Biophysical Properties of Immune Receptor Proteins at the Membrane Interface

Abstract

Mainstream approaches to examining the biophysical properties of integral membrane proteins (IMPs) possess severe limitations, mostly as a consequence of the instability of IMPs in solution. As a result, it has become common practice to characterize only their more soluble, extracellular domains, thus compromising the intrinsic properties of the native IMP. For signaling proteins such as immune receptors, an assessment of the full-length protein in its native membrane environment is critical for revealing the true function of the receptor. Tethered bilayer membranes (tBLMs) serve as the ideal system for reconstituting receptor-lipid complexes, as they are amenable to surface characterization methods such as surface plasmon resonance (SPR) and neutron reflectivity (NR). With the development of these receptor-lipid systems, a better understanding of the complexities of immune receptor signaling mechanisms, which up until now include theories of allosteric effects and receptor clustering, may well be within reach.The studies presented here showcase the design of anchoring transmembrane domains (TMDs) into lipid bilayers. The critical event of TMD insertion was examined using a mimic peptide of the TMD of the T cell co-receptor CD4, along with a hydrophobic and polar peptide which have both been characterized previously in lipid environments using circular dichroism and tryptophan fluorescence. Conducive for these studies, biotin tags were placed at the N-terminus of each peptide to evaluate the proper formation of a peptide-lipid complex using streptavidin capture. Relative insertion efficiency of these peptides were monitored using SPR and tryptophan fluorescence, followed by NR measurements to characterize their orientation relative to the tBLM. By laying the major groundwork of constructing peptide-tBLM systems, we are progressing convincingly towards novel protocols for reconstituting fully-active immune receptor proteins into physiologically relevant lipid systems to aid our studies of immune response signaling.