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Third, screens are being developed that look at the many potentially active sites on each of the
genome fragments. These screens often evaluate selected catalytic activity of the particular sites. As
we look at the large proteins and DNA fragments we need to be aware that the presence or absence
of small possibly important molecules is critical to the site activity. Again, knowing the precise
identity of the site in question is critical. We also need to be aware, that the particular site testing
may be the wrong test and may not unlock the true site function or capabilities.
Fourth, simple language. Because of the need for the involvement of diverse groups, common
language problems will develop. This is true even between seemingly like medically related spe-

cialities. Thus, it is important that we define our terms as we work with other colleagues in related
fi elds.
Fifth, we need to recognize important driving or overriding factors. Structure is one of these.
It is being found that structural similarities may be employed as one (notice only one) factor in
determining site activity. Thus, it is important that precise three-dimensional site geometry is
know including surrounding geometries. This is time and instrument intensive and short cuts are
being developed, but as always caution must be exercised. Creation of shared structural data banks
is occurring. Computer modeling efforts are particularly useful in helping solve such structural
problems.
Sixth, if the vast array of sites and site-important molecules is not enough, protein–protein inter-
actions are part of most cellular processes, including carbohydrate, lipid, protein, and nucleic acid
metabolism, signal transduction, cellular architecture, and cell-cycle regulation. In fact, many of
the major diseases are believed to involve a breakdown in such protein–protein interactions. These
include some cancer, viral infections, and autoimmune disorders.
Techniques to discover the identity of such protein–protein interactions are evolving. One

approach involves protein affinity chromatography. Here, the purified protein of interest is immo-

bilized on a solid polymer support and proteins that associate with them are identified by electro-

phoresis and MALDI. There exists a wide number of modifications to the affi nity chromatography

approach. For instance, a number of proteins can be fixed to the support in such a manner to look at
target molecule interactions as well as nucleic acid–protein interactions, and so on. You can also run
through a variety of possible binders and select the ones that bind most strongly for further study.

Thus, affinity chromatography is a powerful and versatile tool in this search.
Finally, a large sea of information is becoming available to us. How do we handle it? What sense
does it make? Again, we will turn to two powerful tools, the computer and ourselves.
10.14 PROTEIN SITE ACTIVITY IDENTIFICATION
Scientists are developing a number of tools to look at the specific interactions that occur within

proteins. As noted above, some deal with the interaction between proteins and genes while others
are more general. Section 10.5 dealt with one such approach.

In general, protein target identification often employs genetic techniques such as expression clon-

ing, expression profiling, screening of yeast mutations, and yeast three-hybrid assays. None of these
techniques works for every situation.
Another approach in identifying the protein targets employs multicellular organisms. This
approach is more aimed at identifying the targets of small molecules. The notion of using genetics
to identify small-molecule targets is not new but work with worms and zebrafish embryos is new

and represents the first multicellular approaches. In the worm approach, tens of thousands of genet-

ically modified worms are exposed to the test molecules. The worms are watched for changes in
their shape. In this genetic suppressor screening, the genes of the affected and unaffected worms are
compared identifying the particular gene, and hopefully site on the gene, that is interacted with by
the target molecule. By knowledge of the activity of the particular affected site, the potential for that
molecule to be active in treating an illness associated with that site is obtained. As a test, the worms
are treated with a drug that is specific for that illness and again, the site of activity identifi ed.

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