Integral membrane proteins (IMPs), constituting nearly 20–30% of the proteome, carry out diverse functions including cellular signaling, transport, adhesion, and catalysis. Owing to these critical roles and their unique accessibility in cells, they also represent about 60% of drug targets. However, because of technical and time challenges, structures of only approximately 200 human IMPs have been determined. Thus, an unmet need currently exists for new methods to interrogate IMPs, especially in their native environment.

Michael Gross, PhD, professor of chemistry and of immunology and medicine, Weikai Li, PhD, associate professor of biochemistry, and colleagues at Washington University School of Medicine, have developed a technique to footprint IMPs anchored to liposomes.1

Gross reports, “IMPs are difficult to characterize by traditional methods of X-ray crystallography and NMR. While cryo-electron microscopy (Cryo-EM) is making advances in this area, like X-ray crystallography, it only provides a static picture of the protein.”

Fast photochemical oxidation

Gross, Li, and Jie Sun, PhD, a postdoctoral research assistant in the Gross laboratory, adapted a fast photochemical oxidation of proteins (FPOP) platform. FPOP involves the irreversible labeling of proteins by hydroxyl radicals. The radicals are generated following exposure of a solution containing a small amount of hydrogen peroxide to an excimer laser (light pulse-emitting gas laser). The tagged protein is subsequently analyzed by mass spectrometry.

“Footprinting based on mass spectrometry is evolving for soluble proteins,” Gross notes. “One often-used method is hydrogen/deuterium exchange. Reactions with free radicals give good coverage because most amino acids can react, even the aliphatic amino acids that are abundant in membrane proteins. Footprinting provides both comparative structure information and protein dynamics at high spatial resolution.”

Li adds, “Moreover, footprinting allows investigation of structural states of membrane proteins in their cellular environment, which is beyond the reach of current structural methods.”

How does the new method work? Gross explains, “Jie Sun, who adapted our idea of applying FPOP to membrane proteins did all the experimental work. Basically, small nanoparticles (NP, 5 microns) of titanium oxide (TiO2) are attached to the phosphates of certain membrane lipids. Upon excitation with UV light (248 nm from a krypton fluoride excimer laser), electronic excitations occur to produce a swarm of hydroxyl-free radicals at the surface of a membrane mimetic (liposome). Simultaneously, the laser flash promotes a chemical reaction of the lipid membranes to kink the lipids, providing a small opening to admit the radicals to the membrane and membrane proteins. Reactions interrogate for us the parts of the protein that are accessible or exposed to the lipid interface.”

Visual validation

To initially characterize the NanoPOMP (NP-promoted photochemical oxidation of membrane proteins) system, the team first validated the fast footprinting conditions using cryo-EM to visualize the attachment of TiO2-NP to liposomes. To evaluate the performance of the system to characterize IMP, the team assessed bacterial vitamin K epoxide reductase (VKOR) bound to liposomes. They optimized the workflow to separate TiO2 and solution contaminants to enhance HPLC and MS analysis. The results verified the successful footprinting analysis of the entire IMP molecule.

The big question was whether the technique could be applied to human proteins. For this series of studies, Gross, Li, and Sun successfully used NanoPOMP to map human glucose transporter 1 (hGLUT1) before and after binding to two of its small molecule inhibitors, D-maltose and cytochalasin B. By comparing the footprinting of ligand-free and -bound states, the investigators demonstrated the new method could provide structural insight into the ligand-free state that is too flexible to be obtained by crystallography.

An enriched toolbox for the future

As for the future, Gross believes the new method will be another tool in the toolbox of structural biochemists and biophysicists. He predicts, “It will complement cryo-EM and X-ray crystallography, which are considerably more difficult to apply, and it will provide protein dynamics, which are not available with standard structural methods. We are working hard to apply it to other cell membrane mimetics including nanodiscs and picodiscs. Our ‘holy grail’ is to apply it to living cells.”

Beyond that, the team also envisions translating the method to whole organisms. Gross explains, “While our overall goal is to footprint proteins in living cells, ultimately, the approach can be applied to small animals like C. elegans (microscopic worms) and zebrafish, which are used as models of human disease.”


  1. Sun J, Liu, XR, Li, S, He, P, Li, W, Gross, ML. Nanoparticles and photochemistry for native-like transmembrane protein footprinting. Nature Communications. 2021; 12:7270
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