Membrane proteins, WRAPped

Salt is highly soluble in water.  At its maximum, water can hold approximately 350 grams of salt per liter – ten times as much as is in ocean water.  But have you ever tried to dissolve table salt in oil?  It doesn’t work. 

Water and fat have distinct chemical properties, and molecules that are soluble in water are generally not soluble in fat, and vice versa. Molecules are characterized according to this characteristic – they can be hydrophilic (water-loving) or hydrophobic (water-fearing). 

Most proteins have hydrophilic exteriors, making them soluble in the water-based solutions that make up living organisms. Proteins that occupy cell membranes, however, are different. Cell membranes are composed of lipid – or fat – molecules. Membrane proteins contain both hydrophobic regions where they interact with membrane lipids and hydrophilic regions where they interact with water-based solutions on either side. These proteins are not soluble in water.

“Membrane proteins govern how cells sense and respond to their environment, making them pivotal to human biology and disease,” said Dr. Ljubica Mihaljevic, a postdoc in Dr. David Baker’s lab in the University of Washington Department of Biochemistry. “Despite their importance, membrane proteins are notoriously difficult to work with because their water-repelling surfaces destabilize them outside the lipid membrane.”

Existing methods to isolate membrane proteins are labor-intensive and require the use of detergents that can destabilize the protein and limit downstream protein analyses. A new study from the Baker lab, published in Science last month, introduces de novo designed WRAPs (water-soluble RFdiffused amphipathic proteins) as a better option for solubilizing and studying membrane proteins.

WRAPs are engineered donut-shaped proteins, that, well, wrap around the hydrophobic regions of membrane proteins, shielding them from surrounding water.  WRAPs contain a hydrophilic surface that makes them, along with the protein they skirt, soluble in water. Each WRAP is custom designed for a specific membrane protein.

“WRAPs let us produce a variety of membrane proteins in soluble form, bypassing detergents entirely, all while preserving the target's native structure and functional properties,” commented David Kim, an HHMI research staff who is a leading author on the study.

Creating a WRAPped membrane protein is a multi-step process. Researchers first design the WRAP to match the dimensions of the membrane protein’s hydrophobic region, then adjust it to conform to the protein’s specific shape and surface chemistry. Next, they generate a gene encoding the WRAP and fuse it to the membrane protein gene. Finally, the engineered hybrid gene is expressed in bacteria and the resulting protein is purified to yield the desired soluble WRAPped membrane protein. 

Using this process, the researchers successfully created WRAPped versions of various well-characterized proteins, including E. coli outer membrane proteins, E. coli protease GlpG, and human chemokine receptor CXCR4. The WRAPped proteins were water soluble and stable. They retained the structural and functional characteristics of the native forms, including epitope specificity, ligand binding capacity, and enzymatic activity.

The team then created a more complicated WRAP, for an eight-subunit protein complex called Mycobacterium smegmatis porin A (MspA). In its native role, MspA forms a pore in the cell membrane that facilitates transport of nutrients and other small molecule in and out of the cell. The complex has also been adapted for long read DNA sequencing in biotechnology. The researchers used cryo-electron microscopy to determine a high-resolution structure of the full WRAPped protein complex. The structure was consistent with previously reported native MspA structures, maintaining both large-scale subunit configuration and small-scale individual fold architecture. This success demonstrates that WRAPs can be applied to both simple and composite proteins, and that WRAPped protein structures accurately reflect the protein’s native form.

The researchers also generated WRAPs for three Treponema pallidum outer membrane proteins. These proteins sit within the membrane of the bacterium that causes syphilis and may be ideal targets for drug and vaccine development. No one has yet been able to detail their structures. Now that the proteins are soluble with WRAPs, gathering this essential structural and antigenic information may be possible.

Moving forward, the team hopes to apply the WRAP technology to advance medicine. Pooja Bandawane, a PhD candidate in the Baker lab, indicated, “We are interested in using WRAPped antigens, like the ones we made for Treponema pallidum, to help isolate monoclonal antibodies and support vaccine development against syphilis. More broadly, we want to extend this approach to other important drug targets.”

The applications of this new technology are expansive, including structural and biochemical characterization of membrane proteins, vaccine design, drug discovery, biotechnological advancement. How far can we go with this nifty new tool? 

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Drs. Neil King and David Baker contributed to this research.

The spotlighted research was funded by Coefficient Giving, the Gates Foundation, the National Institutes of Health, Howard Hughes Medical Institute, the Curci Foundation PhD Fellows Program, and the Erwin Schrodinger Postdoctoral Fellowship.

Mihaljevic L, Kim DE, Bandawane PD, Eisenach HE, Borst AJ, Courbet A, Weidle C, Carr KD, Bettin E, Liu Q, Trejos AT, Majumder S, Kokane S, Stevens A, Muratspahic E, Schlichthaerle T, Exposit M, Li X, Lamb M, Azcarraga Murray AN, Ravichandran R, Williams EC, Hu S, Stuart L, Grillova L, Thomson NR, Landreh M, Chang P, Giacani L, Caimano MJ, Hawley KL, King NP, Baker D. 2026. Membrane protein solubilization and structure determination using de novo-designed proteins. Science. doi: 10.1126/science.adr3817