Biochemistry
My
research interests are mainly biochemical and biophysical
but also include areas such as surface chemistry and
simple microfabrication which are used to facilitate
and enhance the biochemical investigations. Though
the focus is different in the various areas, the information
acquired and the techniques used strongly complement
one another.
In a human nucleus there are billions of DNA base
pairs and thousands of proteins, yet particular proteins
(transcription factors) are able to locate and bind
tightly to a sequence of DNA that may be only 10 base
pairs long - an incredible feat. There are many different
DNA-binding proteins which employ many different strategies
for achieving high-affinity, high-specificity binding.
The nucleus, however, has certain characteristics which
influences the binding of all transcription factors
regardless of the recognition strategy which they employ.
These basic characteristics and their influence on
a transcription factor’s ability to find its
DNA target site efficiently form the basis of the problems
in which we are interested. One of the major properties
of the nuclear environment is crowding – there
are a large number of macromolecules in a small volume.
We are interested in understanding the effect this
crowding has on the binding of transcription factors
to DNA and their ability to discriminate between specific
and nonspecific DNA. We are approaching the problem
several ways. We are using standard biochemical techniques
such as electrophoresis and fluorescence spectroscopy
but we are also taking advantage of other technology
such as the ability to control surface chemistry and
the ability to fabricate items at the micrometer scale
(the scale of a nucleus). The combination of even simple
techniques from multiple fields leads to something
that exceeds the some of its parts and allows us to
ask and to answer questions in new and better ways.
The ability to control surface chemistry is important
because as you move to smaller and smaller scales,
such as intracellular volumes or even macromolecular
aggregates, the surface area to volume ratio increases
dramatically and the properties of a molecule at a
surface can differ significantly from its properties
in solution. If you assemble a monolayer of carboxylic
acid groups at a surface, the effective pKa can be
five units higher than in solution. This means that
at a neutral or physiological pH, almost all of the
acid groups are still protonated. Researchers have
measured this pKa shift with various indirect techniques.
I would like to use some very simple self-assembled
monolayer techniques and one-dimensional NMR to directly
probe this phenomenon. In reality, very few surfaces
(proteins, membranes, noncrystaline materials) have
homogenous chemical properties and the real strength
of the technique is the ability to probe surfaces with
mixed hydrophobic, acidic, and basic groups - something
that would be difficult to do with the other techniques
now employed.
A third area of interest is in protein arrays. The
idea here is to use a simple system that will allow
flexible formation of protein arrays quickly and cheaply
so that they can be used to answer specific research
questions (as opposed to large scale predetermined
arrays that a company may sell for screening purposes).
We are working on combining some existing techniques
to achieve this goal and to answer specific questions
about protein-protein interactions. |
Steven
J. Metallo