Synthesis and structure-function relationships of tetraether bisglyceryl bolalipids for development of a high-throughput membrane protein biosensor
Abstract
A convergent and efficient synthesis of a bolalipid library to investigate structure-property relationships is reported. The synthesis of C20BAS, C32BAS, C36BAS and C32phytBAS series was accomplished on the multi-gram scale in eight linear steps and 5% overall yield starting from glycerol. Key steps in the synthesis are: (1) intermolecular Grubbs metathesis and (2) highly efficient one pot installation of the phosphocholine headgroups in the last step. For biosensor purposes, the gel-to-liquid crystalline phase transition (Tm) has to be < 23°C to avoid protein denaturation. Differential scanning calorimetry and 31P NMR chemical shift anisotropy were used to study the Tm of the hydrated bolalipids. For C 20BAS Tm = 16°C, C32BAS and C36BAS Tm > 85°C, and C32phytBAS Tm = 14°C. This suggests that the possible candidates for the biosensor are C20 BAS and C32phytBAS. Pulsed-field gradient NMR (PFG-NMR) and fluorescence recovery after photobleaching (FRAP) were used to determine the bolalipid membrane lateral diffusion coefficients (D) and establish a relationship between D and the physical stability of their membranes. The two independent techniques give similar results for C 20BAS at 23°C (DPFG-NMR = 1.8 ± 0.6 x 10 -8 cm2/s; DFRAP = 1.5 ± 0.1 x 10 -8 cm2/s). DFRAP = 0.2 x 10-8 cm2/s for C32phytBAS; the slower diffusion is due to an increase in molecular area induced by methyl branching. The impact of bolalipid structure on Ste14p activity, an isoprenylcysteine carboxyl methyltransferase from S. cerevisiae, was monitored by an in vitro vapor diffusion assay in reconstituted 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% C32phytBAS. Immunofluorescence microscopy confirmed the presence of His-Ste14p in all pure bolalipid vesicle dispersions. Since His-Ste14p activity is not affected by either the Tm of the lipid or the temperature of the assay, the low activity observed in C20BAS membranes is attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of His-Ste14p (28 Å for C20BAS vs ∼40 Å His-Ste14p). These results suggest that the C32-phytBAS is the best candidate for supported membrane biosensor applications targeting His-Ste14p.
Degree
Ph.D.
Advisors
Thompson, Purdue University.
Subject Area
Biochemistry|Organic chemistry
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