WHITE PAPER | No. 1 | December 2015 Detecting Conformational Change in Peripheral Membrane Proteins Bound Directly to Supported Lipid Bilayers Ben Moree, Biodesy, Inc., South San Francisco, CA Peripheral membrane proteins are key regulators of signal transduction pathways at cell membranes. These signaling pathways are activated by conformational changes that occur in peripheral membrane proteins upon ligand binding. Currently, the ability to measure ligand induced conformational change in peripheral membrane proteins under physi- ological conditions is difficult. Here, we discuss a novel approach for detecting and measuring conformational change in peripheral membrane proteins when attached directly to physiologically relevant supported lipid bilayers. Introduction system for the investigation of protein conformational changes by second-harmonic generation (SHG) that uses an Cell membranes are fluid lipid bilayers composed of a integrated immobilized metal affinity capture (IMAC) variety of lipids and associated integral and peripheral Ni-NTA lipid for the tethering of His-tagged proteins to membrane proteins. Structurally, bilayer membranes are SLBs. A major advantage of this approach is that the SLBs composed of two monolayers of lipids, also known as are biomimetic, maintain native protein structure and func- leaflets, in which the hydrophobic fatty acids tails are tion, and they have been used extensively in many oriented toward the center of the bilayer between the biochemical and cell biology experiments6-8. hydrophilic lipid head groups. The most abundant class of lipids in the bilayer is the phospholipid family. Phospholip- Second-harmonic generation (SHG) is a nonlinear optical ids are distinguished from one another through the incor- technique in which two photons of equal energy are com- poration of different lipid head group modifications and bined by a nonlinear material or molecule to generate one commonly include lipids such as phosphatidylcholine (PC), photon with twice the energy9,10. As SHG is a surface selec- phosphatidylserine (PS), phosphatidylethanolamine (PE), tive technique, it requires tethering of proteins to a surface and phosphatidylinositol (PI). Interestingly, membranes in an oriented manner. Once tethered to a surface, a have an asymmetric distribution of lipids in the inner and labeled second-harmonic-active protein irradiated by a outer leaflet of the bilayer that differentiates and special- fundamental light source produces an SHG signal whose izes each leaflet for important physiological processes1. In intensity depends sensitively on the tilt angle of the dye particular, PS and PI are both found primarily in the inner with respect to the surface normal. When the protein cytoplasmic leaflet where they recruit peripheral mem- undergoes a conformational change upon ligand binding, brane proteins via interaction with the lipid head groups to this causes a change in the time- and space-averaged orien- the inner membrane. Once recruited to the inner mem- tation of the second-harmonic-active moiety leading to a brane and activated by extracellular cues, these proteins change in the intensity of light. This yields a real-time mea- regulate diverse signal transduction pathways that control surement that reports directly on a probe’s change in orien- cellular processes such as transcription, cell division, differ- tation at one or more labeled residues in a protein with high entiation, and apoptosis2,3. angular sensitivity11-15. To better understand the role of lipid bilayers in these cellu- We were interested in the potential role of lipid bilayer lar processes, model lipid bilayer systems have been devel- specialization to capture labeled proteins to SLBs for the oped in vitro. One such model system is the supported lipid purpose of conformational change measurement by SHG. bilayer (SLB)4,5. In the SLB system, small unilamellar To explore this possibility, Biodesy developed new bilayer vesicles (SUVs) are deposited onto a glass substrate in an formulations that contain specific phospholipid classes to aqueous solution where they spontaneously form fluid lipid directly capture full-length peripheral membrane proteins bilayers. Biodesy™ recently developed a commercial to SLBs without the need for a His-tag and Ni-NTA lipids. WHITE PAPER | No. 1 | Page 2 We demonstrate that once bound to the SLBs, the full- truncated version of the protein lacking the N-terminal length peripheral membrane proteins are competent to lipid-binding domain (∆N-Protein) was deposited onto lipid bind ligands and undergo conformational change as bilayers containing either PI and PIP. As seen in Figure 2, detected by SHG and the Biodesy Delta system. This new addition of the truncated protein to either surface did not approach should be broadly applicable for both drug produce an increase in the SHG signal intensity compared discovery and mechanistic studies of peripheral membrane to the bilayer-only controls. In combination with the previ- protein function under physiologically relevant conditions. ous results, these data suggest that the full-length periph- eral membrane protein binds the Biodesy surface specifi- Results cally through an interaction between the N-terminal lipid- To explore the ability of the Biodesy SLB platform to binding domain and PIP-containing lipids on the SLB mem- capture proteins without the use of IMAC techniques, we brane surface. began by testing the ability of physiologically mimetic bilayer formulations to capture untagged full-length Figure 2 The Periph- eral Membrane peripheral membrane proteins to the SLB surface. In Protein Requires the collaboration with a pharmaceutical company, our first Lipid Binding Domain target was a clinically relevant peripheral membrane for Capture to the protein that has previously been shown to associate specifi- Biodesy SLB surface. cally at membranes containing phosphatidylinositol phos- The SHG intensity of the N-terminally phate (PIP) but not at membranes containing PI. We modi- truncated protein is fied our commercial SUV formulations, including replace- not enhanced over the ment of our Ni-NTA lipids with either PIP lipids or control baseline bilayer-only lipids containing PI. Using these new SUV formulations, we signal upon deposition of the truncated prepared modified SLBs on Biodesy plates, deposited full- protein onto SLBs length peripheral membrane protein on both the PIP and containing either PI or control PI bilayers, and measured SHG signal using the PIP lipids. Biodesy Delta system to evaluate protein capture on the SLB surface. As shown in Figure 1, SLBs containing PIP or We next wanted to explore whether the peripheral mem- Figure 1 Peripheral Mem- brane protein, when bound to a physiologically relevant brane Protein Capture biomimetic bilayer surface, would undergo a conforma- Directly to the Biodesy tional change upon the addition of appropriate compounds. Bilayer Surface. The SHG Using the Biodesy Delta system, compounds were injected signal of the protein is enhanced over the at saturating doses and the change in SHG intensity was baseline bilayer-only monitored five minutes after injection. As shown in Figure signal upon deposition of 3, the addition of each compound to the peripheral mem- protein onto bilayers brane protein sample resulted in a change in SHG intensity containing either PI or PIP that was significantly different than the injection of the lipids. No enhancement of the SHG signal is seen vehicle alone (Student’s t-test, p< 0.05). As the intensity of when protein is deposited the SHG signal is highly dependent on the average angular onto the control PI lipids. orientation of the second-harmonic-active probe relative to the membrane plane, these results suggest that the mea- sured changes in SHG intensity correspond to conforma- PIP2 captured the full-length peripheral membrane protein tional change induced by compound binding at the attach- whereas the control, non-phosphorylated PI bilayer, was ment site of the second-harmonic-active probe on the unable to capture the protein. These data demonstrate the protein. In addition, these results demonstrate that the specific capture of the untagged, full-length peripheral new capture methodology is amenable to the study of membrane protein to the Biodesy SLB surface through the protein conformational change with the Biodesy Delta addition of PIP lipids to the SUV formulation. system. As a control for the specificity of the protein binding to the As another demonstration of the power of this approach, surface, we performed an additional experiment in which a WHITE PAPER | No. 1 | Page 3 we developed a membrane-binding assay using the Biodesy then prepared on Biodesy plates, protein was deposited Delta system to measure the effects of compound binding onto the PS SLBs, and SHG signal was measured on the on the ability of the protein to associate with the SLB Biodesy Delta. As shown in Figure 5, addition of the full- length kinase to the PS SLB surface results in an enhance- Figure 3 Quantification of the change in SHG intensity ment of the SHG signal over the control PS bilayer-only of a peripheral membrane signal, demonstrating capture of the kinase to the SLB protein upon compound surface in the absence of IMAC tethering. injection. A change in SHG intensity corresponds to We next sought to validate that the SLB captured mem- conformational change at brane protein was competent to undergo conformational the label site of the protein. change when bound to its physiologically relevant bilayer DMSO