Researchers at Stanford University have attached proteins to gold nanoparticles to develop a color-coded test for observing protein folding. When the protein attached to gold is folded in solution, the solution is red; when the protein is unfolded, the solution is blue.
The new color test does not reveal anything new about the chemistry of protein folding, the scientists said, but it presents a novel, cheap, and simple way of observing the process.
“It does quite a gorgeous color change,” said Richard Zare, a professor of chemistry at Stanford who led the study, which appears in the March issue of the Journal of Chemistry and Biology. “That has the smell of being something practical.”
One possible application for a gold-nanoparticle color-test for observing protein folding and unfolding might be to look for the formation of specific antibodies, Zare proposed.
“When you’re exposed to something, your body tends to generate antibodies. You could look for these antibodies by diluting blood and adding gold nanoparticles — something simple like that,” said Zare.
“But this is fundamental research,” he stressed. “I’m not running a company and I’m not looking to leap into making this into a commercial process.”
Zare was using surface plasmon resonance to study a protein called cytochrome c when he got the idea to attach the protein to gold nano particles. Surface plasmon resonance occurs when light is reflected off of thin metal films. A fraction of the light energy that is incident at a sharply defined angle can interact with delocalized electrons in the thin metal film, thus reducing the reflected light intensity. The SPR phenomenon can be exploited to measure biomolecular interactions, such as the interactions between proteins or the interaction between proteins and DNA, in real-time, in a label-free environment.
One day in his lab, Zare had attached Cyt c, a well-studied protein that is important in the process of creating cellular energy, to a flatbed gold surface and was studying the way it denatures and renatures. He decided to investigate what would happen when the protein is attached to gold particles measuring about 19 nanometers in diameter.
“Gold nanonparticles have been used to color things for a long time. In fact they’re used in the red stained glass windows in cathedrals,” said Zare. “So it came naturally to me that they could be used in a color-coded test to observe the conformational changes of a small protein.”
With the help of postodoctoral fellow SoonWoo Chan and graduate student Matthew Hammond, Zare created a liquid solution containing gold nanoparticles saturated with Cyt c.
The initial batch of gold-cytochrome solution had a rosy red hue and a pH value of 10 — about the same as an over-the-counter heartburn medication. But when drops of hydrocholoric acid were added to denature the protein, the solution began to change color, turning purple when the pH reached 5.8, and light blue at pH4, which is close to the acidity of wine.
Lab analysis revealed that when Cyt c began to unfold as a result of the increasing acidity, gold nanoparticles coated with Cyt c began clumping together. This caused the solution to quickly change from red to blue.
Further experiments showed that when the pH was raised back from 4 to 10, the blue solution turned reddish once again — a strong indication that some Cyt c molecules had refolded into their original three-dimensional shape.
Walter Englander, a professor of biochemistry and biophysics at the University of Pennsylvania, said that while Zare’s research did not reveal anything that was previously unknown before about the chemistry of Cyt c, the gold-nanoparticle-Cyt-c system might well find practical applications in the fields of effluent streams, commercial production, or aquatic systems.
“Whether or not [Cyt c’s practical applicatons] are generalizable to other protein structure change systems remains to be seen,” said Englander.
Zare said that he and his research group intend to follow up on their Cyt-c-gold-nanoparticles work by seeing if the color-change test works for other proteins that are attached to gold nanoparticles.
“Attaching proteins to gold nanoparticles is a really simple process, and it’s really inexpensive,” said Zare. “At first it may sound like, ‘Oh, my god, it’s gold so it must cost a fortune,’ but in fact gold plating is relatively cheap and gold nanoparticles are really cheap.”
Before jumping to find applications for the new color test, Aichun Dong, an associate professor in the department of chemistry and biochemistry at the University of Northern Colorado, cautioned that the color change may not in fact have been due to the unfolding and folding of the Cyt c protein.
“There may be alternative interpretations to their data,” Dong told ProteoMonitor.
Dong suggested that when the pH of the Cyt-c-gold nanoparticles solution was lowered from 10 to 4, that could have caused acidic side chain groups of Cyt c to protonate, resulting in charge repulsion between the molecules. This repulsion could have caused the gold nanoparticles coated with Cyt c to aggregate together, regardless of whether or not Cyt c was folded.
Moreover, Dong pointed out that most studies in the literature show Cyt c unfolding between a pH of 2.0 and 3.0, while Zare’s group reported that the color change due to unfolding occurred at around pH 5.7.
“This value is simply too high to be linked to an acid-induced unfolding of Cyt c,” Dong said. “The authors failed to provide sufficient evidence to support their conclusion that there is an acid-induced unfolding of Cyt c.”
Zare said that more study is needed, but he believes that gold nanoparticles will eventually be an inexpensive and useful way for testing Cyt c and other, more complicated proteins.
“To the best of our knowledge, gold nanoparticles have not been previously used to investigate the folding and unfolding of proteins,” he said.