Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Vibrations key to efficiency of green fluorescent protein

12.11.2009
Charting femtosecond energy flow could aid redesign of molecules to improve light capture

University of California, Berkeley, chemists have discovered the secret to the success of a jellyfish protein whose green glow has made it the darling of biologists and the subject of the 2008 Nobel Prize in Physiology or Medicine.

The researchers' study of green fluorescent protein (GFP) and the structural changes it undergoes when it fluoresces is the cover story of the Nov. 12 issue of the journal Nature.

GFP has replaced many dyes in biological studies because it is non-toxic and, when attached to a gene and inserted into an organism, serves as a bright, glowing confirmation that the gene has hit its target. Obtained originally from a bioluminescent Pacific Ocean jellyfish, the protein has been mutated and engineered to absorb and emit various colors.

The UC Berkeley chemists used extremely short laser pulses – 20 millionths of a nanosecond, or 20 femtoseconds – to take snapshots of GFP to determine the structural changes it undergoes when it fluoresces. Only a rapid, strobe-like laser can freeze atoms vibrating 100,000 times every nanosecond, or a hundred trillion times a second.

Their study not only sheds light on how GFP works, but also proves the value of new, ultrafast, pulsed laser techniques, specifically a method called femtosecond stimulated Raman spectroscopy, that take snapshots fast enough to freeze vibrating molecules and thereby distinguish the rapid steps involved in chemical and atomic reactions.

One of the researchers' goals is to understand the processes of absorption and emission of light in such detail that the light-absorbing molecule can be redesigned to more efficiently capture sunlight in photovoltaics, or solar cells.

"If you want to understand how a reaction occurs, you need to look at it as the chemical bonds change structure, which occurs over tens of femtoseconds," said Richard Mathies, UC Berkeley professor of chemistry and dean of the College of Chemistry. "With a femtosecond laser and Raman spectroscopy, we can see all the steps in the proton transfer reaction in the excited state of GFP."

The transfer of a positively-charged hydrogen atom – a bare proton – along a reaction chain in GFP generates a green flash of light. The laser snapshots show that when the light absorber, or chromophore, nestled in the middle of the protein barrel absorbs an incoming photon of blue light, it starts vibrating, and the electrons start sloshing around the chromophore until it is aligned just right for the proton to hop via a water molecule to a nearby amino acid in the protein. From there, it continues down the reaction chain, creating a state with a negatively charged chromophore that emits green light.

"A lot of people have studied green fluorescent protein for many years and found out that proton transfer in the excited state emits a very efficient flash – for every 100 blue photons going in, 80 green ones come out," said first author Chong Fang, a UC Berkeley postdoctoral fellow in the Department of Chemistry. "This experiment shows why it is so efficient with vivid atomistic details."

Previous studies had shown that after the chromophore absorbs blue light, it undergoes proton transfer, and green light is emitted. In the current study, Mathies, Fang and their colleagues could actually resolve the early stage of this proton transfer reaction, taking snapshots of the vibrational wagging of the chromophore skeleton in sync with the electron cloud in the chromophore sloshing back and forth. However, the wagging oscillation might have stopped after a few picoseconds, when the chromophore and its vicinity are aligned just right for the proton to hop off down the reaction chain, and the whole protein shines bright green – which it does in its own good time, in about 3 nanoseconds.

"We don't need the wagging oscillation to persist throughout the proton transfer process, we only need it to position the chromophore rings correctly relative to the rest of the reaction chain, and that initiates the processes leading to a flash of green light," Fang said.

In the past five years, Mathies and his colleagues have used femtosecond stimulated Raman spectroscopy to investigate similar atomic motions in large, light-absorbing molecules including rhodopsin, the visual pigment in the eye; bacteriorhodopsin, the light-capturing pigment in photosynthetic bacteria; and phytochrome, a light-sensing pigment found in plants and bacteria. This technique probes a range of vibrations – tens of femtoseconds to 1 picosecond (one-trillionth of a second) – that is important in chemical reactions, but until now largely inaccessible.

"This is something I've wanted to do for 40 years, ever since I came to Berkeley in 1976, but I didn't have the ability," Mathies said. "I had to develop the tools to get to tackle this challenging problem."

Femtosecond stimulated Raman spectroscopy on GFP involves hitting the protein molecule with an approximately 80 femtosecond pulse of ultraviolet light, which excites many vibrational modes in the molecule, and then a one-two punch of picosecond red and femtosecond white light to stimulate Raman emission. The spectrum of emitted signals tells researchers the vibrational modes of various parts of the molecule. If the molecule is in the middle of a reaction, the emitted light at different time delays tells the researcher the various steps the molecule goes through during the reaction.

"Now, we can get very, very high resolution structure down to 10-25 femtoseconds," Mathies said.

Mathies compares proton or electron transfer – the key event in absorption or emission of light – to a worker's trip from office to home, which can involve any number of routes. But previous techniques with mere picosecond resolution provided only a blurry picture, and often just the start and end points. With ultrafast femtosecond spectroscopy, he said, "you can see all the steps along the way, whether the worker goes down the stairs or out the window."

Coauthors of the Nature paper include UC Berkeley graduate students Renee R. Frontiera and Rosalie Tran. The work was supported by the Mathies Royalty Fund.

Robert Sanders | EurekAlert!
Further information:
http://www.berkeley.edu

More articles from Life Sciences:

nachricht The balancing act: An enzyme that links endocytosis to membrane recycling
07.12.2016 | National Centre for Biological Sciences

nachricht Transforming plant cells from generalists to specialists
07.12.2016 | Duke University

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Significantly more productivity in USP lasers

In recent years, lasers with ultrashort pulses (USP) down to the femtosecond range have become established on an industrial scale. They could advance some applications with the much-lauded “cold ablation” – if that meant they would then achieve more throughput. A new generation of process engineering that will address this issue in particular will be discussed at the “4th UKP Workshop – Ultrafast Laser Technology” in April 2017.

Even back in the 1990s, scientists were comparing materials processing with nanosecond, picosecond and femtosesecond pulses. The result was surprising:...

Im Focus: Shape matters when light meets atom

Mapping the interaction of a single atom with a single photon may inform design of quantum devices

Have you ever wondered how you see the world? Vision is about photons of light, which are packets of energy, interacting with the atoms or molecules in what...

Im Focus: Novel silicon etching technique crafts 3-D gradient refractive index micro-optics

A multi-institutional research collaboration has created a novel approach for fabricating three-dimensional micro-optics through the shape-defined formation of porous silicon (PSi), with broad impacts in integrated optoelectronics, imaging, and photovoltaics.

Working with colleagues at Stanford and The Dow Chemical Company, researchers at the University of Illinois at Urbana-Champaign fabricated 3-D birefringent...

Im Focus: Quantum Particles Form Droplets

In experiments with magnetic atoms conducted at extremely low temperatures, scientists have demonstrated a unique phase of matter: The atoms form a new type of quantum liquid or quantum droplet state. These so called quantum droplets may preserve their form in absence of external confinement because of quantum effects. The joint team of experimental physicists from Innsbruck and theoretical physicists from Hannover report on their findings in the journal Physical Review X.

“Our Quantum droplets are in the gas phase but they still drop like a rock,” explains experimental physicist Francesca Ferlaino when talking about the...

Im Focus: MADMAX: Max Planck Institute for Physics takes up axion research

The Max Planck Institute for Physics (MPP) is opening up a new research field. A workshop from November 21 - 22, 2016 will mark the start of activities for an innovative axion experiment. Axions are still only purely hypothetical particles. Their detection could solve two fundamental problems in particle physics: What dark matter consists of and why it has not yet been possible to directly observe a CP violation for the strong interaction.

The “MADMAX” project is the MPP’s commitment to axion research. Axions are so far only a theoretical prediction and are difficult to detect: on the one hand,...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

ICTM Conference 2017: Production technology for turbomachine manufacturing of the future

16.11.2016 | Event News

Innovation Day Laser Technology – Laser Additive Manufacturing

01.11.2016 | Event News

#IC2S2: When Social Science meets Computer Science - GESIS will host the IC2S2 conference 2017

14.10.2016 | Event News

 
Latest News

NTU scientists build new ultrasound device using 3-D printing technology

07.12.2016 | Health and Medicine

The balancing act: An enzyme that links endocytosis to membrane recycling

07.12.2016 | Life Sciences

How to turn white fat brown

07.12.2016 | Health and Medicine

VideoLinks
B2B-VideoLinks
More VideoLinks >>>