Imaging technologies give researchers an atomic-level view

As technologies have advanced over time, our level of understanding about the biology of humans and other species has progressed through our ability to study tissue, then cells and now biology at an atomic or molecular level. At St. Jude, new ideas and technologies are embraced and used to pave the way for future scientific discoveries. 

Nucleic acids and proteins are important biologic components that play essential roles in the form and function of our bodies. Today, numerous techniques are available to scientists to unravel the atomic details of these biomolecules to understand these potential drug targets better and further enhance the drug discovery pipeline. Some of the most popular methods used are cryo-electron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy and fluorescence resonance energy transfer (FRET), among others. 

Leveraging magnetic properties to “see” the very small

Everything in the universe has a magnetic pull, from colossal planets to tiny subatomic particles such as protons and electrons. Except for hydrogen, which only has a single proton and electron, atoms in biomolecules are mostly magnetically inert or non-reactive. When the inert atoms within these biomolecules are swapped with magnetically reactive counterparts, called isotopes, they become responsive to a magnetic field. Doing so allows scientists to harness the immense power of magnetic fields to explore the intricate details of the structure and dynamics of biomolecules.

Exploiting the magnetic features of atomic cores, or nuclei, to obtain structures of large molecules is the fundamental basis of NMR. The technology is non-invasive and non-destructive, making it ideal for biological research. With the rapid progress of technology, the invention of new NMR methodologies facilitated studies of increasingly complex biomolecules and assemblies. St. Jude is home to what was, at the time of its installation in 2019, the world’s largest NMR.

Using this powerful instrument, high-resolution NMR has enabled scientists led by Babis Kalodimos, PhD, St. Jude Department of Structural Biology chair, to capture invisible states of Abl kinase, a protein dysregulated in leukemia and other cancers. Kinases are enzymes responsible for kickstarting reactions within cells. The elaborate structural insights of this work may lead to the development of novel therapeutics to treat childhood cancer. 

Protein kinases are responsible for a process called phosphorylation; the master on/off switch of most signaling pathways. The Kalodimos lab has discovered that the transition of Abl kinases between active and inactive structural states does not follow one set path, but rather can occupy two distinct inactive states, one physiologically relevant, and one ‘invisible’ state with no apparent biological relevance. 

Published in Science, the work explained how mutations to the kinase can push it towards a conformation for which administered drugs, such as the cancer drug imatinib, cannot bind, leading to drug resistance. 

Trapping the Abl kinase in its inactive conformation is challenging with most standard tools available to structural biologists because it is primarily found in its active conformation. However, the use of NMR has made it feasible for St. Jude scientists to observe the protein in its ‘invisible’ conformations, which provides a new alternative methodology to understand disease mutants and develop novel therapeutics specific to each individual kinase.

Cryo-EM helps shed light on form and function

Cryo-EM is a rapidly emerging imaging technique in which large biomolecules are quickly frozen within thin, glassy ice. Images of the frozen biomolecules are captured from multiple projections across different angles. Cryo-EM is advantageous over other imaging techniques because it captures complex biomolecules such as membrane proteins and ion channels across different conformational states.

The introduction of cryo-EM has revolutionized structural biology by providing structural insights into numerous attractive yet challenging biological targets. The technology has provided a rapid revolution in the rate and scope at which scientists can study the molecular mechanisms of disease and is already aiding in the development of better drugs. 

St. Jude has a world-class Cryo-EM center with highly qualified staff scientists who partner with researchers in studying biomolecular complexes. Recently, Chia-Hsueh Lee, PhD, St. Jude Department of Structural Biology, captured images of complex transporters and ion channels involved in neuronal communication. Published in Nature, their team solved the structure of vesicular monoamine transporter 2 (VMAT2) at multiple different conformational states, which enabled them to decipher its function and interaction with inhibitors related to alternative conformations. Their findings nicely illustrate that different inhibitors can bind alternative conformations of these neuronal transporters.

Lee’s team also resolved a cryo-EM structure of Spns2, a transporter for sphingosine-1-phosphate (S1P), which is involved in regulating immunity and blood vessel formation.  Dysregulation of this protein is involved in chemoresistance and can lead to cancer progression and metastasis. Reported in Cell, their six cryo-EM structures of Spns2 include two intermediate states, which provide an overview of the transportation events occurring through this transporter protein.

Single-molecule resolution enables research breakthroughs

Components of biological systems are always in constant motion. Proteins, lipids and nucleic acids all move to exert their action. Sometimes, this motion happens at a speed beyond our imagination — within a nano- or pico-second. Trapping the motion of a single biomolecule and its interaction with its counterparts is challenging, and traditional imaging techniques often fall short. Focusing attention on individual molecules rather than as an aggregate can help researchers view this motion, capturing the conformational changes in action. 

Researchers at St. Jude can utilize single-molecule imaging using different fluorescence techniques, such as FRET, which allows researchers to examine the dynamic propensity of biomolecules under different environmental conditions. Using this technique, Scott Blanchard, PhD, St. Jude Department of Structural Biology, studied the structure of early translocation events occurring in bacterial ribosomes during peptide synthesis. This helped the researchers trap bacterial ribosome complexes in multiple conformational states to better understand what happens during protein synthesis. 

Published in Nature, the work benefitted from the design of new fluorophores, fluorescent chemicals used to observe biomolecules, and the development of new microscopic instrumentation to explore structural details for this protein synthesis machinery with improved resolution and better signal-to-noise ratio. Combining this FRET imaging technique with mainstay techniques such as cryo-EM and molecular dynamics studies enabled the researchers to unravel the mechanism behind the early stages of protein synthesis.

Obtaining an atomic-level view

Creating the therapies of tomorrow requires scientists today to probe the intricacies of human biology on an atomic level. At St. Jude, researchers are deploying techniques such as cryo-EM, NMR and FRET to do just that: increase our basic understanding of the structure and function of biologic components. This work lays the foundation upon which future therapeutics will be conceived, designed and, hopefully, brought forward to benefit patients. It all starts with a single molecule.