Archaeal-type bipolar lipids, known as bolalipids, self-assemble into monolayers that are more rugged than conventional bilayer membranes while retaining properties expected to support membrane protein activity. We report an efficient synthesis of a bolalipid library to investigate the impact of bolalipid structure on the activity of a histidine-tagged membrane protein, isoprenylcysteine carboxyl methyltransferase (His-Ste14p) from S. cerevisiae. This protein is responsible for the proper localization and transformation ability of oncogenic Ras proteins, making it a target for chemotherapeutic agent discovery. The convergent synthesis of the bolalipid library was designed to involve common synthetic intermediates and the commercially available cis-1,3-O-benzylidene glycerol as the starting material. The synthesis of the bolalipid library components [C₂₀BAS, C₃₂BAS, C₃₆BAS and C₃₂-phytBAS] was accomplished in multi-gram scale, eight steps and 5 % overall yield. Key synthetic steps are: intermolecular Grubbs metathesis of terminal olefinic glycerol backbone monomers and a highly efficient installation of the phosphocholine head groups. The impact of bolalipid structure on His-Ste14p activity was monitored by an in vitro vapor diffusion assay in reconstituted bolalipid/E. Coli polar lipid vesicle dispersions. Protein activity was lost in vesicles composed of 75-100 mol % C₂₀BAS, but retained in vesicles with 0-50 mol % C₂₀BAS and 0-100 mol % C₃₂-phytBAS. Immuno-fluorescence microscopy confirmed the presence of His-Ste14p in all bolalipid vesicle dispersions. His-Ste14p activity was not found to be affected by either the phase state of the lipids or the temperature of the assay. Thus, the low activity observed in membranes with high mol % of C₂₀BAS is attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of His-Ste14p. These results suggest that the C₃₂-phytBAS is the best candidate for supported membrane biosensor applications targeting His-Ste14p and show the significance of varying the length of the transmembrane chains to match the dimensions of different membrane proteins.