Our research emphasizes the area of structural bioenergetics, which studies the molecular basis of biological energy transduction processes. The coupling of cellular energy metabolism to environmental energy sources occurs primarily through consumption of nutrients (redox energy) and absorption of light. These energy sources must then be converted into biologically usable forms, such as ATP and concentration gradients, that are required to drive biosynthetic reactions, electrical signaling, and other metabolic processes. Energy transduction processes mediate these interconversions of different energy forms. A major goal of our group is to characterize the structures and mechanisms of transduction systems involved in membrane transport, mechanosensation, and oxidation-reduction processes, including the development of methodologies to study these processes.
As an example of the latter, we have been working on approaches to characterize the structures of membrane proteins in the presence of a transmembrane gradient, since the functions of many membrane proteins are intimately coupled to the generation, utilization, and/or sensing of transmembrane gradients. Despite advances in the structure determination of membrane proteins, the high-resolution structural analysis of membrane proteins in a biological membrane is uncommon and in the presence of a functionally relevant gradient remains a challenge. This stems from the fact that the primary two- and three-dimensional ordered specimens employed in structural studies of membrane proteins by x-ray crystallography and electron microscopy (EM) lack closed membrane surfaces, making it impossible to establish physiologically relevant transmembrane gradients.
The ideal method for determining high-resolution structures of membrane proteins would require small amounts of material and be compatible with a bilayer environment. While EM has these capabilities, many membrane proteins are too small for EM single-particle analysis, and so it is essential to study assemblies containing multiple copies of the protein of interest to collect data with adequate signal to noise. For membrane proteins, EM structure determinations have typically utilized two types of ordered two-dimensional (2D) arrangements of membrane proteins in membranes, either 2D crystals or tubular crystals, which have been challenging to prepare. As an alternative, we have been developing methodologies for the self-assembly of lipids and membrane proteins into a distinct type of ordered 2D assembly of lipid-embedded membrane proteins, namely the generation of polyhedral arrangements of membrane proteins that would result in their symmetrical distribution around the closed surface of a proteoliposome. An important property of such membrane protein polyhedral nanoparticles (MPPNs) is that they could have closed surfaces that could potentially support transmembrane gradients for structural and functional studies.
We have prepared MPPNs for the Escherichia coli mechanosensitive channel of small conductance (MscS) whose structure was previously determined by our group. MscS is a heptameric channel with 21 transmembrane helices (3 from each subunit) and a large cytoplasmic domain. From initial studies, we were able to establish that these MPPNs have a diameter of ~20 nm, and contain 24 MscS channels in an octahedral arrangement. By using cryo-electron microscopy to process a total of 4,564 MPPNs, we were able to determine their structure to ~1-nm resolution, which allowed us to model the inner and outer helices of the transmembrane pore. The arrangement of the helices more closely resembles the nonconducting conformation than the open-state structure, although some differences in the positioning of the outer helices relative to the nonconducting structure are evident. These results establish that membrane proteins are capable of assembling into MPPNs that are amenable to high-resolution structure analysis by single-particle cryo-electron microscopy.
The self-assembly of membrane proteins into polyhedral shells demonstrates a potentially powerful method for studying the structure and function of membrane proteins in a lipid environment. By providing a closed surface that separates the interior from the external solution, a significant potential advantage of MPPNs could be the ability to establish, for example, pH, voltage, osmotic, and concentration gradients across the membrane to activate various types of gated channels and transporters. We have also designed and fabricated microfluidic devices for high-throughput screening of conditions for MPPN formation. MPPNs may allow a variety of perturbations to be achieved (such as pH, voltage, osmotic, and concentration gradients) that cannot be achieved with other membrane protein assemblies and will potentially allow us to activate various types of gated channels and receptors so that active conformational states can be structurally investigated.
This work was supported in part by a grant from the National Institutes of Health.
As of March 19, 2014