Previously, our group successfully characterized the effect of various lipids on the stalk formation in pure lipid multibilayers by means of x-ray scattering. In this model system, the energy required for stalk formation was supplied by the osmotic pressure of the aqueous vapor. Despite its remarkable simplicity and efﬁ ciency, the sample environment of this method was distinct from the fusion sites in vivo. This work aims to extend the x-ray study of stalk formation to more physiological conditions. To this end, efforts on three levels have been devoted, namely the lipid level, the peptide level and the protein level. On the lipid level, PEG solutions rather than aqueous vapor were used as the stressor to promote stalk formation in pure lipid bilayers. On the peptide level, D-β-peptides were incorporated into model lipid multibilayers to investigate its transmembrane behaviors. Lastly, on the protein level, a novel solvent-free protocol was developed, and SNAREs, a family of proteins which mediate vesicle fuion, were reconstituted into the multibilayers via the micelle-vesicle-multibilayer pathway.