For instance, X-ray crystallography, often used to determine protein
structures, requires separating the molecules from their membrane
environment. Because part of cytochrome b5 sticks to the membrane,
such separations involve breaking the molecule at the sticking point,
which happens to be the part that controls its interaction with
cytochrome P450. So while crystallography can offer some information
on structure, it can't provide insights into exactly what goes on
between P450 and b5 during their cozy, membrane-bound encounters,
However, the technique his lab uses - solid
state NMR spectroscopy - can produce detailed
images of proteins in the membrane environment, not only revealing
molecular structure but also showing how a particular protein nestles
into the membrane. Cytochrome b5 presented a challenge even to that
versatile method, though, because the molecule has three parts that
all behave differently: the rigid, sticky portion that buries into the
cell membrane, a highly mobile, water-soluble portion, and a less
mobile "linker" that connects the other two parts.
But by tweaking their technique, the researchers were able to get
high-resolution images of all three portions.
"The challenge was something like having a room full of people and
trying to get good photos of every one of them," said Ramamoorthy, an
associate professor of chemistry and Biophysics. "With one picture,
you probably can't do it. But if you say, 'Everyone over age 50 stand
up,' and you take one picture, and then you ask for another age group
and take another picture, and so on, you have a better chance."
By spinning their samples (or aligning the molecules in the magnetic
field), the researchers were able to differentiate parts of the
molecule based not on age group, as in the photo analogy, but by
mobility. "With the techniques we designed, we were able to observe
the rigid portion separately from the highly mobile and less mobile
portions," Ramamoorthy said.
In the first part of the work, published in the Journal of the
American Chemical Society in May, the researchers described the
membrane-spanning segment of cytochrome b5, revealing for the first
time its helical shape and the way it tilts in relation to the
membrane. In the new work published in BBA Biomembranes, they
determined that once both molecules are bound in the membrane,
cytochrome b5 modulates the motion and the structure of cytochrome
P450. More work is in progress to determine the detailed
high-resolution structures of these two proteins.
Ramamoorthy's team also is studying other membrane-associated proteins,
a group that includes many biologically important molecules.
"These proteins are involved in all major diseases, everywhere in the
body, and are therefore primary targets for pharmaceutical
applications," Ramamoorthy said. "In my opinion, solving the
structures of membrane proteins should be the highest priority for
structural biologists in the coming years."
Ramamoorthy collaborated on the most recent work with Lucy Waskell, a
professor of anesthesiology and a physician at the Department of
Veterans Affairs Medical Center.
A leader in this area of research, Ramamoorthy has organized several
major international symposia on the field at the University of
Michigan, edited a special issue in the journal BBA-Biomembranes,
published a number of papers in leading journals, and brought out a
book on NMR Spectroscopy of Biological Solids. Ramamoorthy said that
this area of research will grow considerably at U-M from implementing
plans to set up a high magnetic field solid-state NMR spectrometer
facility and an NIH-funded program.