Engineering Proteins for Electrooptical Biomaterials - Advances in

19 Engineering Proteins for Electrooptical Biomaterials Patrick S. Stayton, Jill M. Olinger, Susan T. Wollman, Paul W. Bohn, and Stephen G. Sligar

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Downloaded by UNIV OF LEEDS on June 18, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0240.ch019

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The Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, 405 North Mathews, Urbana, IL 61801

Protein engineering techniques have been used to manipulate the optical and electron transfer (ET) properties of heme proteins in ways that apply to the construction of biomolecular electronic devices. Two important aspects have been directly addressed. First, genetic engineering techniques have been used in conjunction with silane-based self-assembly systems to construct close-packed protein monolayers on optical substrates. The resulting structures have been characterized with immunological and surface-enhanced resonance Raman spectroscopy techniques. Recombinant DNA techniques have also been used to study the role of electrostatic forces in proteinprotein interactions and may be used to gain control over the assembly of multicomponent protein thin films. In addition to these assembly considerations, protein engineering techniques can be used for a second purpose: the manipulation of optical and ET properties of heme proteins. Control over the heme absorption transition energies, redox potentials, and redox center composition can be realized through a combination of genetic engineering and protein chemistry techniques. These experiments are aimed at producing multifunctional protein components with designed assembly and functional properties.

T H E USE OF BIOLOGICAL MACROMOLECULES AS BUILDING BLOCKS in the construction of molecular devices has recently generated much enthusiasm. The exquisite selectivity in the recognition of small molecules has made proteins a natural choice for sensor design. Additionally, many protein systems are electrooptically active, and even express degrees * Corresponding authors. 0065-2393/94/0240-0475$08.00/0 © 1994 American Chemical Society

Birge; Molecular and Biomolecular Electronics Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

MOLECULAR AND BIOMOLECULAR ELECTRONICS


476

of state-dependent switching (termed allostery by the protein chemist). Although several systems such as bacteriorhodopsin and the photosynthetic reaction center are receiving close attention as candidates for molecular devices (I), the proteins that nature has provided do not often have the exact physical and chemical properties required for use as molecular building blocks. For this reason, recombinant D N A technology is a useful collaborative technique, providing the essential synthetic control over protein structure-function relationships. Because the synthetic goals for protein-based materials naturally parallel many of the ultimate aims in nonbiological molecular device construction, we endeavor here to review concomitantly both the general features that make heme-containing proteins particularly interesting as potential electronic device components and to point out how protein engineering and chemistry techniques provide the means to manipulate and optimize key features in biomolecular assemblies. The optical and electron conducting properties associated with heme proteins make them attractive candidates for biomolecular electronic device fabrication. These inherent optical and electrical properties can be manipulated through the site-specific alteration of protein structure by using recombinant D N A techniques. Intertwined with the functional aspects, and indeed a strong determinant of certain properties, is the element of macromolecular assembly structure. The generation of highly ordered structures requires an understanding of the forces and potentials that control the specificity of protein-protein and protein-surface interactions and that could be manipulated to control in-plane ordering and poling. As a first step in the development of ordered protein assemblies, a genetically engineered heme protein system has been used to yield oriented protein arrays through judiciously positioned and unique reactive surface sites. Control over the molecular orientation of protein components in thin film assemblies should lead to a myriad of applications in the biomaterial field. A central player in our attempts to develop an understanding of the physical, chemical, and biological principles governing the assembly of supramolecular protein assemblies has been cytochrome b . This maeromolecule is a bis-imidazole-ligated heme protein, for which high-resolution crystal and NMR structures of the water soluble domain are available (2, 3) (Figure 1). A bacterial over-expression system, based on an easily manipulated synthetic gene construction, has been developed (4). This system permits the production of large quantities of recombinant protein in both the membrane-bound or water-soluble form (4). Cytochrome b exhibits good overall stability and has a fairly symmetric globular structure that should lead to efficient packing at planar surfaces. In functional terms, cytochrome b is also quite versatile, forming stable and active electron transfer (ET) complexes with a variety of proteins 5

5

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Proteins for Electrooptical

Biomaterials

477

T8

Figure 1. Schematic 3D cytochrome b structure. The positions of cysteines introduced through site-directed mutagenesis techniques and the conserved carboxylate binding contacts are noted. 5

including cytochrome c, myoglobin, hemoglobin, and cytochrome P450 (5-8). These properties make cytochrome b a particularly attractive candidate for biomaterial studies. 5

Assembly of Protein Thin Films

Single-Component Monolayers. We are interested in developing the means to control the structure of organized assemblies of biological macromolecules in both two and three dimensions. Figure 2 presents a schematic outline for controlling two-dimensional (2D) crystallization events. The first step involves the tethering of a maeromolecule to a surface in a nonrandom orientation. The scheme for accomplishing the orientation of a biological maeromoleeule involves the introduction of a unique amino acid reactive side-chain at a specific site on the water-accessible surface of the protein (9). In particular, because cytochrome b lacks any sulfur-containing amino acids, site-directed mutagenesis techniques were used to produce four mutants in which a single highly reactive and specific thiol functional group was introduced 5



478

MOLECULAR A N D BIOMOLECULAR ELECTRONICS

Figure 2. Macromolecular assembly of heterogeneous protein thin films. Shading patterns represent complementary interaction surfaces.

at four independent points on the protein surface. These cysteine residues replaced threonine 8 (mutant T8C), threonine 65 (T65C), threonine 73 (T73C), and serine 85 (S85C) (Figure 1). In addition, two double mutants T65C,T8C and D66C,E38C, which present binary thiol reactivity, have been made. Substitution of cysteine residues, with their nucleophilic thiolate functionality, allows for anchoring to a solid substrate derivatized with a complementary electrophilic reactant, typically a silyl ether terminated with an alkyl halide (for the formation of a thioether surface linkage). These reaction schemes are outlined in Figure 3. l,Br,SH

+

$$

0-SI-0-S1-0-S1-0

I

0

0

° I

pH 7.5

PYRIDINE HEMOCHROME ASSAY

85% CLOSE-PACKED MONOLAYER 2

(900 Â /PROTEIN MOLECULE)

Figure 3. Protein derivatization and surface coverage. Cytochrome b is reacted with alkylhalide- or alkylthiol-derivatized glass substrates in 50 mM KPi, pH 7.5, and dried under nitrogen. The degree of surface coverage is then determined by measuring the heme content of the protein assemblies through the use of a pyridine hemochrome assay. 5


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Glass substrates were prepared by treatment with a silane coupling agent, either 3-iodo- or 3-bromoproyltrimethoxy silane. A thiopropyl analogue of these silanes was used to estimate surface coverage through a spectrophotometric titration using dithionitrobenzoate (DTNB). This method yields a quantitation of the surface thiols, for which a coverage area of ca. 65 Â /moleeule was measured. Molecular models show that the size of an individual alkylsilane should be dominated by the crosssectional area of the alkyl chain, which in the all-trans configuration is ca. 25 À . Assuming that the alkyl-halide linkers exhibit similar reactivities, the surface coverage is estimated to be around 40%. The protein is subsequently incubated with the derivatized substrates at p H 7.58.0. The slides are then washed in nanopure water and dried under nitrogen. Very little physisorbed cytochrome b is ever observed under these conditions, presumably because the protein has a very hydrophilic surface and a large net anionic charge inhibiting hydrophobic and electrostatic interactions with the covered or bare substrate (control experiments with wild-type protein also demonstrate the requirement for the surface thiol). To assess the coverage of the protein monolayer, we have relied on quantitation of the heme prosthetic group concentration. The pyridine-heme chromagen assay (10), which uses spectrophotometric measurement of the well-characterized pyridine-heme adduct, yields a coverage of ca. 750-900 À /molecule. Because the crystallographic dimensions of cytochrome b are 25 X 25 X 30 Â, the surface coverage ranges between 85-100%. Because of the large size of the macromolecule relative to the linker, protein coverages approaching 1.0 can be obtained from surfaces on which the linker molecules are present at significantly lower coverages. 2


2

5

2

5

Low-resolution characterization of the surface-bound protein was conducted by monitoring antigenic activity (Figure 4). A thiopropylderivatized surface was reacted with the genetically engineered T 8 C mutant, and the resulting substrates were exposed to polyclonal antibodies directed against cytochrome b . Cross-reactivity was followed with I-labeled protein A , which yields a radiometric signal proportional to the primary anti-cytochrome b antibody concentration. The inset to Figure 4 tabulates the results of controlled experiments that clearly demonstrate the presence of cytochrome b in these assemblies. The use of polyclonal antibodies precludes any high-resolution conclusion about structure retention, but does confirm that chemisorbed (not removed by buffer washes) protein is present along with the heme prosthetic group monitored by the pyridine-heme chromagen assay. Significantly more structural detail has been obtained through the use of surface Raman spectroscopy. As background to the current work, two aspects of surface Raman scattering at dielectric interfaces have been extensively studied (J J, 12). The first aspect is the construction of 5

125

5

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480


Figure 4. Immunological assay for cytochrome b . Protein monolayers were treated with an α-cytochrome b polyclonal antibody preparation and sub sequently with I-labeled protein A to detect surface-bound antibody. Con trol measurements demonstrate the presence of chemisorbed cytochrome b on the optical substrates. 5

s

125

5

structures that permit the surface chemistry required for linker derivatization, while still retaining the necessary enhancement of Raman sig nals associated with the chemisorbed molecules. In addition, analyses to extract molecular-orientation information from polarize-scattering measurements have been developed. Structures consisting of initial Ag or Au islands (ca. 50 Â) deposited over masked S i 0 substrates are subsequently overlaid with 50-100 À of S i 0 spacer. Adsorbates are probed with excitation radiation coupled into the structure just above the critical angle for total internal reflection (calculated for the metal-free interface) as shown in Figure 5. Detailed experiments, including measurements of chemical resistance, Auger spatial mapping and depth profiling, coverage dependences of Raman signals, and studies of the signal intensity versus spacer layer thickness, demonstrate that the majority of the Raman signals are located at the S i 0 surface and not in defect channels to the metal surface. These defects are not a crucial consideration for the protein assemblies because of the large size of the molecules relative to all but the largest defects. Our primary interest was in characterizing the state of the heme prosthetic group. The heme oxidation state is characterized by transitions in the region 1340-1370 c m " , the spin state by bands in the region 1480-1510 c m , and the core size, reflecting doming or nonplanarity, in the region ca. 1550-1600 c m " (13). These marker bands are summarized in the frequency correlation diagram shown in Figure 6, and the spectra for chemisorbed monolayers of T 8 C and T65C are shown in Figure 7. Both proteins exhibit very similar marker band energies. If we assume that the positions of the marker 2

2

2

1

- 1

1



19.

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STAYTON ET AL.

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Figure 5. Schematic of the surface structures used for total internal re flection Raman scattering experiments.

Low»6

}

High,5

Fe

(II)

Fe

(III)

Low,6 Fe Fe

J

1330

1340

V

4

High.siS

(111)

(II)

I

I

1350

1360

Region

}

High,6

L

1370

•ι

ι

I

1470

1480

1490

V

3

I 1500

L 1510

Region

Figure 6. Heme protein marker band positions. Solution-derived resonance Raman band frequencies associated with the chemical and electronic states of the heme-prosthetic group. H represent the range ofRaman shifts observed for the given heme state in a number of heme-protein systems (IS). These markers can be used to assign the spectra of Figure 7.




482

Raman Shift ( c m ) - 1

Figure 7. Surface-enhanced resonance Raman spectra for T8C and T65C cytochrome b assemblies. The structure configurations shown in Figure 5 were used to obtain the fingerprint region spectra with laser excitation at 457.9 nm. s

bands are unaffected by binding to the substrate, then the following observations about the state of the heme proteins can be made (by comparison to the solution spectra). The oxidation state marker band is characteristic of the expected oxidized ferric heme, although the reduced species can be generated through a photoreduction process under sufficiently strong laser fluences. The spin state marker positions are most consistent with assignment as six-coordinate, high-spin heme, where the cytochrome b prosthetic group is present in solution as six-coordinate, low-spin heme. We are currently undertaking a more detailed study of the surface Raman spectra to more definitively assign the spin-spin state. Several phenomena could explain these observations, including a slight alteration in the heme pocket bonding interactions, a distribution of high-spin, 5-coordinate and low-spin, 6-coordinate species (argued against by the width of the observed bands), or simply a slightly shifted band position for the spin state marker in the surface environment. 5

Multieomponent Assemblies. As previously mentioned, cytochrome b is particularly attractive as a component in heterogeneous macromolecular assemblies due to its ability to specifically recognize a number or to partner heme proteins. Control over the assembly of a multieomponent protein thin film ultimately depends on control over the general process of protein-protein molecular recognition. Thus, we are interested in defining and manipulating the protein surfaces involved 5


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Proteins for Electrooptical Biomaterials

in the packing of two proteins during complex formation, and subse quently in tiling schemes to propagate order radially (Figure 2). Protein surfaces can be readily manipulated by using genetic engineering tech niques to control electrostatic properties and the distribution of hydrophilic and hydrophobic surface side chains. O f particular importance to surface design is the need to separate the contributions of electrostatics and nonpolar interactions and to develop the computational method ology to predict native and altered electrostatic potential surfaces. Again, site-directed mutagenesis has been invaluable in this endeavor. To map the electrostatic potential surface around cytochrome b , we have used a series of 15 surface point mutations that change the number and po larity of surface charged amino acid side chains (14,15). These mutations together with the change in the charge number relative to wild-type protein are shown in Table I. Although detailed self-consistent field theories are needed to describe accurately the electrostatic fields around a biological macromolecule at all points in space and for all solvent and protein conditions, it turns out that to manipulate protein-protein rec ognition events, a two-continuum dielectric-based calculation can pro vide an excellent picture of electrostatic surfaces (16,17). To check the accuracy of this model of macroscopic electrostatics, we use the heme as a reporter group and calculate the effect of each surface charge mu tation on the redox potential of the heme prosthetic group (18). Figure 8 shows the excellent agreement between experiment and theory and gives confidence that modeling can lead experiment in the de novo gen eration of docked potential surfaces to control assembly in planar arrays. 5


483

Table I.

Surface Point Mutations

Cytochrome b Mutants

ACharge

Eo'(mV vs. ΉΗΕ)

Q13E S64D

-1 -1

-9 -13

D66S E11Q E37Q E56Q E44Q E48Q D66S D60N

+1 +1 +1 +1 +1 +1 +1 +1

+1 -7 -5 -3 -1 +1 +1 +3

E44Q, E48Q E43Q, E44Q D66K E44K

+2 +2 +2 +2

+5 +7 +5 +1

E44Q, E48Q, D 6 0 N

+3

+13

5



484

f

ο

LU

Engineering Proteins for Electrooptical Biomaterials - Advances in

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