Research in Brief: The What, Why, and How

a detailed closeup of an internal protein region depicting azide binding to a binding site.

In Research In Brief: The What, Why, and How, we explore new research from the UW–Madison Department of Biochemistry to learn more about the world around us — and inside us. In this edition: The twists and folds of proteins determine their structure and function. But, it can be tricky to determine the biochemical properties of regions hidden within these folds without destroying the proteins themselves. Scientists have identified a molecule that may open opportunities for assays that could unlock secrets held within proteins’ folds.

WHAT you need to know

Picture a television. There’s a screen on the front, some buttons along one side, vents in the back so it doesn’t overheat, and inputs for specific cords on the other side. The connections between all of these — what makes the television work — are nestled within a black box — literally and figuratively. If you open the black box and line up all of the television’s mechanical components, it may not be obvious how they work together to make a functional television. To truly understand how the television works, you need more information about the purpose of each component and how it functions in concert with other components.

Scientists have faced a similar challenge when studying proteins: the external components of a protein’s structure are evident, but it’s difficult to see the ‘inside’ without pulling the protein apart, which results in biomolecular chains and fragments that no longer fit together.

Proteins are comprised of chains of amino acids. Although human proteins are made using only twenty different amino acids, the chains are often hundreds of units long, resulting in seemingly endless possibilities. The order of the amino acids — a protein’s primary structure — is only part of what determines the shape, and the function, of the protein. Interactions among amino acids along the chain result in the twists, folds, and coils that give each protein a distinct tertiary structure which, in turn, determines how it interacts with other molecules. Long regions of hydrophobic, or ‘water-fearing’, amino acids, for example, allow a protein to sit comfortably within a cell or organelle’s lipid-based membrane.

WHY it matters

Within the folds and twists of a protein are small, hidden pockets with their own distinct properties. For example, when a protein is twisted into its three-dimensional structure, the chain of amino acids may fold in such a way that hydrophobic amino acids cluster together to form hydrophobic pockets. These pockets, protected within the protein’s structure and insulated from the environment around the protein, create regions with properties different than the rest of the cell.

Identifying hydrophobic microenvironments has become a key aspect of therapeutic drug development because they provide possible targets for drug binding. Many drugs are water-soluble and hydrophobic regions, which are protected from a cell’s watery environment, can keep drugs from dissolving prematurely. Similarly, hydrophobic regions can be essential binding sites for research techniques and drug delivery pathways that require proteins and molecules to interact with high affinity.

UW–Madison scientists are developing new tools to pinpoint the exact location of hydrophobic microenvironments within a protein’s folds without damaging the protein itself.

HOW our scientists made progress

More than two thirds of the gases that make up the air we breathe is composed of the nonreactive nitrogen molecule, N2. Simply adding one more nitrogen atom to this gas results in the solid, charged compound called azide. Thanks in part to its reactivity, azide is widely used in chemical and biological research. Researchers in the Sussman Lab have now identified a new use for this compound: the small, negatively charged azide molecule is attracted to proteins’ hydrophobic microenvironments and be used to locate and identify these hydrophobic regions without disrupting the structure of the protein.

This image depicts, from left to right: a region in a protein, azide added to the protein’s environment, azide attaching to a binding site within a protein.
This image depicts, from left to right: a region in a protein, azide added to the protein’s environment, azide attaching to a binding site within a protein.

A form of azide called an azido radical establishes a strong covalent bond with the amino acids around hydrophobic regions. Radicalized molecules, such as the azido radical, are highly unstable on their own but have a high affinity for creating strong, stabilizing bonds.

The scientists explored azide’s affinity for binding to hydrophobic microdomains in model proteins as well as dozens of proteins in the model plant organism, Arabidopsis thaliana. Their new research indicates that the radicalized molecule’s ability to bind to proteins is more universal than was previously understood.

These findings will be investigated further to determine whether there are residues or other byproducts of the binding process that might impact how therapeutic drugs interact with proteins, with an eye toward using azide to assist in drug delivery pathways.

Written by Renata Solan.

This edition of Research in Brief: The What, Why, and How, and all images, are based on the following publication:

Minkoff, B., Burch, H., Wolfer, J., and Sussman, M. Radical-Mediated Covalent Azidylation of Hydrophobic Microdomains in Water-Soluble Proteins, ASC Chem. Biol., 2023 Jul 18, 18(8).

This research was funded by awards from the Department of Defense Threat Reduction Agency (HDTRA1-16-1-0049), National Science Foundation (2010789-IOS), and Department of Energy (DE-FG02-8ER13938).