Registrieren

Proteins and peptides play critical roles in membrane binding and in mediating a variety of cellular processes. Although numerous membrane-interacting proteins and peptides have been identified, the molecular factors that influence their underlying mechanisms of binding to lipid membranes and conformational dynamics remained unexplored. To further understand these complex interactions, biomimetic models that closely resemble the environment of natural cell membranes are of considerable importance. In this thesis, Attenuated Total Reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used in combination with biomimetic membranes to study calcium-dependent lipid binding protein (CaLB), a previously uncharacterised Arabidopsis C2 domain protein and Arabidopsis ALG-2 interacting protein X (ALIX). For CaLB, membrane binding specificity and calcium ion (Ca2+) dependency were assessed using solid-supported lipid bilayers (SSLBs) and small unilamellar vesicles (SUVs) composed of either pure POPC or a lipid mixture of PI(3)P and POPC. The CaLB protein showed an increased binding affinity to membranes containing one percent PI(3)P and at calcium concentrations in the micromolar regions. Secondary structure analysis through IR spectra indicated minor conformational changes upon lipid membrane binding, with a slight increase in helical and disordered regions. Similarly, ALIX protein displayed a preference for PI(3)P containing membranes under similar experimental conditions with optimum binding being observed at Ca2+ concentrations in the millimolar range. Secondary structure analysis of ALIX revealed a more significant change in the secondary structure elements, including an increase in β-sheet and disordered regions upon membrane binding. To further improve the biomimetic properties of the lipid membrane model, circular dichroism (CD) spectroscopy was used to access the interaction of the ALIX protein to large unilamellar vesicles (LUV) composed of PI(3)P and POPC in a ratio of 1 to 99. Vesicles allow for buffer solutions to be on both sides of the lipid membrane, enabling the membrane to be in a more fluidic environment to study the conformational dynamics of ALIX. The results demonstrated that, at a Ca²⁺ concentration of 20 µM, ALIX undergoes notable conformational changes upon binding to the lipid membranes. While CD spectra provide information on the conformation changes of ALIX binding to membranes, complementary to this FTIR spectroscopy can deliver additional informations by detecting specific bond vibrations. To address this, nanocavities were developed using electron beam lithography (EBL) to create a platform capable of supporting tethered lipid membranes for surface-enhanced infrared absorption spectroscopy (SEIRA). The pores of these nanocavities were filled with aqueous buffer and their exterior surfaces were functionalised with self-assembled monolayers (SAMs), which were characterised using cyclic voltammetry (CV). By tuning the microcontact printing parameters, the time and pressure conditions were optimized, yielding effective surface coverage of SAM. Following the confirmation of successful tethering of DOPC lipid membrane, the platform was used to study the interactions between the LAH4 antimicrobial peptide and these membranes. As both membrane systems showed pH-dependent responses to LAH4, the interactions on the tethered membrane were not as robust compared to the SSLB system. Specifically, exposure to LAH4 at pH 9 caused considerable damage in the tethered system, causing membrane destabilisation and even collapse at lower pH. In contrast, SSLB exhibited only minor damage at pH 9. These differences underscore the fundamental role of the membrane underlying support structure in modulating peptide-membrane interactions.