The tools of biochemistry have finally caught up with
lactose repressor protein. Biologists from Rice
University in Houston
and the University of Florence in Italy this week published new
results about "lac repressor," which was the first known genetic
regulatory protein when discovered in 1966.
Using cutting-edge techniques, the scientists tied together
two segments within individual molecules of lactose repressor protein. They
then measured the ability of these tethered molecules to form DNA loops to
determine how flexibility within the protein influences the extent to which
these loops can form. The results appear online this week in the Proceedings of
the National Academy of Science.
"It's become increasingly clear that many proteins are
highly flexible and able to form different types of structures when they
interact with something else, often another protein or DNA," said study
co-author Kathleen Matthews, Rice's Stewart Memorial Professor of Biochemistry
and Cell Biology, who began studying lactose repressor protein in 1970.
"That's true for lactose repressor in binding to DNA, making it a good
candidate to learn more about the process of DNA looping because it's a
relatively simple and well-studied protein."
With proteins, it is impossible to separate form from
function; they do what they do because of their shape. That said, it is also
unusual for scientists to get a clear picture of what a protein looks like in
its native environment. For example, the general structure of lactose repressor
has been known for some time, but questions have remained about how it flexes
and moves inside a living cell.
Lactose repressor is a V-shaped bacterial protein that has
two arms connected by a central hinge. Each arm has a sticky tip that's
designed to grab hold of DNA. When each arm "sticks" to a different
site within a single DNA molecule, a loop forms, creating a "pinched-off"
section of DNA. The combination of protein binding and the loop prevent the
machinery that encodes proteins from copying the DNA, so in essence, lactose
repressor "turns off" the nearby genes.
Lactose repressor draws its name from the genes that it
blocks -- genes that encode enzymes used to transport and metabolize lactose in
bacteria. If a bacterial cell happens to be where lactose is plentiful, lactose
repressor binds to a derivative of lactose that prevents high-affinity binding
of repressor to the DNA. The cell is then able to manufacture the enzymes
needed to convert the lactose into food. If no lactose is present, the protein
clamps onto the DNA and inactivates the process of copying the lactose genes so
the cell doesn't waste energy making the enzyme.
In 2007, the University
of Florence's Francesco
Vanzi visited Matthews' lab to learn new techniques for purifying and assaying
samples of lactose repressor. The protein has a limited shelf life, and Vanzi,
who was preparing to do single-molecule studies on the protein, needed to find
out how to make it on-site in his lab.
"While he was here, we talked about various ways to fix
the two arms of the protein with cross-linkers," Matthews said. The idea
was to bind the arms together with chemical manacles that would limit the
movement around the hinge of the "V." Vanzi, Matthews and Rice
postdoctoral fellow Hongli Zhan wound up using three sets of manacles, or
tethers, including longer and shorter chemical tethers; they also used some
reversible tethers to allow return to the protein's original state. The
researchers chose two different binding sites, one that provided some degree of
flexibility in opening the structure and one that kept the arms bound in the
more-closed "V" position characteristic of the structure determined
for the protein in crystals.
The team found that the more they restricted the flexibility
of the arms, the less likely the protein was to create DNA loops by binding at
two sites.
DNA looping was measured directly by employing
single-molecule techniques in the biophysics laboratory at the European
Laboratory for Nonlinear Spectroscopy (LENS). The LENS team was led by
Francesco Pavone. Lab postdoctoral researcher Danielis Rutkauskas adapted the
methods to optimally measure the looping behavior of lactose repressor with
different degrees of structural constraint.
"Our findings are important, but there is clearly
more work to be done," Vanzi said. "We've found that limiting
flexibility indeed limits protein function in this case. Now we need to apply
our methods more broadly to see if this applies in other cases as well. We are
also very excited about the dawn of single-molecule biophysical methods being
applied inside living cells. Interdisciplinary approaches at the edge between
physics and biochemistry are proving very powerful, and the months I spent at
Rice were a blast."
The research was supported by the European Union, the
Italian Space Agency Project MoMa, the National Institutes of Health and the
Welch Foundation.
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