Single molecule probes of membrane structure: Orientation of BODIPY probes in DPPC as a function of probe structure

Abstract

Single molecule fluorescence measurements have recently been used to probe the orientation of fluorescent lipid analogs doped into lipid films at trace levels. Using defocused polarized total internal reflection fluorescence microscopy (PTIRF-M), these studies have shown that fluorophore orientation responds to changes in membrane surface pressure and composition, providing a molecular level marker of membrane structure. Here we extend those studies by characterizing the single molecule orientations of six related BODIPY probes doped into monolayers of DPPC. Langmuir–Blodgett monolayers transferred at various surface pressures are used to compare the response from fluorescent lipid analogs in which the location of the BODIPY probe is varied along the length of the acyl chain. For each BODIPY probe location along the chain, comparisons are made between analogs containing phosphocholine and smaller fatty acid headgroups. Together these studies show a general propensity of the BODIPY analogs to insert into membranes with the BODIPY probe aligned along the acyl chains or looped back to interact with the headgroups. For all BODIPY probes studied, a bimodal orientation distribution is observed which is sensitive to surface pressure, with the population of BODIPY probes aligned along the acyl chains increasing with elevated surface pressure. Trends in the single molecule orientations for the six analogs reveal a configuration where optimal placement of the BODIPY probe within the acyl chain maximizes its sensitivity to the surrounding membrane structure. These results are discussed in terms of balancing the effects of headgroup association with acyl chain length in designing the optimal placement of the BODIPY probe.

Ganglioside Influence on Phospholipid Films Investigated with Single Molecule Fluorescence Measurements

“…This approach has been used previously to probe DPPC membranes as a function of relative humidity, the addition of additives such as cholesterol, and membrane surface pressure. 31–34 …”

“…These measurements have been used to characterize changes in membrane structure due to surface pressure, the addition of additives such as cholesterol, and ambient humidity levels. 31–34 Here we use this approach to probe changes in DPPC structure at the molecular level with the addition of GM1.…”

Single molecule optical measurements of orientation and rotations of biological macromolecules

“…Defocused imaging has been applied to a number of biological systems, including the orientation tracking of bifunctional rhodamine attached to calmodulin on myosin V [97], observation of endocytosed vesicles within living cells [111], estimation of the torque produced during the 360° rotations of F 1 -ATP synthase [113], recording the orientation of carbohydrate- binding modules on cellulose microfibrils [116], determination of lipid orientation during membrane packing [36, 117] and raft formation [37]. …”

Changes in Order Parameters Associated with Ceramide-Mediated Membrane Reorganization Measured Using pTIRFM

“…5,48 However, several acyl-chain-labeled BODIPY PCs have been reported to adopt two conformations in supported monolayers and bilayers, one in which the fluorophore is aligned with the acyl chains and one in which the fluorophore loops back to interact with the polar headgroups. 31,32 For example, Dunn and co-workers have used single-molecule fluorescence to show that there are approximately equal amounts of the two orientations for a chainlabeled BODIPY-PC in DPPC bilayers and that the fraction of probes aligned parallel to the acyl chains increases with increasing Chol content. 32 It is possible that a mixture of conformations for BODIPY-PC contributes to the lower ⟨P 2 ⟩ values obtained for the L o phase of DOPC/ESM/Chol bilayers with this probe.…”

“…5,6,26 Similarly, the average orientation of fluorophores in supported membranes can be obtained by measuring the orientation of a large number of single molecules by defocused pTIRFM. 31,32 Although pTIRFM provides information that is analogous to order parameters measured by methods such as NMR, FTIR, and X-ray scattering, 33−38 it is important to note that different techniques report on different aspects of membrane order.…”