structural changes, conserved across many similar proteins, are also
necessary for the function of various other proteins, such as the
rhodopsin pigment responsible for low-light vision in the human eye.
‘A light-sensing protein from a salt-loving, sulfur-forming microbe has proved key to developing methods essential to advanced drug discovery, understanding human vision and other biomedical applications.’
When triggered by a flash of blue light, PYP undergoes a series of
structural changes that occur within milliseconds, forming many
intermediate structures along the way. Over nearly three decades,
researchers have used techniques such as spectroscopy and
synchrotron-based measurements to identify each intermediate formed
within this tiny time scale.
This light-sensing protein from a salt-loving, sulfur-forming microbe
has proved key to developing methods essential to advanced drug
discovery, understanding human vision and other biomedical applications.
In a review published this week in Structural Dynamics
, by AIP
Publishing, physicist Marius Schmidt of the University of
Wisconsin-Milwaukee presents a history of decades of research of this
microbe and the many new technologies that have enabled these
Being able to spot these reaction intermediates is an important step
in drug development because the same methods can then be applied to
study reactions that are of biomedical importance, Schmidt explained.
"For example, you can see how a cancer-related enzyme that catalyzes
a specific reaction works," he said. "Every new intermediate structure
we identify could be a potential drug target to manipulate that
Unlike these other proteins, however, PYP is small and easy to
produce in large quantities, making it ideal for experimental studies of
protein structure. In 1995, researchers determined the structure of the
PYP protein using crystallography at 1.4 angstrom resolution - that's
about the size of individual atoms. At first, most investigations
employed spectroscopy-based approaches to understand the speedy
light-catalyzed structural changes in PYP.
Early structure-based investigations relied on the synchrotron and
synchrotron-based beamlines, which use a single X-ray pulse, as light
sources to study protein crystals. These experiments produced
diffraction patterns to reveal reaction intermediates formed just 100
picoseconds, less than one-billionth of a second, after the reaction up
to the end of the photocycle. But observing earlier time points was a
The first time series of data revealed intermediates at the 100
nanosecond to 100 millisecond phase of the PYP reaction, but also posed
an analytical challenge. Because intermediates form and decay so
quickly, a sample at any given point carries a mix of intermediate
structures. How could scientists tell them apart?
"Until the early 2000s, how to untangle this mixture was an unsolved
problem," Schmidt said. "But PYP provided the first datasets from which
one can try to do this."
The solution stemmed from a component analysis method known as
singular value decomposition (SVD), which has been applied to
time-resolved crystallography by Schmidt and his colleagues.
"[SVD] can conveniently be used to extract the structure of pure
intermediates from a mixture," Schmidt said. "It has really proved to be
a central method in analyzing these data and the one that has the most
applications so far."
Until 2013, researchers had elucidated the PYP photocycle with a
resolution of 100 picoseconds; the faster time-scale proved elusive. The
advent of the X-ray free electron laser (XFEL), a new kind of light
source, helped resolve this issue. By using the XFEL and SVD analysis,
researchers have now identified earlier processes as well, revealing the
fundamental, crucial steps in the cis- to trans- isomerization of PYP
on the femtosecond and picosecond time scales.
Similar reactions, which are essential for human vision, also occur
when light strikes the retinal pigment rhodopsin. "It would have been
super exciting to see this isomerization happen in real time using
XFEL," Schmidt said. "If we can see it in PYP, we can perhaps visualize
it in rhodopsin as well. PYP has proved to be a role model for other
reactions that feature cis- trans- isomerization."