Coated substrates employing oriented layers of mutant heme proteins and methods of making and using same

The present invention is directed to coated substrates having a coating of biological macromolecules, preferably proteins, which are capable of being immobilized on a substrate surface and have a marker. These proteins usually are mutant proteins obtained by mutagenesis of the gene encoding a random positioning protein. When a mutant protein molecule is immobilized on the substrate, the marker of the mutant protein molecule is in a select spatial relationship with both the substrate and the markers of adjacent protein molecules. A substrate coated with an oriented layer of the mutant proteins exhibits improved or different properties when compared to a substrate having a randomly positioned layer of proteins thereon.

BACKGROUND OF THE INVENTION 
This invention relates to coated substrates employing oriented layers of 
biological macromolecules (e.g., proteins) which have a preferential 
binding site that is capable of coupling with functional groups on the 
surface of a substrate. In particular, the present invention relates to 
substrates coated with mutant protein molecules having, at a select 
position in the amino acid sequencer either an amino acid or the side 
chain of the amino acid replaced to create the preferential binding site, 
which is in a select spatial relationship to a marker on the mutant 
protein. The mutant protein molecules are immobilized on a substrate in a 
desired pattern, which includes uniform immobilization over the entire 
surface of the substrate. Due to the spatial relationship between the 
marker and preferential binding site, the markers of the mutant protein 
molecules of the layers are in a select spatial relationship with both the 
substrate and the markers of the adjacent mutant protein molecules. 
Surface-immobilized proteins have had a great impact in many fields of 
basic research, including many industrial and medical technologies. Most 
of the research in this area has been directed towards controlling the 
overall activity of hybrid biomaterials, for example, through reversibly 
affecting the surrounding matrix materials or by immobilizing related 
proteins in close proximity to provide multi-step enzyme processing. 
Systems which utilize a layer of proteins coupled to inorganic carriers are 
well known for many purposes, including antigen or antibody purification, 
assaying and detecting biological reactions and sensor operations. 
In such systems, the surface of the carrier is often treated to provide an 
intermediate coupling agent. For example, U.S. Pat. No. 3,652,761 to 
Weetall teaches utilizing silane coupling agents. U.S. Pat. No. 4,071,409 
to Messing et al. teaches using polymeric isocyanates and discloses other 
coupling agents. 
Such previous systems possessed limited usefulness. It was determined that 
a significant advance would be realized if the immobilization process 
itself specified assembly and regulated function, which allows selective 
control of molecular recognition events or molecular activity. Because 
many molecular recognition processes, such as protein-protein 
interactions, are controlled through specificity in complementary reactive 
surfaces, controlling the orientation of immobilized proteins can be a 
straightforward means of manipulating assembly and function. Similarly, 
molecular activity can also affected by orientation of the molecule on the 
substrate. 
Proteins for use with this invention can have a marker such as a heme 
prosthetic group, an enzyme active site, a ligand or epitope binding site, 
a particular amino acid sequence or the like. The efficacy and usefulness 
of systems employing such proteins is greatly improved where the marker of 
the protein molecule is positioned in a particular, select relationship to 
the substrate surface and the markers of adjacent protein molecules. 
Heretofore, random protein and marker positioning on a substrate surface 
could only be obtained because the vast majority of proteins have more 
than one reactive residue. These proteins are random positioning proteins. 
In other instances, some protein molecules have only one reactive residue 
on its surface and therefore will naturally be oriented relative to the 
substrate surface and adjacent proteins. However, the position of the one 
reactive residue limits the protein to be oriented in only one particular 
manner. Such a protein is not suitable if a different orientation is 
desired. Proteins that do not have a residue that is reactive with the 
coupling agent could not be previously utilized. 
Random positioning of protein molecules on a substrate surface can be 
deleterious in a number of applications. For instance, in systems which 
utilize proteins that transfer electrons or electromagnetic radiation 
between adjacent proteins, random positioning of these protein molecules 
in the layer inhibits transfer between proteins and propagation through 
the layer as compared to systems where proteins are mutated so that the 
marker of the each immobilized protein molecule is in a select spatial 
relationship with both the substrate surface and the markers of adjacent 
protein molecules. Random positioning also can reduce the absorption of 
light by the layer of proteins as when each protein requires the light to 
be incident in a direction parallel to a light absorptive marker of the 
protein. Additionally, random positioning can adversely influence the 
binding and interaction characteristics of immobilized protein. 
Industry has needs which can be satisfied by coated substrates which have 
orientated layers of proteins. Such substrates can be produced by 
immobilizing mutant proteins on a substrate--these mutant proteins have a 
marker in a desired and select spatial relationship with a preferential 
binding site, which binds the protein to functional groups of a substrate. 
OBJECTS OF THE INVENTION 
It is therefore an object of this invention to provide coated substrates 
having a coating of biological macromolecules immobilized in an oriented 
manner thereon; 
It is another object of this invention to provide a coated substrate where 
mutant protein molecules employed on the substrate have a marker and a 
preferential binding site; 
It is still another object of this invention to control molecular 
interactions or recognition events by controlling the orientation and 
positioning of the protein binding or interaction structure; 
It is a further object of this invention to provide a coated substrate 
wherein the mutant protein molecules are immobilized on the substrate via 
their preferential binding sites so that the markers of the mutant protein 
molecules are in a select spatial relationship with both the substrate and 
the markers of adjacent mutant protein molecules; 
It is still a further object of this invention to provide a method of 
producing the coated substrates described above; 
Other objects, features, and advantages of the present invention will be 
apparent from the accompanying description drawings, and sequence data. 
SUMMARY OF THE INVENTION 
The present invention is directed to coated substrates having a coating of 
biological macromolecules, preferably proteins, which are capable of being 
immobilized on a substrate surface. These proteins usually are mutant 
proteins obtained by mutagenesis of the gene encoding a random positioning 
protein, which often is a protein in its native state. However, a random 
positioning protein is not necessarily limited to native proteins; 
synthetic genes encoding proteins or polypeptides which closely correspond 
to native proteins may also be used. Mutant proteins may also be obtained 
by altering the side chain of a given amino acid. Mutagenesis changes the 
amino acid of the encoded protein at a select position. This mutation 
creates a preferential binding site in the resulting mutant protein; the 
preferential binding site has a reactive amino acid residue which is 
capable of coupling with functional groups on the substrate surface. The 
preferential binding site is in a select spatial relationship with a 
marker of the mutant protein. When a mutant protein molecule is 
immobilized on the substrate, the marker of the mutant protein molecule is 
in a select spatial relationship with both the substrate and the markers 
of adjacent protein molecules. 
When the preferential binding site has an amino acid residue capable of 
forming a covalent bond, the functional group on the substrate surface is 
a nucleophilic or electrophilic functional group and coupling is by a 
covalent bond that is formed therebetween. Alternatively, when the 
preferential binding site has a charged amino acid residue, the functional 
group is charged functional group having the opposite charge and coupling 
is by electrostatic forces that are formed therebetween. Alternatively, 
the preferential binding site can have a specific ligand, e.g. biotin; 
thus, functional group would be a specific, noncovalent binding partner, 
e.g., avidin or streptavidin. 
As stated above, the mutant proteins each have a marker. Such markers 
include: heme prosthetic groups, enzyme active sites, ligand or epitope 
binding sites, particular amino acid sequences and the like. The entire 
substrate surface can include a plurality of immobilized mutant proteins 
wherein each marker of each mutant protein molecule has the same spatial 
relationships in regard to the substrate surface and the markers of 
adjacent mutant protein molecules. This would be a coating with a uniform 
protein pattern. Alternatively, different portions on the substrate 
surface can have groups of mutant protein molecules immobilized in 
different select spatial relationships. In this alternative arrangement, 
the markers of some adjacent protein molecules will not be in the same 
spatial relationship with each other. Moreover, all of the immobilized 
protein molecules will not have their markers in the same select spatial 
relationship with the substrate. This type of coated substrate can be 
obtained by immobilizing different mutant proteins on the substrate. This 
would be a coating with a varied protein pattern. A varied protein pattern 
can also be obtained where only portions of the substrate have protein 
immobilized thereon. 
The select position for mutation can be on or beneath the protein surface 
provided mutation at the select position affects the protein surface to 
create the preferential binding site. The select position and the position 
of the preferential binding site can therefore be different or the same. 
Mutation, also referred to as mutagenesis, creates a preferential binding 
site that preferably is unique to the protein surface by replacing the 
amino acid at the select position with another amino acid or by replacing 
the side chain of the amino acid at the select position. The removed side 
chain preferably is replaced with a different side chain that produces a 
different amino acid or different binding capabilities. In addition, 
certain mutations can result in changes in the marker properties even if 
the protein is not mutated at the positions of the marker. Moreover, 
mutation can be undertaken in the marker of the random positioning protein 
to change the properties of the marker in the resulting mutant protein. 
Furthermore, mutation of the random positioning protein can be used create 
or introduce a marker in the resulting mutant protein. 
The random positioning protein can have more than one site capable of 
coupling with a functional group. In this situation, the protein is 
mutated so that an amino acid at the coupling site that is not preferred 
is replaced with an amino acid that is not capable of coupling, or the 
side chain thereof is replaced, to leave only the residue at the 
preferential binding site available to couple. Alternatively, the protein 
can be mutated at an amino acid near enough to the coupling site to 
prohibit the amino acid residue at the nonpreferred coupling site from 
coupling. Therefore, the select position and the position of the 
preferential binding site can be different. 
The random positioning protein can have an amino acid residue at the 
position where the preferential binding site is located that is not 
capable of coupling with the functional group. In this situation, the 
protein can be mutated at an amino acid near the position of the 
preferential binding site to affect the amino acid at the position of the 
preferential binding site and thereby create the preferential binding 
site. Here again, the select position and the preferential binding site 
are different. Alternatively, an amino acid, or the side chain thereof, at 
the position of the preferential binding site is replaced to create the 
preferential binding site. Here, the select position and the position of 
the preferential binding site are the same. 
The mutant protein can have more than one preferential binding site as when 
the protein surface is symmetrical with respect to the spatial 
relationship between the marker and the preferential binding sites. 
Therefore, use of any of the preferential binding sites of such a protein 
having symmetry results in desired orientation of the markers in relation 
to the substrate surface and the markers of adjacent protein molecules. 
The gene can be mutated a second time to obtain a double mutant protein 
having two binding sites. The additional or second binding site can be 
present when one binding site is preferred because it has a greater 
affinity for the functional group of the substrate or when one of the 
binding sites is blocked until after the residue of the preferential 
binding site couples with the functional group. Having two binding sites 
is useful to provide a second monomolecular layer of mutant protein 
molecules on the first monomolecular layer of mutant protein molecules. 
The mutant protein of the second monomolecular layer can also have two 
binding sites so that one binding site is available to bind with mutant 
protein molecules to form a third monomolecular layer. By using mutant 
proteins having two binding sites, a plurality of oriented, monomolecular 
layers can be sequentially built upon the substrate surface. 
Furthermore, having more than one binding site can be useful when it is 
desired to bind a ligand or reporter group to the proteins in the layer. 
The ligand can affect the transfer of electrons in the layer and this 
change can be noted to determine the concentration of ligand present as 
analyate in a medium. 
Representative protein types include metallo-proteins (such as heme 
proteins), enzymes, antibodies, antigens, and the like. In theory, any 
protein could be employed with this invention; however, in practice some 
proteins may prove difficult to immobilize or be of a nature where 
maintenance of an orientation is unlikely (e.g., where a protein can 
assume random conformations spontaneously). Furthermore, some proteins may 
denature or otherwise assume inactive conformations when immobilized. 
Thus, it must be recognized that although every type of protein should be 
amenable to this invention, every protein member of a given protein type 
might not function with this invention. 
Preferred proteins include heme proteins. Preferred mutant heme proteins 
include mutants of cytochrome b.sub.5 that have the amino acid threonine, 
e.g., at position 73, 65 or 8, replaced with cysteine to produce, e.g., a 
T73C mutant, a T65C mutant or a T8C mutant, respectively. These mutations 
place a unique thiol group at the designated position (73, 65 or 8 
respectively). When the T65C and T8C mutant heme proteins are coupled to a 
substrate, the heme prosthetic group of each protein is aligned about 
40.degree. to about 60.degree. from the normal to the substrate surface. 
Other preferred mutant heme proteins have their preferential binding site 
at position 85, 75 or 33. Alternatively, the heme protein can be mutated 
so that the spatial relationship between a plane dissecting the prosthetic 
heme group (the "heme plane") is at any predefined angle from the 
preferential binding site. 
The heme proteins can be mutated so that the spatial relationship between 
the heme plane and the preferential binding site is such that when the 
protein is immobilized on a substrate, the plane is oriented at an angle 
in the range of about 40.degree. to about 60.degree. from the normal to 
the surface of the substrate. Alternatively, the spatial relationship can 
be selected so that the angle is chosen from the group consisting of about 
0.degree. about 90.degree. and about 180.degree. or at a variety of angles 
therebetween. 
The mutagenesis of the protein to replace an amino acid or a side chain can 
be accomplished by conventional mutagenesis techniques that can result in 
complete synthesis of both DNA strands. Preferred mutagenesis techniques 
include cassette-style site-directed mutagenesis, polymerase chain 
reaction (PCR) mutagenesis and the like. To replace side chains, known 
methods of mutation can be employed. However, other mutagenesis techniques 
may be employed with departing from the scope and spirit of this 
invention. 
The resultant mutated gene codes for the mutant protein. The mutated gene 
is conventionally isolated, inserted into a high copy number plasmid that 
directs high level heterologous protein production in the host cell and, 
after the desired density is achieved, a conventional purification 
technique is utilized to obtain the mutant proteins. 
Suitable substrates are those conventionally utilized in industry for 
incorporation into electronic, optical and like devices. The substrates 
are composed of conductive metals, or, preferably, inorganic materials 
such as siliceous materials and metal oxides. If the mutant protein is to 
be immobilized for use in chromatography columns and the like, substrates 
such as sepharose or other synthetic or natural polymeric materials may be 
used. 
The substrate surface can have functional groups that couple with the 
reactive amino acid residue at the preferential binding site of the mutant 
protein. Alternatively, substrates which do not possess appropriate 
functional groups can be provided with such functional groups. A 
derivatization reagent can be employed to provide functional groups as 
long as the substrate surface has surface groups that are reactive with 
the derivatization reagent. As previously discussed, the functional group 
can be a nucleophilic or electrophilic functional group, a charged 
functional group, or a specific binding functional group. 
The derivatization reagents have two different kinds of reactivity, i.e., 
they have a group that is reactive with a surface group of the substrate 
and a functional group that couples with the reactive amino acid residue 
of the mutant protein. 
The choice of the derivatization reagent can control the hydrophobicity of 
the surface which affects the orientation of the protein and thereby the 
spatial relationship of the marker to the substrate and the markers of 
adjacent mutant proteins. The chain length of the derivatization reagent 
can provide flexibility to obtain various orientations of the mutant 
protein. 
The mutant protein can be applied to the substrate as a solution by 
immersion, by spin coating, by application with a doctor blade and by 
similar techniques. Once applied, the mutant protein is then incubated on 
the substrate surface for a time period effective to form the 
monomolecular layer. 
When the mutant protein of the first monolayer is a double mutant protein, 
a second oriented monolayer can be formed thereon by the above-described 
method of applying and incubating. Additional monomolecular layers can be 
formed by using a double mutant protein in the previous layer. 
The coated substrate can be utilized in biochemical, electronic, optical 
and similar devices. Representative applications of the coated substrate 
include use as a chromatography system, potentiometric sensor, 
amperometric sensor, phase grating, spatial light modulator, variable 
integrated optical waveplate, integrated optical sensor, directional 
electron transfer device, a ligand concentration measuring device, and the 
like.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Although this invention can be practiced in many different forms, there are 
shown in the Figures and will be described in detail presently preferred 
embodiments of the invention. It should be understood, however, that the 
present description is to be considered as an exemplification of the 
principles of this invention and is not intended to limit the invention to 
the embodiments illustrated. 
The mutant protein preferably used in the present invention has an amino 
acid residue at a preferential binding site provided by replacing an amino 
acid, or the side chain of the amino acid, at a select position along the 
amino acid sequence of a random positioning protein. The mutant protein 
also has a marker at or beneath the protein surface that is in a select 
spatial relationship with the preferential binding site. 
The term "biological macromolecule" refers to molecules of biological 
origin. Included are proteins, nucleic acids, lipids, polysaccharides, 
hormones, transmitters, factors and the like and any combination of the 
above. 
The term "marker" refers to structures on the biological macromolecules. 
Where the biological macromolecule is a protein, the marker can be a heme 
prosthetic group, enzyme active site, ligand or epitope binding site, 
particular amino acid sequence, or the like. This marker can be naturally 
occurring on the or can be provided by mutation of the protein. 
Furthermore, mutation may be undertaken to alter the properties of the 
marker or provide a marker altogether. 
The term "oriented" as used in its various grammatical forms, indicates 
that the spatial relationship between the marker of each biological 
macromolecule (e.g., mutant protein molecules) in the same layer is 
substantially the same, and that the spatial relationship between the 
markers and the substrate surface in the same layer is substantially the 
same. 
The term "random positioning protein" as used in its various grammatical 
forms, refers to a protein that either does not have a binding site in a 
select spatial relationship with marker or has a binding site in an 
undesired spatial relationship with the marker, i.e., the binding site is 
at a nonpreferential position. A random positioning protein is often a 
protein in its native state; however, random positioning proteins are not 
necessarily so limited. 
The term "select position" in regard to proteins, refers to the position 
along the amino acid sequence where mutation is to be undertaken. The 
select position can be on the surface of the protein or in the interior of 
the protein, i.e., beneath the protein surface. In either case, mutation 
at the select position affects the protein surface by creating a 
preferential binding site or making a binding site at a nonpreferential 
position nonbinding in the resulting mutant protein. 
The term "select spatial relationship" concerns the spatial relationships 
between the immobilized biological macromolecules (e.g., heme proteins), 
the markers on such macromolecules (e.g., the heme prosthetic group), and 
the substrate. A select spatial relationship is a preferred relationship 
which will endow the coated substrate with the desired properties. Where 
the structure and/or sequence of the biological macromolecule is at least 
largely known, the select spatial relationships can be predetermined; 
thereby, resulting in a wholly rational approach to coated substrate 
construction. Mutagenesis of the genes encoding random positioning protein 
provides a preferential binding site in the resulting mutant protein at 
the select position or at another position by replacing the amino acid at 
the select position. Replacement of a side chain of an amino acid at the 
select position may also be undertaken, usually by known translational 
mutation methods, to create the preferential binding site in the resulting 
mutant protein. 
When the random positioning protein does not have a preferential binding 
site, one can be introduced at the select position by replacing the amino 
acid, or the side chain thereof, at the preferential binding site. In this 
situation, the select position and the preferential binding site are at 
the same position along the amino acid sequence. Alternatively, the 
protein can be mutated at a select position that is not at the 
preferential binding site provided mutation affects the protein to create 
the preferential binding site. In this situation, the select position and 
preferential binding site are at different positions. When the random 
positioning protein has more than one binding site, a binding site that is 
not preferred can be removed by replacing the amino acid, or the side 
chain thereof, at the select position which is not the preferential 
binding site. Alternatively, the mutant protein can be obtained by 
mutation at a select position that is not at the nonpreferred binding site 
nor at the preferential binding site provided that the mutation only 
affects the protein at the nonpreferred binding site. 
Representative random positioning proteins that can be subjected to 
mutagenesis techniques to produce the mutant proteins of the present 
invention include protein types such as metallo-proteins, enzymes, 
antibodies, antigens, ligands, structural proteins, adhesion proteins and 
the like. 
Presently preferred randomly positioning proteins include heme proteins, 
which are proteins that have iron-containing ring compounds. 
Representative heme proteins are cytochromes b, c, a and P-450, 
hemoglobin, myoglobin and the like. A preferred cytochrome is cytochrome 
b.sub.5 which is produced from a gene that was chemically synthesized in 
vitro, is produced in bacteria and is identical or at least substantially 
similar to the 89 amino acid water soluble part of rat liver cytochrome 
b.sub.5. 
The average molecular weight of the mutant protein can vary considerably. 
Preferably, the molecular weight is greater than about 5,000 daltons. More 
preferable, the molecular weight is in the range of about 10,000 to about 
10 million daltons; however, the invention can be practiced with proteins 
outside of these ranges. 
The random positioning proteins discussed herein have a sequence and 
structure which is at least largely known. Knowledge of the sequence and 
structure permits a rational approach to selecting and obtaining the 
mutant proteins. When the sequence and structure of the protein are known, 
a mutation can be undertaken to create a preferential binding site in a 
known location, which will cause the marker of the immobilized mutant 
protein to be in a predetermined spatial relationship with both the 
substrate surface and the markers of adjacent immobilized mutant proteins. 
A predetermined spatial relationship is a type of select spatial 
relationship. Structure of proteins can be predicted based on sequence 
data and can be confirmed by well-known studies such as structural nuclear 
magnetic resonance and X-ray crystallography. However, it is important to 
note that the practice of this invention is not limited to proteins which 
have a sequence and structure which is at least largely known. The minimum 
information that is required is enough sequence data to allow one of skill 
in the art to introduce mutations to create a preferential binding site. A 
series of mutations can be undertaken in a given random positioning 
protein or other biological macromolecule to create a variety of mutant 
proteins. These mutant proteins can thereafter be immobilized on a 
substrate. The spatial relationships between the marker of a given protein 
molecule with the substrate and the markers of adjacent protein molecules 
can then be determined or estimated by observing the properties of the 
coated substrate. If the coated substrate having particular mutant 
proteins has the desired properties, then it is reasonable to conclude 
that mutant protein molecules so employed afford a spatial relationship 
which is appropriate and therefore select. Thus, a select spatial 
relationship can be obtained without knowledge of the entire sequence and 
structure of the random positioning protein and the mutants obtained 
therefrom. Although this approach may require a further degree of 
experimentation, it can be readily performed by one of skill in the art 
and is not undue. 
Mutagenesis techniques for replacing the amino acid are conventional. A 
preferred mutagenesis technique is cassette-style site-directed 
mutagenesis which is disclosed in Wells, et al. "Cassette-mutagenesis: an 
efficient method for generation of multiple mutations at defined sites" 
Gene, 34 315-23 (1985). 
Another preferred mutagenesis technique is polymerase chain reaction (PCR) 
mutagenesis. An illustration of this technique is provide in EXAMPLE 1, 
below. 
A representative mutagenesis technique is the "Oligonucleotide-directed in 
vitro mutagenesis system, version 2, Code RPN1523, " of Amersham 
International Plc. 
Alternatively, the random positioning protein can be mutated by replacing 
the side chain of the amino acid at the select position by conventional in 
vitro translation methods as disclosed in Maniatis, T. et al., eds. 
"Molecular Cloning" Cold Spring Harbor Laboratory, New York (1982). 
Suitable techniques for mutating, cloning and expressing the protein are 
disclosed in Wu, R., ed., "Recombinant DNA Parts A, B and C", Methods in 
Enzymology, Volumes 68, 100 and 101, Academic Press, New York, 1979, 1983 
and 1984. 
The gene produced by mutagenesis codes for the protein. The mutated gene is 
isolated and introduced into a host cell utilizing a suitable vector 
system for the particular host. Suitable vector systems include all 
vectors with Escherichia coli (E. coli) or broad host range origins of 
replication, e.g., pUC, RSF1010 and the like, and contain strong 
promoters, e.g., lac, tac, T7, lambda and the like. 
Suitable means for expressing the mutant protein include yeast, viral, 
Bacillus, Pseudomonas, Xenopus, mammalian, insect, and like expression 
systems. 
A preferred host cell is E. coli. A particularly preferred E. coli is E. 
coli TB-1 commercially available from BRL Laboratories, Bethesda, Md. 
which will be used in the following discussion of the parameters of a 
representative expression system for a mutant cytochrome b.sub.5 protein. 
A representative culture media for the E. coli is a conventional "Luria 
Broth" which contains 5 grams (g) of Yeast Extract (commercially available 
from Difco or Sigma Chemical Co.), 10 g of casein hydrosylate 
(commercially available from Difco) and 10 g of sodium chloride per liter 
of distilled and deionized water (water). 
The E. coli and the media are introduced into a suitable bioreactor such as 
a rotating drum reactor, a bubble column reactor, or the like. 
The growth temperature preferably is in the range of about 35.degree. to 
45.degree. C. Low oxygen tension preferably is utilized with mutant heme 
proteins since heme biosynthesis is normally repressed under strong 
aeration. The cells can be grown to an optical density of about 4 to about 
6 under these low aeration conditions. However, optical densities above 10 
can be achieved. 
The mutated gene is purified by a conventional purification technique. 
Representative purification techniques are disclosed in Scopes, "Protein 
Purification Principles and Practice", Springer-Verlag, New York (1982) 
and Deutscher, M. P., ed., "Guide to Protein Purification" Methods in 
Enzymology Volume 182, Academic Press, New York 1979. 
The mutant protein can be immobilized upon the surface of a substrate in a 
monomolecular oriented layer because each mutant protein has a 
preferential binding site. 
The substrates are made of materials that are conventionally utilized in 
the biotechnology, chemical, electronics, optics, and other industries. 
However, suitable materials that are discovered in the future can also be 
utilized. The substrate is typically a wafer of a siliceous material or a 
metal oxide. Alternatively, the substrate can be a conductive metal, a 
polysaccharide such as Sepharose, or a functionalized mineral-derived 
material like mica or graphite. The substrate materials can be provided as 
pure crystals or can be applied to a conventional base material by a known 
process such a sputter coating, chemical vapor deposition, evaporation 
coating and the like. 
The thickness of the substrate can be varied over a wide range and is 
primarily dependent upon the application for the coated substrate. 
Preferably, the substrate has a thickness in the range of about 100 
micrometers (.mu.m) to about 1 millimeter (mm). 
Representative substrate materials include titanium zinc glasses, i.e., 
glasses containing large concentrations of titanium and zinc, fused 
quartz, oxidized silicon wafers, i.e., wafers exposed to steam oxidation 
to produce a thin film layer of silicon oxide on the surface of the 
silicon, aluminum oxide, indium tin oxide, tin oxide, copper oxide, lead 
oxide, silver oxide, nickel oxide, silicon nitrides, silver, gold and the 
like. 
The substrate surface has thereon functional groups that are capable of 
coupling with the reactive amino acid residue at the preferential binding 
site. The functional group either occurs naturally on the surface, i.e., 
the functional group is a reactive functional group that is part of the 
substrate material, or can be provided by a derivatization reagent. 
The substrate material has on its surface an effective number of either 
functional groups capable of coupling with the amino acid or surface 
groups, e.g., hydroxyl or amine groups, to react with the derivatization 
reagent and thereby provide an effective number of functional groups. An 
effective number of functional groups is that which provides the desired 
density of mutant proteins in the oriented monomolecular layer. 
The functional group can be a nucleophilic functional group that is capable 
of coupling with the amino acid residue by forming a covalent bond. 
Alternatively, the functional group can be a charged functional group that 
is capable of coupling with the amino acid residue having the opposite 
charge by electrostatic forces that arise therebetween, or a specific 
binding molecule capable of forming strong, noncovalent interactions with 
the amino acid residue. 
The derivatization reagent can be any compound that has a reactive group 
capable of reacting with surface groups of the substrate surface and a 
functional group capable of coupling with the reactive amino acid residue 
at the preferential binding site. For example, when the preferential 
binding site has a cysteine residue the functional group of the reagent 
can be capable of forming a thioether, disulfide, thioester or 
metal-sulfur bond. 
Representative derivatization reagents include organomercury compounds, 
e.g., p-mercuriobenzoic acid and methyl mercuric iodide; organic compounds 
with an activated double bond, e.g., styrene; maleimide derivatives, e.g., 
N-ethylmaleimide and maleic anhydride; alkyl halides, e.g., 
3-bromopropyltrimethoxysilane, 3-iodopropyltrimethoxysilane, 
3-acryloxypropyltrimethoxysilane and 3-mercapto-propyltrimethoxysilane; 
metal complexes, e.g., gold, silver, copper, iron, zinc, cobalt, 
molybdenum, manganese, arsenic, and cadmium ions in aqueous solution; 
organodisulfides, e.g., bis-dithiothreitol and dithionitrobenzoic acid; 
organoisothiocyanates, e.g., fluorescein isothiocyanate and phenyl 
isothiocyanate; nicotinamide derivatives, e.g., nicotinic acid, 
nicotinamide and nicotinamide adenine dinucleotide; aldehydes and ketones, 
e.g., benzaldehyde, glyceraldehyde and methylethlylketone; lactones and 
lactams, e.g., penicillin, gluconolactone, glucuronolactone and uracil 
lactam; quinones, e.g., benzoquinone, naphthoquinone and anthraquinone; 
any compound with a double bond, whether or not it has beta-unsaturation, 
in the presence of ultraviolet (uv) or higher energy radiation, e.g., 
diazirenes, carbenes, and nitrenes; other organothiols, e.g., other 
cysteine-bearing proteins, mercaptoethanol and mercaptopyruvic acid; 
phosphoesters with high energy phosphate bonds, e.g., adenosine 
triphosphate, cyclic adenosine triphosphate, guanosine triphosphate, and 
cyclic guanosine triphosphate; and the like and any mixtures or 
combinations thereof. 
Furthermore, the selection of the derivatization reagent can affect the 
hydrophobicity and/or ionic content of the surface which is useful in 
fine-tuning the orientation of the mutant protein and thereby the select 
spatial relationship between the marker of a given mutant protein with 
both the substrate and the markers of adjacent mutant protein molecules. 
The hydrophobicity and/or ionic content of the surface can exert a force 
on the mutant protein that shifts the orientation of the protein. Thus, 
the orientation of a mutant protein can be varied by varying the 
derivatization reagent. For example, the marker of a mutant protein may 
orient at a 60.degree. angle with respect to the surface normal when the 
surface is hydrophilic. However, when the surface is highly hydrophobic, 
the marker may orient at a 55.degree. angle. Thus, tailoring the surface 
chemistry can provide a method of fine-tuning the orientation of the 
protein. 
The derivatization reagent can be selected to have a relatively short chain 
length which can provide a degree of flexibility in obtaining various 
orientations. Preferred chain lengths range from about 1 to 30 carbons. 
Flexibility can be provided by the minimization or alleviation of steric 
hindrances that often occur when longer chain length derivatization 
reagents are utilized. The steric hindrances are caused by the interaction 
of the longer chains and can change the orientation of the protein as 
compared to when the steric hindrances are not present. Although this 
change in orientation is desirable in some situations, it is undesirable 
in many situations, especially those situations wherein the orientation is 
expected to change in response to a stimulus. 
The coated substrate can be made by the following procedure. The surface of 
the substrate is scrupulously cleaned prior to application of the mutant 
protein or the derivatization reagent. Cleaning of the surfaces of 
substrates of the type which are commonly used in the electronics and 
optical industries is preferably performed by immersing the surface, 
preferably the entire substrate, in three successive hot, e.g., about 
160.degree. to about 180.degree. C., sulfuric acid baths. The immersion 
time in each bath is about three minutes. The surface is then rinsed with 
water. Optionally, the surface is then dipped in buffered hydrofluoric 
acid followed by a second water rinse. Next, the surface is immersed in a 
hot, e g , about 60.degree. C., mixture of ammonium hydroxide: hydrogen 
peroxide in a volume ratio of 4:1. Subsequently, the surface is rinsed in 
water and blown dry with a dry, inert gas, e.g., nitrogen, that is 
filtered. The substrate having a clean surface can be stored in a vacuum 
desiccator or water until needed. 
The derivatization reagent can be applied by refluxing the clean substrate 
in a dry chloroform solution of the derivatization reagent for a time 
period of about 1 to about 4 hours. 
The mutant protein is preferably applied to the substrate by immersing the 
surface in an about 10 micromolar (.mu.M) protein solution in an aqueous 
phosphate or aqueous Tris (hydroxymethyl) aminomethane buffer. The surface 
is then incubated in the solution for a time period effective to achieve 
full monomolecular layer coverage. Preferably, this time period is at 
least 3 hours and, more preferably, is in the range of about 6 to about 24 
hours. 
Alternative techniques for applying the mutant protein in solution to the 
substrate surface include spin coating, horizontal flow, substrate pulling 
and doctor blade techniques. The concentration of the protein solution 
varies depending upon the application technique and the specific 
processing conditions. Preferably, an about 10 .mu.M to about 1 millimolar 
(mM) protein solution in a previously described buffer is utilized. After 
the protein solution is applied it is incubated on the surface for the 
effective time period previously disclosed. 
In the spin coating technique the protein solution is applied to the 
surface of the substrate after which the substrate is held in a chuck, 
preferably by reduced pressure, and is spun at a relatively high 
rotational speed, e.g., about 1,000 to about 10,000 rpm, for a relatively 
short time period, e.g., about 5 to about 60 seconds. 
In the horizontal flow technique a horizontal substrate surface first is 
coated with a solution of the protein, the substrate is rotated to 
position the surface in a vertical position and the majority of the 
solution is permitted to drain off of the surface. 
In the substrate pulling technique the substrate is immersed in a tank 
containing the protein solution and is slowly removed from the tank as by 
a motor-driven pulley or conveyor belt system. 
In the doctor blade technique the protein solution is applied to the 
surface of the substrate, a sharp edged blade is positioned at a desired 
height, e.g., about 50 to about 500 .mu.m, above the surface, and excess 
solution is removed by contact with the blade as the substrate is moved 
under the blade or the blade is moved over the substrate. 
After incubation, the surface can be rinsed to remove multiple layers of 
mutant protein held to the surface by weak physiosorption forces and 
thereby obtain the desired oriented monolayer of mutant proteins. The 
rinsing step can include rinsing with pure buffer for about 10 minutes 
followed by rinsing with water for about 10 minutes. 
The thickness of a monomolecular layer is dependent upon the dimensions of 
the individual mutant protein molecules. For example, a monomolecular 
layer of the mutant heme protein cytochrome b.sub.5 has a thickness of 
about 30 Angstroms (.ANG.). 
The coated substrate of the present invention has many applications 
including as sensors and conductive devices. The energies sensed or 
conducted include electricity and electromagnetic radiation (especially 
light). Representative applications include potentiometric sensors, 
amperometric sensors, bipolar transistors, semiconductor or other 
electrodes, piezoelectric crystals, thermoelectrical crystals, 
charge-coupled devices, opto-electronic devices such as integrated optics 
and waveguide sensors, nonlinear optical devices, fiber optic devices, 
surface plasmon resonance-based sensors, magnetic sensors and the like. 
More particularly, the aforementioned mutant heme proteins can be utilized 
as a phase grating, spatial light modulator, variable integrated optical 
waveplate, integrated optical sensor, directional electron transfer device 
and the like. Some of these uses are discussed hereinbelow. 
As illustrated in FIGS. 1 and 2, a coated substrate 10 can be used as a 
phase grating 11 wherein a substrate 12 has on a substrate surface 14 
oriented, monomolecular mutant protein layers 16. The heme prosthetic 
group of the proteins is preferably oriented substantially parallel to the 
surface 14. Next to the layers 16 are open spaces 18. 
The phase grating is generated by arranging the layers 16 in an alternating 
fashion on the substrate surface 14; thus, causing the imaginary portion 
(absorption) of the refractive index to vary in a periodic fashion. This 
alternating fashion produces a surface 14 that has absorbing regions 
adjacent to nonabsorbing regions. The absorbing regions are coated with 
the oriented protein layer 16 while the nonabsorbing regions are not 
coated (like open spaces 18) or are coated with a nonabsorbing protein or 
other nonabsorbing material. The size of the regions are preferable in the 
range of about 0.05 to about 5 .mu.m wide, about 100 to about 1,000 um 
long and about 30 to about 300 .ANG. thick. The size of adjacent regions 
can be alike or different and can be varied to adjust the coupling 
properties, e.g., to improve the coupling efficiency at a specific 
wavelength or in a given direction, of the phase grating. 
To achieve an absorbing situation, which would be desired for the phase 
grating application, the light used would be polarized such that its 
electric vector is parallel to the surface, i.e., traverse electrical 
radiation would be utilized. Alternatively, if the protein is oriented 
such that the absorbing group is primarily oriented perpendicular to the 
surface, the radiation would be chosen to be transverse magnetic 
polarized. Phase gratings are typically utilized as analogues to mirrors 
in integrated optical devices and as couplers, which are components that 
switch light into and out of the thin film waveguide. 
FIG. 3 is a schematic representation of a typical sequence utilized to 
produce the phase grating 11. A silicon dioxide substrate 12 has applied 
thereto a conventional photoresist coating 28. A desired pattern of the 
phase grating is conventionally exposed in the photoresist coating 28 to 
produce exposed areas 22. Development of the exposed areas 22 results in 
the substrate surface 14 being revealed in the developed areas 23. A 
derivatization reagent is applied to the substrate surface 14 to produce a 
derivatized surface 24. The functional groups of the derivatization 
reagent couple with the amino acid residues of the mutant proteins to 
produce the monomolecular oriented layers 16. The remainder of the 
photoresist coating 28 is conventionally removed to produce a coated 
substrate 10 having monomolecular layers 16 with open spaces 18 
therebetween. 
FIG. 4 is a schematic representation of a spatial light modulator 26 
utilizing a coated substrate 10A. On a substrate surface 14A are 
positioned two opposed, spaced electrodes 28 having an oriented protein 
layer 16A therebetween. A phase grating 11A, or other coupling structure, 
can be present to couple light into the protein layer 16A of the spatial 
light modulator 26. The orientation of the immobilized proteins can be 
manipulated by imposing external perturbations, e.g., AC or DC electric 
fields, or the presence of a phase grating radiation pulse. Furthermore, 
manipulation can also be effected by the presence or absence of salts or 
chaotropic agents in a solvent on the spatial light modulator or the 
presence of small molecule ligands that bind to the heme groups. 
An individual protein 30 in the protein layer 16A of the spatial light 
modulator 26 is illustrated in FIGS. 5A and 5B. As shown in FIG. 5A, when 
the electrical field is off, the protein 30 is in a position in which the 
interaction with the propagating light radiation field is strong because a 
schematically represented plane 32 dissecting the heme prosthetic group 
("the heme plane") is substantially parallel to the surface 14A. In 
contrast, as shown in FIG. 5B, when the electric field is on, the 
interaction with the propagating light radiation field is weak because the 
heme plane 32 is substantially perpendicular to the surface 14A. That is, 
when the electric field is off, the proteins are in an absorbing 
configuration (FIG. 5A), and when the electric field is on the proteins 
are in a nonabsorbing configuration (FIG. 5B). Alternatively, the mutant 
proteins can be tailored so that when the electric field is off, the 
proteins are in a nonabsorbing configuration and when the electric field 
is on the proteins are in an absorbing configuration. Thus, the spatial 
light monitor can be utilized in signal generation/processing since the 
presence or absence of light can stand for a 0 or a 1, which is a basis 
for digital logic. 
When the coated substrate is utilized as a variable integrated optical 
waveplate it has a physical structure similar to that shown in FIG. 4. An 
important function in integrated optics for which waveplates are used is 
the incorporation of mechanisms for manipulating the polarization state of 
the light in the device. The mutant heme protein can act both as an 
absorber and as a birefringent material, i.e., the refractive index of the 
protein is different in all three spatial directions. Thus, if the mutant 
heme protein is coated onto the substrate and the radiation is propagated 
at a wavelength at which the heme is not strongly absorbing, then changing 
the orientation of the protein changes the refractive index affecting by 
the light propagating in the protein layer. The change in refractive index 
consequently changes the phase delay of the light for each of the 
orthogonal polarization components. This would require that the 
propagating radiation (light) have its electric vector not oriented along 
the optic axis of the oriented protein monolayer. A difference between the 
spatial light monitor and the variable integrated optical waveplate is 
that for the waveplate careful attention would be paid to the polarization 
state of the light and radiation would be propagated at a nonabsorbing 
wavelength. The relative retardation of the two components desired can be 
varied by simply changing the length over which the radiation sees a 
specific protein orientation. 
When the coated substrate is utilized as an integrated optical sensor, the 
specific ligand-binding properties of the proteins are taken advantage of. 
For example, a synthesized myoglobin (hereinafter "wild-type") closely 
corresponding to sperm whale myoglobin, the only difference being that the 
product of the synthetic gene retains the N-terminal initiating 
methionine, is mutated twice to produce a H64L/A125C double mutant. The 
H64L/A125C designation means that the mutant was obtained by mutations 
which resulted in a leucine at position 64 rather than a histidine and a 
cysteine at position 125 rather than an alanine. This double mutant is 
thereafter immobilized on the substrate surface to produce an oriented 
layer. The mutant myoglobin protein binds carbon monoxide preferentially 
to oxygen whereas the "wild-type" myoglobin binds both carbon monoxide and 
oxygen with near equal affinity. When the ligand is bound, the wavelength 
of strongest absorption shifts dramatically. The shift is up to a 40 
nanometer (nm) in favorable cases. The transmission of a laser light of 
the appropriate wavelength is monitored through the sensor. When some of 
the myoglobin proteins bind carbon monoxide, their wavelength of maximum 
absorption will shift and the transmitted light intensity will change. 
As illustrated in FIG. 6, the integrated optical sensor can be implemented 
in a double-beam configuration in which two lasers (not shown) are used. 
One laser beam 34 has the wavelength that is transmitted by the heme 
protein with no bound ligand, and the other laser beam 36 has the 
wavelength that is transmitted by the heme protein with the bound ligand. 
The substrate surface 14B has an uncoated area 38 and a region 40 having 
an oriented, monomolecular double mutant heme protein layer. Two pairs of 
fiber optic probes 42 and 44 are mounted close to the substrate surface 
14B. Fiber optic probes 42 measure the transmission of light having the 
wavelength transmitted by the unbound protein and fiber optic probes 44 
measure the transmission of light having the wavelength transmitted by the 
bound protein. The ratio of transmission of the light from the two lasers 
is used as a signal as illustrated in FIG. 7. Curve 46 is for the 
absorption versus wavelength for the transmission of light through the 
unbound protein. Curve 48 is for the absorption verses wavelength for the 
transmission of light through the bound protein. 
Alternatively, the integrated optical sensor can be implemented in a single 
beam configuration (not shown) in which the light initially is completely 
transmitted by the layer. Binding of carbon monoxide then shifts the 
absorption away from the wavelength of the propagating radiation and this 
shift is measured. 
The rate of transfer of an electron through a heme protein is dependent 
upon the path the electron is traveling. Furthermore, the rate of electron 
transfer also is dependent upon the orientation of the protein to which 
the electron is being transferred. Thus, by adjusting the orientation of 
the protein through which the electron is to be transferred and of the 
protein to which the electron is to be transferred the rate of electron 
transfer can be adjusted. 
For example, the fastest direction of electron transfer of a heme protein 
is parallel to the heme plane. Thus, as illustrated in FIG. 8A, a protein 
diode 50 can be produced when a substrate 12A preferably is an 
electrically conductive material. A heme protein 30C having a heme plane 
32C is immobilized on the surface 14C oriented so that the heme plane 32C 
is not perpendicular to the surface 14C when the diode 50 is turned off. 
Thus, electron transfer is slow in the direction perpendicular to the 
substrate surface 14C. However, as illustrated in FIG. 8B, when the diode 
50 is turned on the protein 30C is displaced so that the heme plane 32C is 
perpendicular to the substrate surface 14C. Thus, electron transfer is 
relatively fast in a direction perpendicular to the substrate surface 14C. 
The following Examples are given by way of illustration, and not 
limitation, of the present invention. 
Although the EXAMPLE 1 utilizes primers, reagents, conditions and the like 
to generate the T73C mutant protein of cytochrome b.sub.5, other mutant 
proteins can be produced using this technique to mutate other proteins 
using different primers, reagents, conditions and the like. Production of 
the aforementioned T8C and T65C mutant cytochrome b.sub.5 is discussed in 
Stayton et al. Biochemistry 28:8201-8205 (1989); Stayton et al. J. 
Biological Chemistry 263:13544-13548 (1988); Beck von Bodman et al. Proc. 
Natl. Acad. Sci. USA 83:9443-9447 (1986). 
EXAMPLE 1: GENERATION OF T73C CYTOCHROME b.sub.5 MUTANT BY A PCR 
MUTAGENESIS TECHNIQUE 
A site-directed mutation was engineered into a gene that codes for the 
desired protein by using three primers in two consecutive rounds of PCR 
amplification. The substrate DNA was the synthetic cytochrome b.sub.5 gene 
cloned into a pUC 19 plasmid. Of the three primers necessary for this, one 
primer was specific for the desired mutation, i.e., the mutagenic primer, 
whereas the other two primers flanked the gene by annealing to the plasmid 
and therefore needed only to be specific for sequences of the vector, 
i.e., the pUC plasmids. Therefore the choice for the latter two primers 
was the sense and antisense sequencing primers, i.e., universal and 
reverse, both of which are commercially available from sources such as New 
England Biolabs. Alternative vectors may also be used and the flanking 
primers would then have to correspond to sequences of the selected 
alternative vector. The mutagenic primer is preferably about 18 to 24 
nucleotides long and code for the desired amino acid substitution(s) plus 
any other base alterations necessary for screening, e.g., colony 
hybridization or restriction site gain or loss. In the first PCR round, 
using the mutagenic primer and one of the flanking primers (the 3' ends of 
the primers need to face one another), a portion of the gene which now 
contains the desired mutation was amplified. This mutated gene fragment 
then served as one primer, along with the other flanking primer, in the 
second PCR round which amplified the entire mutated gene. Subsequent 
digestion with the appropriate restriction enzymes facilitated ligation of 
the mutated gene into the vector of choice for direct screening, 
sequencing and expression. 
The mutagenic primer had the nucleic acid sequence 5' 
GATGATGTAACATTTCGACAGTTC 3' (SEQ ID NO:1). This primer was an anti-sense 
primer; however, mutations can be accomplished using mutagenic sense 
primers. The mutagenic primer was stored in ammonium hydroxide which was 
removed and the DNA resuspended in water, ethanol precipitated, washed, 
dried, and brought up in water. 
A. PRIMARY PCR 
To a 500 microliter (.mu.l) eppendorf tube was added the following reagents 
in this order: 83 .mu.l water (to make 100 .mu.l total); 10 .mu.l 10x 
Polymerase Buffer (supplied by the Vent polymerase manufacturer); 2 .mu.l 
dNTP's (10 mM in each dNTP); 1 .mu.l mutagenic primer, described above; 1 
.mu.l of commercially available universal primer (10 .mu.M stock); 1 .mu.l 
template, i.e., cytochrome b.sub.5, 30 ng total; and 2 .mu.l Vent 
polymerase (2 units) commercially available from New England Biolabs. The 
reagents were mixed thoroughly by finger flicking the tube, spun down and 
placed in a PCR machine, i.e., a commercially available DNA Thermal Cycler 
from Perkin-Elmer, Cetus. The reagents in the tube were subjected to 15 
heating cycles in the PCR machine. One heating cycle consisted of 2 
minutes at 94.degree. C., 2 minutes at 50.degree. C. and 2 minutes at 
72.degree. C. 
A 20 .mu.l aliquot was removed for observation and isolation by 
conventional electrophoresis on a 1.2% TAE agarose gel. Geneclean 
(Geneclean II kit, commercially available from Bio 101, LaJolla, Calif.) 
was used to isolate the primary PCR band, and hence the primary PCR 
product, from the gel. 
B. SECONDARY PCR 
To a 500 .mu.l eppendorf tube was added the following reagents in this 
order: 62 .mu.l water; 10 .mu.l 10X Polymerase Buffer (supplied by the 
Vent polymerase manufacturer), 2 .mu.l dNTP's, 10 mM in each dNTP 20 .mu.l 
of the above Genecleaned primary PCR product; 1 .mu.l reverse sequencing 
primer (10 .mu.M stock); 3 .mu.l template plasmid, i.e., cytochrome 
b.sub.5, 90 ng total: and 2 .mu.l Vent polymerase. 
The reagents were mixed thoroughly by finger flicking the tube, spun down 
and subjected to the 15 heating cycles in the PCR machine as described in 
conjunction with the primary PCR. 
C. LIGATION AND TRANSFORMATION 
The secondary PCR reaction was ethanol precipitated by adding 10 .mu.l of 
3M sodium acetate and 200 .mu.l ethanol at 70.degree. C. for 15 minutes. 
The pellet was washed with 70% ethanol and dried. After reconstitution the 
DNA was digested with Pst I and Eco RI restriction enzymes in a solution 
of 86 .mu.l water, 10 .mu.l 10X restriction buffer, 2 .mu.l Pst I, and 2 
.mu.l Eco RI at a temperature of 37.degree. C. for a time period of 1 
hour. 
Similarly, 5 .mu.l pUC19C (5 .mu.g) was digested using a solution of 81 
.mu.l water, 2 .mu.l Pst I, and 2 .mu.l Eco RI at a temperature of 
37.degree. C. for a time period of 1 hour. 
The reaction products were ethanol precipitated, washed, and dried to 
obtain pelleted products. The pellets were combined in 15 .mu.l water, 4 
.mu.l 5X ligase buffer and 1 .mu.l T4 DNA ligase and the resultant product 
was incubated at 16.degree. C. overnight to produce the ligation mixture. 
Competent cells were prepared by resuspending a TB-1 E. coli culture (0.3 
OD) in 100 mM calcium chloride. 
Transformation of the ligation mixture was conducted by incubating the 
ligation mixture with 200 .mu.l of competent cells on ice for 20 minutes. 
This mix was then heat shocked at 37.degree. C. for one minute and 1 ml of 
Luria Broth media added. After 40 minutes at 37.degree. C., 100 .mu.l was 
plated on X-Gal plates After overnight incubation of 37.degree. C., 
colonies were visible on the plates. 
D. SELECTION AND SCREENING 
White colonies were picked and used to inoculate 5 ml overnight cultures 
for miniprep plasmid purification. Miniprep isolation of plasmid yielded 
templates for sequencing. Dideoxynucleotide sequencing using a commercial 
sequencing kit resulted in the confirmation of successful mutant 
generation T73C cytochrome b.sub.5. The cells were red indicating that 
holo cytochrome b.sub.5 was produced. Frozen stocks of the T73C mutant 
were prepared and used to ferment. 
E. FERMENTATION BIOREACTION AND PURIFICATION 
Fermentation (bioreaction) of the T73C mutant was conducted in a New 
Brunswick 12 liter fermentation vessel and in 6.times.3 liter shaker 
flasks which yielded 70 g of wet cells. 
The T73C mutant protein was purified utilizing the following process. The 
plate colonies were cultured and introduced into two 5 ml cultures at a 
temperature of 37.degree. C. for a time period of 16 hours. The 5 ml 
cultures were then introduced into 250 ml cultures with 50 mg Ampicillin 
and grown at 37.degree. C. for 16 hours. The 250 ml cultures were 
introduced into a 25 liter fermentor and maintained therein under growth 
conditions for a time period of 16 to 24 hours. Cells were then harvested 
and subsequently frozen at a temperature of -70.degree. C. Next, the cells 
were thawed at room temperature in lysis buffer and stirred in the lysis 
buffer at a temperature of 5.degree. C. for a time period of 16 hours. The 
lysate was then centrifuged at a temperature of 5.degree. C. at 10,000 
.times.G for a time period of 20 minutes. The pellet and supernatant were 
separated and the supernatant was saved. The pellet was resuspended in 
lysis buffer at a temperature of 5.degree. C. and stirred for a time 
period for 2 hours. This lysate was centrifuged at a temperature of 
5.degree. C. at 10,000 .times.G for a time period of 20 minutes. The 
resultant pellet was discarded and the supernatant saved. The supernatants 
were pooled and then passed through an ascending DEAE cellulose column 
(0-0.25M salt gradient). A concentrate first column pool was obtained by 
ultra filtration and subjected to gel filtration on a Bio gel p-60 resin 
followed by passing through an ascending DEAE cellulose column (0-0.25M 
salt gradient). The resulting product, purified T73C cytochrome b.sub.5 
mutant protein, was then evaluated for purification using SDS-page gel 
electrophoresis. 
EXAMPLE 2: PREATION OF A COATED SUBSTRATE WITH AN ORIENTED LAYER OF 
PROTEINS 
A substrate was treated with the derivatization reagent 
bromopropyltrimethoxysilane (hereinafter "silane") and then coated with 
T8C mutant heme protein. 
The substrate utilized was lab craft microscope cover glass, commercially 
available from Curtis Matheson Scientific, Inc., catalog No. 266-809, 1 oz 
that were 24.times.60 mm in dimension and had a #1 thickness. To clean the 
substrate, three baths of sulfuric acid which had been heated to a 
temperature of about 180.degree. C. for about one hour were prepared. The 
substrate was immersed in each bath for a time period of about 3 minutes 
before being moved to the next bath. After the third bath, the substrate 
was rinsed with water for a time period of 1 minute. Then, the substrate 
was immersed in a hydrogen peroxide/ammonium hydroxide mixture that had a 
volume ratio of about 1:4, respectively. The mixture had been heated to 
about 50.degree. C. for a time period of about 20 minutes prior to 
immersion of the substrate. The substrate was immersed in the mixture for 
a time period of 3 minutes, removed therefrom, rinsed with running water 
for a time period for 1 minute and then dried with dry nitrogen. 
The cleaned substrate was then annealed in preparation for the linear 
dichroism experiment by placing the substrate in a conventional tube 
furnace in a flowing high purity argon stream, turning the heat control of 
the furnace to high and switching the furnace off when it had reached a 
temperature of about 500.degree. C. The furnace with the substrate therein 
was permitted to cool about 3 hours before the substrate was removed and 
stored in a moisture-free environment. 
Next, the substrate was treated with silane and dry, distilled chloroform. 
The substrate, 300 ml of chloroform and 3 ml of the silane were introduced 
into a conventional reflux vessel. The contents of the vessel were heated 
to a temperature in the range of about 55.degree. C. to about 60.degree. 
C. for a time period of about 2.5 hours. Additional chloroform was 
introduced into the vessel to maintain coverage of the substrate. At the 
end of this time period, the contents of the vessel were permitted to cool 
for a time period of about one-half hour. The substrate was then rinsed in 
chloroform and dried with nitrogen. 
An oriented layer of the T8C mutant heme protein was then formed on the 
substrate by the following procedure. A sample chamber was made using 
poly(tetrafluoroethylene) (commercially available as "Teflon"). The 
interior of the chamber had a depth of about 30 mm and a width of about 24 
mm and was capable of holding half of the substrate and about 2.5 ml of a 
mutant protein-containing solution. Thus, half of the substrate was coated 
in the chamber. 
A 300 .mu.M stock of the T8C mutant heme protein was diluted to about 20 
.mu.M utilizing a pH 7.5, 50 mM potassium phosphate buffer to produce the 
mutant protein-containing solution. 
The chamber was filled with the mutant protein containing solution and half 
of the derivatized substrate was inserted therein. A beaker was positioned 
over the chamber to limit evaporation. The substrate was maintained in the 
chamber for a time period of about 3 hours at about room temperature, 
i.e., about 20 to about 30.degree. C. The substrate was removed from the 
chamber and placed in a small beaker of the potassium phosphate buffer for 
a short time period. Then, the substrate was rinsed with water and dried 
with nitrogen. 
The heme of the protein was utilized to quantatively measure the amount of 
protein present. This measurement required that the heme be released into 
a solution and complexed with pyridine to form a complex that could be 
assayed after being reduced with a reducing agent, such as dithionite. A 
release solution was prepared by admixing in a suitable vessel 3 ml of the 
50 .mu.M potassium phosphate buffer, 0.5 ml pyridine and 0.25 ml of a 1M 
solution of sodium hydroxide. About 2 ml of the release solution was 
introduced into a clean chamber and then the coated substrate was 
introduced into the chamber. The release solution was pipetted onto the 
upper parts of substrate that were not immersed. The substrate was removed 
and the release solution containing complex was equally divided and placed 
into two 1 ml cuvettes. The cuvettes were optically balanced on a Hewlett 
Packard 7470 Spectrophotometer and then a grain of dithionite was added to 
the sample cuvette and the spectrum was taken. 
The extinction coefficient (.epsilon..sub.mm 557-574) for the pyridine-heme 
complex is about 34.4 mM.sup.-1 cm.sup.-1. The number of moles of the 
pyridine-heme complex, which equals the number of moles of heme group and 
also the number of moles of protein, on the substrate was calculated 
utilizing the following equation: 
##EQU1## 
The actual number of heme molecules was calculated utilizing the following 
equation: 
EQU (0.14 .mu.Moles/1000 ml).times.2 ml.times.(1 mole/10.sup.6 
.mu.Moles).times.6.02.times.10.sup.23 molecules/1 
mole)=1.7.times.10.sup.14 heme molecules per monolayer. 
The theoretical number of protein molecules on the substrate, assuming a 
close packed protein layer, was calculated as follows: 
EQU (2.times.30 mm.times.24 mm).times.(10.sup.14 .ANG..sup.2 /1 
mm.sup.2).times.[1 cytochrome b.sub.5 /(30 .times.2 
.ANG..sup.2)]=2.times.10.sup.14 cytochrome b.sub.5 protein molecules per 
monolayer. 
The numeral 2 in the multiplier "2.times.30 mm.times.24 mm" is necessary 
because both the front and the back of the substrate were coated. 
The percent coverage is determined by dividing the actual number of heme 
molecules by the theoretical number of protein molecules that should be 
present in a close packed monolayer. Thus, for the first substrate the 
actual percent coverage is: 1.7/2.times.100 =85% actual coverage. 
A second substrate prepared utilizing the above procedure had a spectrum 
reading of 0.003 A and therefore the concentration of the release solution 
containing the complex was 0.087 .mu.M. For the second substrate, the 
percent coverage was 61%. 
EXAMPLE 3: A CYTOCHROME b.sub.5 IMMOBILIZATION SYSTEM 
The cytochrome b.sub.5 immobilization system depicted in FIG. 9 is a means 
for examining and controlling protein-protein interactions. These 
interactions are of crucial importance to important biological processes 
such as electron transfer and vectorial proton transfer; thus, 
manipulating macromolecular specificity at interfaces is an important step 
toward controlling function and self-assembly in a wide variety of 
immobilized protein systems. 
The system schematically depicted in FIG. 9 employs the cytochrome b.sub.5 
/cytochrome c electron transfer pair. Immobilized T8C cytochrome b.sub.5 
(50) is shown interacting with cytochrome c (55). Molecules representing 
the E38C immobilization and the D66C or T65C immobilizations are depicted 
nearby for comparison purposes. The E38C and D66C immobilizations were 
obtained using the E38C/D66C double mutant discussed at length below. 
Extensive biochemical, biophysical, theoretical and genetic investigations 
have provided a detailed understanding of the interaction surfaces for 
both cytochromes b.sub.5 and c. The complementary binding interface 
between the two molecules places their heme edges in close proximity for 
fast electron transfer between the redox centers. 
As shown in FIG. 9, three cytochrome b.sub.5 mutants were employed, namely: 
the previously-discussed T8C and T65C mutants and the double mutant 
D66C/E38C. The D66C/E38C double mutant has aspartic acid replaced by 
cysteine at position 66 and glutamic acid replaced by cysteine at position 
38. 
The D66C/E38C double mutant was generated by a method which was somewhat 
different than the method used to produce the T8C and T65C mutants, 
described above. To generate the D66C/E38C mutant, two oligonucleotide 
primers encoding the position 66 mutation and position 38 mutation were 
used to amplify the gene segment that contains the mutations. The E38C 
mutagenic primer has the sequence of 5' TTCCTCGAATGCCACCCC 3' (SEQ ID 
NO:2); the D66C mutagenic primer has a sequence of 5' GTTCTCGAGCGCAGGTACTA 
3' (SEQ ID NO:3). This PCR product was used in a second PCR reaction in 
conjunction with a universal sequencing primer, which is commercially 
available from sources such as New England Biolabs, to amplify the 3' end 
of the mutated gene (the C-terminal end of the protein). This PCR product 
was used with the reverse universal sequencing primer, which is also 
commercially available from sources such as New England Biolabs, to 
amplify the entire gene. This final PCR product was digested with the 
appropriate restriction enzymes and ligated back into the vector. 
The T65C provides a preferential binding site that is close to the 
cytochrome b.sub.5 /cytochrome c binding interface, yet apparently remains 
sufficiently spatially distinct to prevent steric interference. The T8C 
mutant provides a preferential binding site on the surface opposite the 
exposed heme edge, at a site distant from the binding interface. Linear 
dichroism measurements of the prosthetic heme group demonstrate that the 
T65C and T8C mutants are differentially orientated at the substrate 
interface when immobilized. The D66C/E38C double mutant has two free thiol 
groups (from the two cysteine residues) which are free for binding; thus, 
the double mutant can be bound on either side of the heme edge. However, 
the percentage of protein bound via either site is presently not known, 
but such knowledge is not required for the practice of this invention. 
Both binding sites in this double mutant are relatively close to, yet 
sterically distinct from the cytochrome b.sub.5 /cytochrome c binding 
interface. 
The system of FIG. 9 can be used for chromatography. Avidin (60) 
immobilized on sepharose 6B (62) was chosen as a support because it has a 
high pI (-10.2), which disfavors nonspecific electrostatic interactions 
between avidin and cytochrome c. The three mutant cytochrome b.sub.5 
proteins (T8C, T65C, and D66C/E38C) were biotinylated (64) with 
N-iodoacetyl-N-biotinylhexylenediamine (Pierce Pharmaceuticals). Excess 
biotin label was removed by passage over a Sephadex G-25 (Pharmacia) gel 
filtration resin. The biotinylated cytochrome b.sub.5 was mixed with 
avidin immobilized on the Sepharose 6B and stirred extensively to obtain a 
homogeneous protein concentration throughout the resin. Chromatography 
columns employing this system were integrated with a Pharmacia FPLC pump 
system. 
Equilibrium binding isotherms were obtained for the three mutants with 
linear potassium chloride elution gradients and corrected for small 
differences in cytochrome b.sub.5 concentration. These isotherms were 
obtained as follows: 0.3 nmoles of each mutant cytochrome b.sub.5 were 
loaded onto a 730 .mu.l column (Pharmacia HR 5/5 containing the cytochrome 
b.sub.5 resin) pre-equilibrated in 1 mM KPi pH=7.0 (3 columns each with a 
mutant plus 1 control column). A linear 0 to 100 mM KCl gradient over 30 
ml was run at a flow rate of 0.75 ml/minute and the elution profile was 
calculated using the area under the elution peak (monitoring absorbance at 
405 nm, with the total area reproducible to within 5%). The midpoint of 
the cytochrome c elution profile was reproducible to a standard deviation 
of +/-1 mM with all three cytochrome b.sub.5 columns. To normalize the 
binding isotherms to cytochrome b.sub.5 concentration, the heme content 
was determined (1:1 heme to cytochrome b.sub.5) A solution of 20% 
pyridine/200 mM NaOH was passed through the column and the effluent 
collected. The heme concentration was determined with the 
pyridinehemochromogen assay using an extinction coefficient of 23.98 for 
.epsilon. (556-538)(Reduced-Oxidized). 
FIG. 10 is a graphical representation of the binding isotherms for the 
cytochrome b.sub.5 mutants. The isotherms were determined by the elution 
profiles generated with the linear KCl gradient. The 3 mutants and control 
are represented in FIG. 10 as follows: T8C (-- . . . --); T65C (-- - --); 
D66C/E38C (. . . ); control, no cytochrome b.sub.5 (---). The striking 
dependence of cytochrome c affinity on the cytochrome b.sub.5 attachment 
site can be readily observed. Consistent with previous measurement of 
differential T8C and T65C orientations at surface immobilization sites, 
these mutants display distinct binding isotherms. Further, the reduction 
in cytochrome c affinity with the T65C and D66C/E38C mutants is in the 
direction expected for orienting the cytochrome b.sub.5 binding surface 
toward the immobilization interface. The T8C data is consistent with an 
orientation which allows for substantial cytochrome c interaction. Control 
measurements of the binding isotherm for cytochrome c-avidin interactions 
(---) show that the T65C and D66C/E38C association constants are slightly 
higher than the non-specific background, indicating that some low affinity 
cytochrome b.sub.5 -cytochrome c interactions are retained with these 
mutants. These results, in combination with previous documentation 
indicating that cytochrome c binds close to, but sterically distinct from 
the T65C and D66C/E38C positions, strongly suggest that orientation 
dependent molecular recognition has been achieved with such a immobilized 
protein system. 
Orientational control of protein-protein molecular recognition at 
interfaces will be important in two crucial interrelated aspects of device 
design, namely protein function and molecular assembly. Protein function 
is often exquisitely sensitive to the spatial relationships of subunits 
and protein partners. Pioneering work, disclosed herein, has demonstrated 
the utility of immobilization as a technique for generating spatially 
localized systems. Specific orientation of macromolecules, e.g., proteins, 
on a substrate is useful in applications such as chromatography columns. 
In such columns, the orientation of the immobilized macromolecule (e.g., 
protein) affects the properties of that macromolecule. These affected 
properties include binding or affinity of the macromolecule to ligands, 
epitopes, binding sites or the like. Thus, altering the orientation of the 
macromolecule will permit differential reactivity evidenced by binding, 
affinity or the like. 
This invention has been described in terms of specific embodiments set 
forth in detail. It should be understood, however, that these embodiments 
are presented for the purposes of illustration and demonstration, and that 
therefore the invention is not limited to such embodiments. Modification 
and variation may be resorted to without departing from the spirit and 
scope of the invention as claimed. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GATGATGTAACATTTCGACAGTTC24 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
TTC CTCGAATGCCACCCC18 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iv) ANTI-SENSE: YES 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GTTCTCGAGCGCAGGTACTA20