Hydrogen bonds are ubiquitous but nowhere more important than in the structure of DNA and RNA, where they join the “stair-step” base pairs across the double strand. It has long been thought that protons transfer in molecules only by means of such hydrogen bonds. Image: Lawrence Berkeley National Laboratory |
When a proton—the bare nucleus of a hydrogen atom—transfers
from one molecule to another, or moves within a molecule, the result is a
hydrogen bond, in which the proton and another atom like nitrogen or oxygen
share electrons. Conventional wisdom has it that proton transfers can only
happen using hydrogen bonds as conduits, “proton wires” of hydrogen-bonded
networks that can connect and reconnect to alter molecular properties.
Hydrogen bonds are found everywhere in chemistry
and biology and are critical in DNA and RNA, where they bond the base pairs
that encode genes and map protein structures. Recently a team of researchers
using the Advanced Light Source (ALS) at the United States Department of
Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) discovered to
their surprise that in special cases protons can find ways to transfer even
when hydrogen bonds are blocked. The team’s results appear in Nature
Chemistry.
Stacking the
odd molecules
A group led by Musahid Ahmed, a senior scientists in Berkeley Lab’s
Chemical Sciences Division (CSD), has long collaborated with a theoretical
research group at the University of Southern California (USC) headed by Anna
Krylov. In recent work to understand how bases are bonded in staircase-like
molecules like DNA and RNA, Krylov’s group made computer models of paired,
ring-shaped uracil molecules, and investigated what might happen to these
doubled forms (dimers) when they were subjected to ionization—the removal of
one or more electrons with resulting net positive charge.
Uracil is one of the four nucleobases of RNA, whose
structure is similar to DNA except that, while both use the bases adenine,
cytosine, and guanine, in DNA the fourth base is thymine and in RNA it’s
uracil. The USC group used a uracil dimer labeled 1,3-dimethyluracil—”a strange
creature that doesn’t necessarily exist in nature,” says CSD’s Amir Golan, who
led the Berkeley Lab team at the ALS. The purpose of this strange creature,
Golan says, is to block hydrogen bonding of the two identical monomers of the
uracil dimer by attaching a methyl group to each, “because methyl groups are
poison to hydrogen bonds.”
The uracils could still bond in the vertical
direction by means of pi bonds, which are perpendicular to the usual plane of
bonding among the flat rings of uracil and other nucleobases. “Pi stacking” is
important in the configuration of DNA and RNA, in protein folding, and in other
chemical structures as well, and pi stacking was what interested the USC
researchers. They brought their theoretical calculations to Berkeley Lab for
experimental testing at the ALS’s Chemical Dynamics beamline 9.0.2.
To examine how the molecules were bonded, Golan and
his colleagues first created a gaseous molecular beam of real methylated uracil
monomers and dimers, then ionized them with a beam of energetic ultraviolet
light from the ALS synchrotron. The resulting species were weighed in a mass
spectrometer to see how the uracil had responded to the extra boost of energy.
“Uracils could be joined by hydrogen bonds or by pi
bonds, but these uracils had been methylated to block hydrogen bonds. So what
we expected to see when we ionized them was that if they were bonded,
they would have to be stacked on top of each other,” Golan says. Instead of
holding together by pi bonds, however, when ionized some uracil dimers had
fallen apart into monomers that carried an extra proton.
Where the
protons come from
“What we did not expect to see was proton transfer,” Golan says. “Surprising as
this was, we needed to find where the protons were coming from. The methyl
groups consist of a single carbon atom and three hydrogen atoms, but methylated
uracil has other hydrogens too. Still, the methyl groups were the natural
suspects.”
To test this hypothesis, the researchers invited
colleagues from Berkeley Lab’s Molecular Foundry to join the collaboration.
They created methyl groups in which the hydrogen atoms—which like most hydrogen
had single protons as their nuclei—were replaced by deuterium atoms, “heavy
hydrogen” atoms with nuclei consisting of a proton and a neutron of virtually
the same mass.
The molecular beam experiment was repeated at the
ALS, and once again some of the methylated uracil dimers fell apart into
monomers upon ionization. This time, however, the tell-tale monomers were not
simply protonated, they were deuterated.
Says Golan, “By looking at the mass of the
fragments we could see that instead of uracil plus one”—the mass of a single
proton—”they were uracil plus two”—a proton and neutron, or deuteron. “This
proved that indeed the transferred protons came from the methyl groups.”
The experiment showed that proton transfer in this
case followed a very different route from the usual process of hydrogen
bonding. Here the transfer involved not just an attraction between molecular
arrangements that were slightly positively charged and others that were
slightly negatively charged, as in a hydrogen bond. Instead it required
significant rearrangements of the two uracil dimer fragments, to allow protons
of hydrogen atoms in the methyl group on one monomer to move closer to an
oxygen atom in the other. Theoretical calculations of the new pathway were led
by USC’s Krylov and Ksenia Bravaya.
The moral of the story, says Golan, is that methyl
groups do not always kill proton transfer. “Granted, this was a model system—what
we did was ionize the uracil systems in the gas phase instead of in solution,
as would be the case in a living organism,” he says. “Nevertheless, we showed
that proton transfer is possible without hydrogen-bonding networks. Which means
there could be unsuspected pathways for proton transfer in RNA and DNA and
other biological processes—especially those that involve pi-stacking—as well as
in environmental chemistry and in purely chemical processes like catalysis.”
The next step: A range of new experiments to
directly map proton transfer rates and gain structural insight into the
transfer mechanism, with the goal of visualizing these unexpected new pathways
for proton transfer.