Research Project
10095937
Integral membrane proteins reside in the biological membrane where they function and intimately interact with lipid molecules. The environment of the biological membrane is dynamic and composed of a rich chemical diversity of lipid molecules. Alongside the complexity of the biological membrane is the growing realization of the important roles of lipid molecules in the folding, structure, and function of membrane proteins. In fact, there is often density in maps of structures determined by X-crystallography and cryoEM that are ascribed to lipids but their identity remains largely unknown. Although a handful of examples exist which provide insight into membrane protein-lipid interactions, how individual lipid molecules influence the structure and function of membrane proteins on the molecular level largely remains poorly understood. What determines the selectivity of membrane proteins towards lipids? How important is the lipid chemistry, such as lipid tail length, stereochemistry and position of unsaturated double bonds, in protein-lipid interactions? Do membrane proteins recruit, through allostery, their own microenvironment? Here, this proposal seeks to address these fundamental questions by developing new tools and reagents to probe membrane protein-lipid interactions using the ammonia channel (AmtB) from E. coli in complex with its regulatory protein GlnK as a model membrane protein system. More specifically, native Mass Spectrometry (MS) technology, whereby non-covalent interactions are preserved in the mass spectrometer, will be employed in combination with new MS approaches pioneered in the Laganowsky group that, unlike other biophysical methods, allow individual lipid binding events to membrane protein complexes to be resolved and interrogated. The proposed studies build off the foundation of previous work where native MS technology is integrated with other biophysical techniques, such as Surface Plasmon Resonance (SPR) and X-ray crystallography, to address fundamental questions regarding membrane protein-lipid interactions. More specifically, proposed studies are aimed at unravelling cooperativity for a considerable number (up to 20) of individual lipid binding events to AmtB by the (i) use of charge-reducing molecules and (ii) synthesis of new detergents engineered for native MS applications. Next, proposed studies pushing the technological limits of MS technology aimed at deducing allostery within heterogeneous lipid binding events to AmtB. Here, these studies move beyond previous work on mixtures of two different lipid headgroups to more complex lipid mixtures, composed of three to four different lipid species, binding to AmtB. Novel approach is proposed to deduce the position-dependent effects of bound lipids on AmtB by using a combination of protein engineering and covalent labeling strategies. Taken together, the results and outcomes from our proposed studies are anticipated to have a significant impact in our understanding of membrane protein-lipid interactions and, more generally, to our understanding of membrane protein biology, especially how changes in the biological membrane may regulate membrane protein physiological function.
