Many diseases are initiated from molecular events that take place at the cell membrane level. The membrane plays a critical role in cell communication and is the launching point for many vital signaling processes that occur within and between cells.
Membrane-associated proteins either span the membrane, or are closely associated with the surface through domain interaction or through a number of posttranslational modifications that allow them to target the membrane.
Many of these proteins present a unique set of challenges to the researcher. Because of the inherent problems associated with designing assays for this class of proteins, researchers are forced to work with only the small cytoplasmic fragments of receptor tyrosine kinase (RTKs) domains. While these soluble fragments are simple to purify, they do not always exhibit proper biological function or normal activity because they have been removed from the fluidic environment of the membrane.
The membrane surface provides a unique organizing influence that allows complex signaling units, often composed of many different proteins, to form and carry out their natural function in a dynamic environment. When removed from the membrane environment, these proteins often exhibit low activity and frequently require the addition of nonphysiological counterions or other molecular crowding agents just to recapitulate some of their activity.
Protein Attachment Technologies has developed a method to facilitate the interrogation of membrane-associated proteins. Template-directed assembly (TDA) makes use of the ubiquitous histidine tag (His-tag) that has gained popularity in protein purification and in structural studies over the last two decades.
The TDA platform is a stable liposome that is decorated with NiNTA headgroups. When users combine His-tagged receptor fragments with TDA’s synthetic liposomes, proper enzymatic function is restored and the ability for the proteins to form multiprotein complexes is obtained. This technology was specifically designed for proteins that normally reside at or near the membrane surface—soluble cytoplasmic kinases anchored to TDA nanospheres show no improvement of activity, as expected.
While TDA is simple in principle, there are procedural and engineering concerns that must be considered. For example, TDA nanospheres are sensitive to EDTA and large concentrations of detergents. Additionally, membrane-associated proteins are frequently sensitive to changes in their juxtamembrane domains and may need to be engineered specifically to function on TDA nanospheres in addition to being N-terminally His-tagged for proper orientation. Thus, some care must be taken when designing proteins and reagents for use with TDA.
The end result, however, is a more biologically relevant system—more complete cytoplasmic domains that interact as they do in the cell, lower concentrations of ions, and proteins that are placed back into the membrane environment. Figure 1 depicts how TDA enables protein assembly.