Protein crystallography makes use of X-ray diffraction to understand the molecular structure of proteins and is responsible for the majority of structural knowledge of biology at the molecular level. However, most of this knowledge has been established – and is limited – by observing static structures of protein molecules. Now, the National Institutes of Health (NIH) has issued a three-year, $936,000 grant to Associate Professor Sarah Perry of the UMass Chemical Engineering (ChE) Department to develop novel microfluidic devices that promise to take this iconic method to the next level and enable visualizing dynamic protein motions using crystallography experiments. 

Perry’s groundbreaking X-ray crystallography technology is aimed at directly probing structural dynamics and evolving the field of structural biology as a whole.

“We are interested in understanding how proteins as molecular machines move,” explains Perry. “In our everyday lives we are used to being able to look at a video of how different things work, from machines that weave fabric to those that create plastic bags. The difference here is that the machine we want to observe is atomic in nature. Therefore, instead of using the normal light that we see with, we have to use X-rays, and instead of being able to look at just a single molecular machine, we have to use crystals that present an ordered array of these molecules so that we can get enough signal.” 

X-ray crystallography is a tool that has long been used to determine the atomic structure of different molecules. In fact, the method has been used to reveal the structure and function of many molecules, including salts, minerals, vitamins, drugs, proteins, and nucleic acids like DNA and RNA. Indeed, the 1962 Nobel Prize in Chemistry was awarded to Max Perutz and John Kendrew for the use of X-ray crystallography to determine the structure of proteins. 

In 1964, Dorothy Crowfoot Hodgkin was the third woman to ever have been awarded the Nobel Prize in Chemistry for her work in using X-rays to determine the structure of vitamin B12, and, in 2009, Ada Yonath became only the fourth woman to win the award for her work on understanding the structure and function of the ribosome – the molecular motor responsible for the synthesis of proteins. 

The Nobel Prize has been given to a number of other scientists in recent years for their work on ever more complex protein systems – studies which have allowed for the design of efficient drugs with fewer side effects. 

An article by Wang-Shick Ryu in ScienceDirect explains that “The underlying principle [of X-ray crystallography] is that the crystalline atoms cause a beam of X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a 3D picture of the density of electrons within the crystal. From this electron-density image, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder, and various other information.”

Now, the NIH is funding Perry to help enable the next generation of structural biology experiments. As Perry explains, “Next-generation structural-biology experiments look to move beyond static observations of structure to a dynamic, time-resolved understanding of function. Think of the difference in appreciation that you have looking at static photographs taken during a sporting event vs. being able to watch video highlights of the game. It is the same with understanding protein structure.” 

These next-level experiments are enabled by a method known as time-resolved serial crystallography, which requires the use of cutting-edge, X-ray, free-electron lasers or microfocus synchrotron beamlines. However, there are two big challenges that Perry is working to overcome. The first of these is the need to individually expose thousands, if not tens of thousands, of microscopic protein crystals to the X-ray beam. 

Perry’s first aim, and the basis for her entire NIH project, will be to develop devices that facilitate the study of large numbers of protein crystals while maintaining their biological activity. This initiative involves the development of innovative devices designed to mimic the native physiological environments in which proteins thrive and perform their essential biological functions. Notably, these devices will cater to specific conditions, such as providing a stable, oxygen-free environment for anaerobic conditions. 

Additionally, Perry aims to create devices capable of stabilizing proteins under elevated temperatures (60-70°C, or 140-160°F), simulating the conditions found in hot springs. This approach will enable the analysis of proteins that flourish in anaerobic or high-temperature environments, thereby enhancing our understanding of their structural and functional characteristics.

“The second big challenge,” according to Perry, “is that, in order to study protein dynamics, you need a way of triggering all of the protein molecules in the crystal to undergo their functional motions at the same time. The best analogy that I have relates to looking at the crowd in a stadium. If everyone is doing their own thing it just looks like a mass of people. However, if somebody gets folks organized to do the ‘wave’ then you can easily see that ‘signal.’ That organization for the wave is the trigger.”

To achieve this kind of triggering, Perry is looking to develop devices that not only protect crystals and deliver them to the X-ray beam, but also enable triggering in response to a variety of different external factors, including light, chemical, and voltage triggers. 

“Many of the time-resolved studies that have been done to date have focused on proteins that respond to light,” Perry explains. “However, these are few and far between. This is where more advanced strategies like chemical triggering or voltage triggering come in. For example, if we can watch how a protein responds when particular drug molecules binds, it could be really impactful in terms of designing the next generation of therapeutics so they are more effective and have fewer side effects. 

Perry's research team is exceptionally well-suited for this investigation, seamlessly integrating expertise in microfluidics, X-rays, and structural biology. With a proven track record, the team has consistently demonstrated its proficiency in developing cutting-edge and enabling technologies. Furthermore, they are partnering with leading scientists at a number of different X-ray sources and structural biologists from around the world to test and validate their devices. 

Perry research group at Stanford 2
Dr. Sarthak Saha, a postdoctoral researcher in the Perry Research Group, and Dr. Ricardo Padua, a collaborator from Brandeis University, conducted experiments at the SSRL synchrotron source at Stanford University.

This history of collaborative innovation reflects the team's commitment to addressing prevailing challenges within the structural biology community, establishing them as leaders in advancing the field.

As Perry concludes, “The long-term goal of this project is to democratize studies of protein structural dynamics. The impact of this work will be improvements in the ability to perform time-resolved studies of protein structural dynamics that will immediately enhance the research capabilities of the structural biology community writ large.” (February 2024)

Article posted in Research