtained on the surface of glucose solution (150). The mutant ferritin, mutagenated at the cadmium ion chelating site by replacing glutamine and aspartate with a neutral amino acid, serine, resulted in an oblique lattice (258). Depending on whether the lattice form was hexagonal, tetragonal, or oblique we obtained three different orientations of ferritin molecules on the supporting substrates (158). A secondary film comprised of the secondary minimum of the electric double layer near the silicon surface was also utilized (151) in the preparation of protein 2D arrays with crystalline order directly on solid substrates. This simple means of fabrication is expected to be employed in various fields of material engineering because of its straightforward way to prepare particulate films with proteins or colloids on solids without any sample transfer (coined molecular coating).

2. A)

Characterization Simulation

The contribution of electrostatic interactions to the stability of crystals was rationalized by a computational simulation performed for reported 3D crystals of three protein species, insulin, basic pancreatic trypsin, and ferritin (160,161). A poker-chip model to study physical aspects of molecular assembly and molecular interactions was developed (162-

16

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165), and phase separation by size of latex particles and ferritin molecules was experimentally found and numerically reproduced (165). B)

Experiment

Important surface structures of protein molecules responsible for interprotein interactions were investigated using nuclear magnetic resonance (157, 166, 167). Crystal forms, the degree of crystallization, and the molecular orientation in 2D protein crystals have been characterized by electron microscopic techniques. Nanometer-scale resolution is required to determine the molecular orientation in protein arrays. Electron microscope crystallography is unique in satisfying this requirement (158, 168). The transmission electron microscope has, however, one drawback of the sample transfer from the primary substrate to a pertinent electron microscope specimen grid, so that direct observation of protein array on the substrate can be carried out with a scanning electron microscope or an atomic force microscope (169-I 71). The color ellipsoscope was developed to monitor the growth in real time of very thin protein array film on liquid or solid substrate surfaces (172). Various video microscope techniques were also developed to monitor the particle array (77-85) or the protein array formation (I 72). 3. A)

Application Protein function

One of the forces driving the development of the engineering of protein arrays is our expectation for their future applications in micro-optics, micro-electronics, and biotechnology. Prompted by the pioneering work of McConnell et al. (243), various lipid and polymer membrane systems for protein immobilization have been developed. This technique is now widely utilized in biosensors, as recently reviewed by Sackmann (I 73). Among them the most successful is the design of receptor surfaces for electro-optical devices (174). Bacteriorhodopsin, due to its photochromism (I 75) and photovaltanic (176) effects, is now viewed as the first engineering protein material. Its arrays will be utilized for photosensors (177) and photomemories (178). Solid surfaces functionalized by thiols or dextrans are also designed to accept the 2D protein arrays. The popular biosensor system that exploits the high sensitivity of surface plasmon resonance is a good example of how adsorped protein molecules can be efficiently used to detect specific biological reactions (I 79-182). Here the basic study of functionaliza-

PROTEIN

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17

tion of the gold surface and the protein adsorption to the coupling reagents have finally found an efficient application. There have yet been very few applications, where the strict crystalline forms in protein arrays play a central role. To date two applications are suggested. One is the use of uniform size of holes in 2D crystals of bacterial S-layers developed on a permeable solid substrate as ultra-membarane filters (183); the other is the use of ferritin arrays as the component of an ultimate memory device (184). The success of both applications depends highly on the fabrication of defect-free 2D crystalline arrays with large areas, which has not yet been done. B) Protein stability As mentioned earlier, the first requisite for a protein array to be active as an engineering material in industry is its robustness. Actually, enzymes became very popular in fermentation technology only after engineers in the field took notice of their being tough and usable under very abnormal conditions such as on a polymer surface (185) or in an organic medium (186). Aspartame, a sweetener replacement for sugar in the diet, for example, can be synthesized using reverse hydrolysis of a peptidase extracted from a microorganism in a binary mixture of water and organic solvents (187). The central issue in enzyme engineering is the preservation of biocatalytic functions in extreme environments. Fortunately, enzymes can preserve their functions even under such non-physiological conditions as in organic solvents (186) or in an immobilized state (188), when they are properly treated. Anhydrous environments or multi-point attachment to immobilizing supports are thought to make enzymes more rigid and stable, thus enabling them to resist any conformational change induced by heat or organic solvent. Acquired protein stability in anhydrous conditions can be partly explained by the thermodynamics of protein unfolding. The difference of free energies between the folded and unfolded states of proteins are much enhanced in favor of the folded state in a completely anhydrous condition, a vacuum, rather than in an aqueous condition, as theoretically proven by a novel thermodynamic approach by Ohbatake and Ooi (189). We estimate that putative denaturation temperature in the vacuum state is as high as 500 K for various popular proteins such as lysozyme and myoglobin. In this context the recent advent of ultrastabilization of protein molecules in a thin film form in the dry state seems very promising. Shen et al. (190) reported that bacteriorhodopsin in purple membrane deposited on a silicon wafer retained its original conformation up to

18

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NAGAYAMA

140°C in the dry state monitored by small angle X-ray scattering. Nicolini et al. reported that the photosynthetic reaction center can preserve its function and structure up to 200°C in the dry state, when the protein film is prepared with the Langmuir-Blodgett film technique, but this is not the case when prepared by the simple treatment of cast film (291, 192). Stimulated by these attractive findings, we tested the stability of protein structure and preservation of protein function with 2D arrays of ferritin molecules in extreme environments. We reproduced the same experimental results that ferritin arrays when completely dried can preserve the ferritin function of iron uptake over 150°C. Ferritin is ideal to test the enhancement of protein stability in the array form, since the peptide part and the incorporated iron have been separately monitored by high resolution electron microscopy. The real emergence and synergism acquired by the crystalline protein array seems to be the ultrastabilization of protein structure and function, a prosperous gift from Nature to protein arrays.

SUMMARY

To adapt proteins, the materials in life, for use as materials in science and technology, we focused not only on the biological aspects (functional aspects) but also on the material aspects as matter (structural and physical aspects). Engineering with protein arrays will develop under such consideration and advance toward stable devices made of protein molecules. The protein arrays with 2D crystalline order provide a primary model of macroscopic protein-based devices. The combination of protein engineering, the leading edge of life science, and array engineering, the leading edge of materials science, will provide clues to the controlled integration of protein molecules to a form of functional supramolecules on proper surfaces.

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