Customizing Proteins—Changing Structure To Change Function
Stephen Fuchs, PhD, joined the Department of Biology in 2012. His interdisciplinary work probes the structure-function relationships of proteins to inform the design of novel biomolecules and explain biological phenomena. His research group includes chemists, biochemists, biologists, and geneticists who approach the study of proteins from many angles.
Fuchs earned his PhD in biochemistry at the University of Wisconsin Madison and worked at the University of North Carolina at Chapel Hill until 2012 as a postdoctoral fellow at the Lineberger Comprehensive Cancer Center and as a fellow at the Parr Center for Ethics. “My basic scientific question is very simple,” says Fuchs. “I’m interested in how protein structure relates to protein function.” Fuchs is currently approaching this question by studying two sources of protein variation that can alter protein structure and function: post-translational modifications (PTMs) of proteins and repetitive protein domains (repeats). PTMs include phosphorylation, methylation, and isomerization, and are known to be key regulators of development, cell division, and disease. Less is known about repeats (repetitive sequences of amino acids), but he believes they play much more important roles in cells than is currently appreciated. Fuchs hypothesizes that cells use a combination of PTMs and variability in repeat number to modulate protein function, thus creating many different, functionally distinct proteins from a single protein-encoding gene.
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Working primarily in the model organism yeast, the Fuchs lab is currently focusing on PTMs of two proteins that are important in DNA transcription: histones and RNA polymerase II. Histone proteins form a core around which DNA is tightly wrapped to pack it into a cell nucleus. Histones have “tails”— long sequences of amino acids that protrude from the core and play important roles in histone structure and function. Histone tails are among the richest sources of PTMs in cells.
Because the PTMs in histone tails can be in very close proximity, they can work together. For example, one region of a histone protein may have two adjacent PTMs that together create a unique binding site for a particular protein. “You can imagine that one protein may recognize two marks [PTMs] and one might recognize only one or the other mark, and this is going to be temporally controlled or spatially controlled to induce a particular cellular response,” says Fuchs. “In many cases one of them is a transcriptional activator, and one of them will be a transcriptional repressor, so you can control whether a gene is on or off based on what marks are present on a given histone at a given point in time.”
RNA polymerase II is the enzyme primarily responsible for mRNA synthesis in eukaryotic cells. “RNA polymerase II has a tail that is made up of a repetitive domain of seven amino acids,” says Fuchs. “The tail serves as a scaffold for the binding of many proteins involved in transcription, and the binding is controlled by the combinations of modifications that exist within the scaffold.” The Fuchs lab has recent data suggesting that although the many repeats of RNA polymerase have the same sequence, they may actually perform discrete functions within the cell. This information could potentially change the way the field explains RNA polymerase interactions during transcription.
Some of the modifications in RNA polymerase II cause structural changes to the protein/DNA complex. Therefore, the Fuchs group is looking at the effects of different structural combinations as well as different combinations of chemical modifications and variations in repeat number. Fuchs decided that the best way to understand how different combinations of all of these modifications affect protein function is to build and test them. “We build them with specific combinations of modifications, array them on glass slides as peptides, and do binding studies,” he says. Using these peptide microarrays, the Fuchs lab can interrogate a large number of peptide variants and ask how neighboring modifications interact with one another to affect protein binding.
Fuchs believes there’s a good chance that repeats are very important in cell surface binding and recognition since proteins expressed on the surface of cells tend to be very repetitive. His research group is creating libraries of peptides containing different combinations of PTMs and variable numbers of repeats for use in experiments on how protein structure relates to protein function. Questions include
- What PTM patterns do particular proteins bind to?
- Can we build proteins that bind specific patterns?
- Can we create modified protein domains to use as diagnostic tools that are more sensitive than currently used antibody-based diagnostics?
In addition to analyzing the functional role of repeats, the researchers in the Fuchs lab would like to understand how variations in repeat number are generated and controlled. They are using the RNA polymerase system to identify the cellular mechanisms that control the dynamic nature of repetitive protein domains. Current work is exploring how proteins change in length under different pressures, offering a potential model for rapid evolution of a protein sequence.
While the group works primarily with yeast, there is potential to make informative inroads in mammals. “The repetitive sequence of RNA polymerase is conserved between yeast and humans, so we can actually test human proteins,” says Fuchs. “One of the particularly interesting target proteins is from influenza virus because it hijacks the cellular polymerase machinery by binding to the RNA polymerase tail, and not much is known about this interaction.” Fuchs is currently collaborating with a group in England on this topic, and he would welcome other collaborations in mammalian systems.
Another current collaboration is with Lenore Cowen’s lab (Computer Science). The two labs recently received a “Tufts Collaborates!” grant to develop computational tools to investigate repetitive protein sequences, a potentially rich area for future collaborations. Fuchs enjoys expansive and interdisciplinary collaborations, and welcomes discussions with interested researchers. His lab members are available to teach collaborators how to use the various tools and techniques employed in his lab at 200 Boston Avenue, including basic peptide synthesis and microarraying.
For more information, please see http://ase.tufts.edu/biology/labs/fuchs.