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Summer 2017

Model Proteins and How They Help Scientists

Using simple proteins to understand how bacteria save energy

Luke Berry

The 3D structure of the NfnI model protein complex used by scientists at BETCy. The two proteins that form the NfnI complex are colored green and blue. The five cofactors present in the NfnI complex are indicated by arrows. Image courtesy of Luke Berry

Members of the Center for Biological Electron Transfer and Catalysis (BETCy), an Energy Frontier Research Center, are working to identify proteins in bacteria that produce energy through a reaction called flavin-based electron bifurcation (FBEB). FBEB is identified by the use of a cofactor (see sidebar) called flavin, which moves electrons to form compounds that — when broken down — will release energy. These compounds, such as NADH, are needed if a bacterium is to grow and reproduce. Understanding how FBEB works will reveal how bacteria save energy in a rather unusual way.

It starts with the proteins

The BETCy team has identified several proteins that perform FBEB. One group of proteins is hydrogenases. These proteins contain three or more large protein subunits, and each subunit incorporates several cofactors. Another group of proteins, called NfnI, also uses FBEB. But NfnI uses different materials than hydrogenases.

Compared with hydrogenases, the NfnI complex is made of two protein subunits (see figure). Overall, NfnI is smaller than the hydrogenase complex. In total, NfnI contains five cofactors, compared with one hydrogenase subunit containing six. The simplicity of NfnI made it significantly easier to understand the basics of FBEB.

The first steps to amassing energy

Using a variety of techniques, the BETCy team detailed the first step in the FBEB reaction. In NfnI, FBEB first occurs through the transfer of two electrons to a flavin (the F in FBEB) located within the protein group NfnI. This flavin now has two additional electrons that it can transfer to distant locations in NfnI, allowing the cell to access high amounts of energy. It first transfers one electron to a molecule called NAD+, causing the formation of NADH. The next electron has too much energy to give to another NAD+ molecule. Instead, the electron is transferred to a small protein called ferredoxin. Normally, ferredoxin has a high energy barrier that makes it difficult to give an electron to it. However, scientists at BETCy determined that with the energy the central flavin has, NfnI can overcome the energetic barrier that normally prevents ferredoxin from accepting an electron. The energy now stored within the ferredoxin is significantly higher than the energy in NADH, allowing ferredoxin to provide the energy for more complicated reactions.

The value of models

Using NfnI for the initial studies allowed the scientists to first focus on a protein that is much easier to study. Proteins come in a staggering variety of structures and folds, and can interact with a wide array of cofactors. For scientists, the core question surrounding proteins is to understand how their structure determines their function. For instance, how does the structure of a protein determine which cofactors will bind to it? However, the structure-function relationship of proteins is famously complex and can make it difficult to study them.

A common practice when studying a complex protein is to identify a model system. A model protein is much smaller than the more complex systems and requires fewer cofactors while also performing the same function. For instance, NfnI and hydrogenase both use FBEB to make energetic compounds for bacteria; however, NfnI is smaller and uses fewer cofactors than the hydrogenases. This made NfnI an ideal target as a model system to begin the study of FBEB.

Learning from proteins

The BETCy’s ultimate goal is to understand how proteins overcome energy barriers that would otherwise be problematic to surpass. That is why the BETCy team has focused on understanding the FBEB reaction seen in many bacteria. The initial studies using NfnI revealed some of the requirements for FBEB. The techniques used in the study of NfnI are now being applied to larger complexes, such as hydrogenases, to fully understand how FBEB works. These studies provide fundamental new insights into electrocatalysis and may also help scientists manipulate more complex biological systems to more efficiently produce renewable fuels and chemicals.

Acknowledgments

This work was supported as part of the Center for Biological Electron Transfer and Catalysis (BETCy), an Energy Frontier Research Center funded by the Department of Energy (DOE), Office of Science, Basic Energy Sciences. Use of the Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC National Accelerator Laboratory was supported by the DOE, Office of Science, Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the DOE, Office of Science, Office of Biological and Environmental Research and by the National Institutes of Health (NIH), National Institute of General Medical Sciences. The Proteomics, Metabolomics, and Mass Spectrometry facility at Montana State University received support from the Murdock Charitable Trust and NIH of the COBRE program. C.E.L., D.W.M. and P.W.K. were supported by the DOE with the National Renewable Energy Laboratory.

More Information

Lubner CE, DP Jennings, DW Mulder, GJ Schut, OA Zadvornyy, JP Hoben, M Tokmina-Lukaszewska, L Berry, DM Nguyen, GL Lipscomb, B Bothner, AK Jones, A Miller, PW King, MWW Adams and JW Peters. 2017. “Mechanistic Insights into Energy Conservation by Flavin-Based Electron Bifurcation.” Nature Chemical Biology 13:655-659. DOI: 10.1038/nchembio.2348

About the author(s):

  • Luke Berry is a graduate student at Montana State University. He is part of the Center for Biological Electron Transfer and Catalysis (BETCy). He studies the biophysical characteristics of the proteins studied by BETCy by examining the changes in structure and dynamics of a protein using mass spectrometry to gain insight into the protein structure-function relationship.

The Value of Keeping It Simple

Model stands in for complex protein, letting scientists learn the basics before diving into the complexities

Scientists are identifying energy-producing proteins in bacteria that could change how we generate biofuels. Image courtesy of Shutterstock

Bacteria and plants do a better job than some industries of turning fuel into energy. To learn from these natural sources, scientists study microbial proteins that harness the energy of “digesting food.” To study a protein with potential, scientists often begin with a simpler version. At the Center for Biological Electron Transfer and Catalysis (BETCy), an Energy Frontier Research Center, a team discovered a model protein, called NfnI. It stands in for a much more complex microbial protein. The simple model and the more complex protein harness energy via a newly discovered and elegant route. The researchers used the NfnI model to determine the key electron transfer steps. These studies provide fundamental new insights into electrocatalysis and may also help scientists manipulate more complex biological systems to more efficiently produce renewable fuels and chemicals. BETCy is led by Montana State University and funded by the Department of Energy.

More Information

Lubner CE, DP Jennings, DW Mulder, GJ Schut, OA Zadvornyy, JP Hoben, M Tokmina-Lukaszewska, L Berry, DM Nguyen, GL Lipscomb, B Bothner, AK Jones, A Miller, PW King, MWW Adams and JW Peters. 2017. “Mechanistic Insights into Energy Conservation by Flavin-Based Electron Bifurcation.” Nature Chemical Biology 13:655-659. DOI: 10.1038/nchembio.2348

Disclaimer: The opinions in this newsletter are those of the individual authors and do not represent the views or position of the Department of Energy.