Biomedical Engineering Reference
In-Depth Information
30
B2
20
B3
10
0
ITO bR ITO
FIGURE 14.2
The bacteriorhodopsin (BR) photoelec-
tric effect is a voltage that is produced
upon absorption of light by the protein.
It consists of three phases, B1, B2, and B3,
which are coupled to events in the BR
photocycle. See text for details.
10
excite = 532 nm
E excite = 500
20
J cm 2
µ
B1
30
200
0
200
400
600
800
1000
Time (
µ
s)
removing the retinal chromophore from one of the three BR monomers in the trimer (pho-
tochemical bleaching using polarized light—see Li et al., 2002 [63]). To utilize the photo-
electric effect for device applications, the protein must be uniformly oriented to maximize
signal magnitude. Signal magnitude scales linearly with the number of oriented layers, at
least for n < 200 (in-house data).
14.2.5
Bacteriorhodopsin Modification Through Genetic Engineering
As indicated above, BR's unique qualifications have facilitated a number of architectures
for a variety of device applications. Yet after decades of effort, only one commercially
viable device has been developed. Although the wild-type protein has a remarkably high
quantum efficiency for the primary event, it was not designed by nature for either holog-
raphy or memory storage. As a result, device applications based on the wild-type protein
are inherently less efficient. For holographic applications, there is an intrinsic trade-off
when considering the two photochromic approaches; the M state is produced with high
efficiency, but its lifetime can only be prolonged to a few minutes. The Q state is perma-
nent, but can only be produced with very low efficiency. The researcher needs tools by
which the protein can be modified toward specific goals for specific applications. To date,
two approaches have been employed: chemical modification and genetic engineering. The
former technique can be done by either direct or indirect means, including synthetic mod-
ification of the retinal chromophore (e.g., 4-keto BR [65,66]) or affecting the protein's envi-
ronmental milieu (e.g., pH and ionic strength). Unfortunately, neither approach is capable
of altering BR's properties enough to make a difference with respect to most applications.
Furthermore, chemical modification of any variety often is done at the expense of cyclic-
ity. The latter approach, through genetic engineering, has proven successful enough to
revitalize interest in bioelectronic applications of BR.
The advent of new tools in genetic engineering has resulted in new approaches to opti-
mizing and enhancing BR's properties. Site-directed mutagenesis is the classical approach,
and has proven to be a powerful technique for elucidating BR's biophysical properties,
especially the proton pump mechanism. This molecular biology technique offers control
of the protein at the genetic level, by enabling individual replacement of specific amino
acids. The resulting mutant protein can be analyzed for new properties—such mutants
provide tremendous insight into structure function studies. Although site-directed muta-
genesis provided tremendous insight into BR, it became clear that this approach alone
would not ultimately be successful for reengineering the protein; despite considerable
understanding of protein structure and function (especially in the case of BR), even the
most powerful supercomputer still does not have the ability to predict de novo the effects
of most individual amino acid substitutions, especially when they are far removed from
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