Watching a protein as it functions

Mar 15, 2013 by Mona Mort
Watching a protein as it functions
The PYP structure in its ground (pG) state: Arrows point to the pCA C2=C3 double bond and the Ca atoms of key residues. Hydrogen bonds are indicated with dashed blue lines. Arg52 is stabilized in its “closed” state via hydrogen bonds to the protein backbone.

(Phys.org) —When it comes to understanding how proteins perform their amazing cellular feats, it is often the case that the more one knows the less one realizes they know. For decades, biochemists and biophysicists have worked to reveal the relationship between protein structural complexity and function, only to discover more complexity. One challenging aspect of protein behavior has been the speed with which they change shape and interact with their neighboring biomolecules. Until recently, researchers have relied on a somewhat static approach, using freeze-trapping to capture protein intermediates at various steps along a biochemical pathway. But exciting breakthroughs now allow us to watch proteins changing in real time.

A research group has developed the necessary infrastructure at the BioCARS 14-ID-B at the U.S. Department of Energy Office of Science's Advanced Photon Source to watch proteins function in real time on the picosecond time scale. Their work brings us many steps closer to knowing how proteins function, or malfunction when leading to disease.

A signaling usually responds to a messenger or trigger, such as heat or light, by changing its shape, which initiates a regulatory response in the cell. Signaling proteins are all-important to the proper functioning of , yet the rapid sequence of events, occurring in picoseconds, had, until now, meant that only an approximate idea of what was actually occurring could be obtained.

Watching a protein as it functions
Front and side views of time-resolved structural changes recorded 100 ps after photoexcitation of PYP. (Lower) Expanded, annotated view of upper insets. The ground-state electron density map is colored magenta, and the 100-ps map is colored green. Where magenta and green overlap, the electron density blends to white. The magenta-to-green color gradient unveils the direction of atomic motion; large-amplitude displacements are indicated with yellow arrows. The stick models correspond to refined structures for the ground state (pG) and the first intermediate (pR0).

The researchers from the National Institutes of Health, the Nara Institute of Science and Technology (Japan), The University of Chicago, and the European Synchrotron Radiation Facility (France) worked to remedy that situation by developing a time-resolved, 150-ps Laue technique that can be utilized to watch structural changes in the photocycle of a protein first discovered in the photosynthetic bacterium Halorhodospira halophila. The protein, called photoactive yellow protein (PYP), is implicated in causing the bacterium to swim away from blue light, which could be genetically harmful, and toward green light, which it needs for photosynthesis.

The team tracked the reversible photocycle of PYP over ten decades of time, from 100 ps to 1 sec. The signaling reaction is triggered by absorption of a photon of light by p-coumaric acid (pCA) chromophore, the non-protein entity that actually absorbs the photon, and which undergoes trans-to-cis isomerization. The bacterium responds to this light signaling within about half a second, indicating how rapidly the biochemical changes that facilitate the response must occur in the cell.

The team identified four major intermediates in the photoisomerization cycle. The first cis intermediate, lasting for only 600 ps, is in an unusual, highly contorted form not previously observed in crystallography experiments and is the precursor to intermediates that had been observed on the time scale of nanoseconds. Additional intermediate steps in the process of structural transition include breaking and making of hydrogen bonds, formation and relaxation of strain, and gated water penetration into the protein's interior. The researchers cross-validated their structural data, utilizing density functional theory calculations to confirm the chemical plausibility of the intermediates.

By tracking structurally the PYP photocycle with near-atomic resolution, the team provided a foundation for understanding the general process of signal transduction in proteins at nearly the lightning speed in which they are actually happening. By extending these techniques to enzymes, it would be possible to perform real-time observations of enzymatic action. Thanks to the team's groundbreaking work, the mechanistic and kinetic complexities with which proteins carry out their sophisticated functions are now much more clearly on view to those who have been eagerly waiting to see.

Explore further: Jumping hurdles in the RNA world

More information: Schotte, F. et al. Watching a signaling protein function in real time via 100-ps time-resolved Laue crystallography, Proc. Natl. Acad. Sci. USA 109(47), 19256 (November 20, 2012). DOI:10.1073/pnas.1210938109

Jung, Y. et al. Nat. Chem. 5, 212 (March 2013). www.nature.com/nchem/journal/v… full/nchem.1565.html

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User comments : 5

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Tausch
not rated yet Mar 15, 2013
All molecular protein structures must have an a priori potential for their eventual shape.
As if every molecular protein structure carries a unique coupling constant - a dress that only has the right shape when worn.
Sorry for the dumb down.
Tausch
1 / 5 (1) Mar 15, 2013
If there is a phase space to every unique coupling constant, then you have a measure for the folding that is independent of mass and energy.
Tausch
1 / 5 (1) Mar 15, 2013
Structures that couple have a least one common phase state independent of mass and energy.
ValeriaT
not rated yet Mar 15, 2013
The four movies accompanying the article will give more illustrative insight into conformal changes of protein.
Tausch
not rated yet Mar 15, 2013
"The team has used a mutant version of the protein to control which of these two pathways is taken. A mutation in PYP known as E46Q weakens the connection between p-coumaric acid and the protein itself and this seems to preclude the process from taking the bicycle pedal route."

Read more at: http://phys.org/n...html#jCp

Yes. If you alter the structure you alter the phase space.
Before you had a phase space that couples to either wave length encountered - blue or green light. Important is the phase space is independent of mass and energy.

I see the phase space of protein structures as the solution to the protein folding problem.

"The researchers cross-validated their structural data, utilizing density functional theory calculations to confirm the chemical plausibility of the intermediates."

Phase space replaces density functional theory. Removing any doubt about the chemical 'plausibility' of the intermediates.

You are kind. Thank you for the links.

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