Monday, 8 December 2014

New research paves the way for nano-movies of biomolecules

An international team, including scientists from Arizona State University, the University of Wisconsin-Milwaukee (UWM), and Germany’s Deutsches Elektronen-Synchrotron (DESY), have caught a light sensitive biomolecule at work using an X-ray laser. Their new study proves that high speed X-ray lasers can capture the fast dynamics of biomolecules in ultra slow-motion, revealing subtle processes with unprecedented clarity. 

                                                                              

                                                                      Ray of Light:
A new generation of ultrafast X-ray lasers is redefining the field of X-ray crystallography, revealing never-before-seen features and dynamic processes.

"This work paves the way for movies from the nano-world with atomic resolution," said Professor Marius Schmidt from UMW, corresponding author of the new paper, which appears in the Dec. 4 issue of the journal Science. 

Study co-author Petra Fromme, professor in ASU’s Department of Chemistry and Biochemistry, echoes the importance of the new study: “This paper is very exciting as it is the first report of time resolved studies with serial femtosecond crystallography that unravels details at atomic resolution,” said Fromme. “This is a huge breakthrough toward the ultimate goal of producing molecular movies that reveal the dynamics of biomolecules with unparalleled speed and precision.” 

A femtosecond is a quadrillionth of a second, an almost unfathomably brief duration. Around 100 femtoseconds are required for a ray of light to traverse the width of a human hair.

The technique of X-ray crystallography allows researchers to probe atomic and molecular structure, by exposing crystals to incident X-rays that diffract from the sample in various directions. Careful measurement of X-ray diffraction angles and intensities allows a three-dimensional portrait of electron densities to be constructed—information used to define atomic structure. 

The technique has been an invaluable tool for investigating the structure and function of a broad range of biologically important molecules, including drugs, vitamins, proteins and nucleic acids like DNA. 
But just as shutter speed determines a camera’s ability to capture action of very short duration, so X-ray lasers must deliver extremely brief pulses of light to capture fine structure and dynamic processes at the atomic level. Some of the phenomena researchers wish to explore take place in mere quadrillionths of a second. A new generation of ultrafast lasers like the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory (used in the current study) are redefining the field of X-ray crystallography. 
The researchers used the photoactive yellow protein (PYP) as a model system. PYP is a receptor for blue light that is part of the photosynthetic machinery in certain bacteria. When it catches a blue photon, it cycles through various intermediate structures as it harvests the energy of the photon, before returning to its initial state. Most steps of this PYP photocycle have been well studied, making it an excellent candidate for validating a new method. 

For their ultrafast snapshots of PYP dynamics, the scientists first produced tiny crystals of PYP molecules, most measuring less than 0.01 millimeters across. The dynamics of these microcrystals were captured in exquisite detail when the world’s most powerful X-ray laser at SLAC was trained on them. Initiation of their photocycle was triggered with a precisely synchronised blue laser pulse.

Thanks to the incredibly short and intense X-ray flashes of the LCLS, the researchers could observe different steps in the PYP photocycle with a resolution of 0.16 nanometers, by taking snapshots of X-ray diffraction patterns. The spectacular time resolution afforded by the technique allows researchers to detect changes in the atomic-scale conformation of PYP molecules as they switch back and forth between light and dark states. 
                                 Part of the ASU team of scientists involved in the work: (from left to right) Christopher Kupitz, Dingjee Wang, Nadia Zatsepin, John Spence, Petra Fromme and Raimund Fromme. Other members of the ASU team (not in the photo) include Bruce Doak, Uwe Weierstall, Ingo Grotjohann, Tzu-Chiao Chao, Mark Hunter and Richard Kirian.
                                                             

The investigation not only reproduced what was already known about the PYP photocycle, thereby validating the new method, it also imaged delicate phenomena in much finer detail. Thanks to the high temporal resolution, the X-ray laser could in principle study steps in the cycle that are shorter than 1 picosecond (a trillionth of a second) - too fast to be captured with previous techniques. The ultrafast snapshots can be assembled into a movie, detailing the dynamics in ultra slow-motion.
“This is far more than a proof of concept for time resolved crystallography. LCLS can use micron size crystals and therefore have an unmatched light initiation efficiency to explore uncharted territory in the dimension of time resolution of molecular reactions,” Raimund Fromme stated, an ASU associate research professor participating in this project.

"This is a real breakthrough," emphasizes co-author professor Henry Chapman from DESY. "Our study is opening the door for time resolved studies of dynamic processes, providing an unprecedented window on subtle transformations at the atomic scale."
John Spence, director of science for the STC at ASU, stresses the importance of studying delicate life processes by means of new tools capable of extreme spatial and temporal resolution:
"When combined with previous work, it is remarkable now to be able to assemble a true molecular movie of the photocycle of this blue light detector in bacteria at atomic resolution, with the intermediate structures appearing and fading in the correct sequence. It is a huge step forward, which will also aid research on artificial photosynthesis,” he says. “It builds on our earlier work at LCLS, and is supported by our NSF Science and Technology Center for the use of X-ray lasers in biology." 
The new research advances the ASU team’s pre-existing investigations, highlighting the first time-resolved serial femstosecond crystallography studies on Photosystem II.  The study on PYP now shows that this method can unravel previously undetected details at the atomic level.
The ASU team involved in this study includes four faculty and their research teams (John Spence, Uwe Weierstall, Petra Fromme and Raimund Fromme) from the Departments of Physics and Chemistry and Biochemistry who are members of the new Center for Applied Structural Discovery at the Biodesign Institute. The ASU team contributed to many aspects of the study, which range from experimental planning to the application of injector technology, growth and biophysical characterization of the PYP microcrystals and data evalution.
The ASU team also includes the graduate students Christopher Kupitz,Chelsie Conrad, Jesse Coe, Shatabdi Roy-Chowdhury, who worked on the growth and biophysical characterization of the PYP crystals at ASU and on site at LCLS, the graduate students Daniel James and  Dingjie Wang, who worked on sample delivery as well as the research scientist Nadia Zatsepin and the graduate student Shibom Basu, who worked on “on the fly” data evaluation.
“Since the sample injector developed at ASU allows for continuous sample replenishment, the X-ray laser always probes fresh, undamaged crystals, allowing us to make molecular movies of irreversible reactions,” says Research Professor Uwe Weierstall. Further, X-ray lasers typically investigate very small crystals that often are much easier to fabricate than larger crystals. In fact, some biomolecules are so hard to crystallise that they can only be investigated with an X-ray laser. 
“This is the highest resolution X-ray laser dataset we’ve worked with – these tiny crystals were of very high quality,” adds research scientist Nadia Zatsepin. “It was very satisfying to see such high resolution electron densities by the second day of our experiment, but to then also see such strong signals from the changes in the structure was even more exciting,” 
The small crystal size is also an advantage when it comes to kick-starting molecular dynamics uniformly across the sample. In larger samples, the initiating optical laser pulse is often quickly absorbed in the sample, which excites only a thin layer and leaves the bulk of the crystal unaffected. 
The PYP microcrystals were perfectly matched to the optical absorption so that the entire crystal was undergoing dynamics, which in turn allows sensitive measurements of the molecular changes by snapshot X-ray diffraction. 
Taken together, X-ray laser investigations can offer previously inaccessible insights into the dynamics of the molecular world, complementing other methods. Using the ultra slow-motion, the scientists next plan to elucidate the fast steps of the PYP photocycle that are too short to be seen with previous methods. 
In the future, ultrafast laser crystallography promises to illuminate a broad range of biomolecules, from light sensitive photoreceptors to other vital proteins.



Written by: Richard Harth
Science Writer: Biodesign Institute
richard.harth@asu.edu