Electrostatic interactions drive molecular assembly and organization in the plasma membrane. Specific protein-lipid interactions, however, are difficult to resolve. Here we report on a unique approach to investigate these interactions with time-resolved fluorescence spectroscopy. The experiments were performed on a model membrane system consisting of a supported lipid bilayer with an asymmetric distribution of PIP2 in the upper leaflet of the bilayer. The bilayer also contained nickel-chelating lipids that bind to a histidine-tagged peptide of interest. Both the peptide and the lipid were labeled with orthogonal fluorescent probes, so that diffusion and binding could be measured with two-color, pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Our PIE-FCCS data showed significant lipid-peptide cross-correlation between PIP2 lipids and membrane-bound cationic peptides. Cross-correlation is a direct indication of lipid-peptide binding and complexation. Together with mobility data, we quantified the degree of binding, which offers new insight into this class of lipid-peptide interactions. Overall, this is the first report of lipid-peptide cross-correlation by FCCS, and provides a new route to quantifying the interactions between proteins and lipid membranes, a key interface in cell signaling.
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Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy
In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10–11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan’s GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan’s REES values are high (9.3–18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4–5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan’s excited state dipole moment, and “solvent” reorientation around Laurdan’s chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan’s lifetime.
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- Award ID(s):
- 1830866
- NSF-PAR ID:
- 10171977
- Date Published:
- Journal Name:
- International Journal of Molecular Sciences
- Volume:
- 20
- Issue:
- 21
- ISSN:
- 1422-0067
- Page Range / eLocation ID:
- 5308
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary Adaptation of lipid membrane composition is an important component of archaeal homeostatic response. Historically, the number of cyclopentyl and cyclohexyl rings in the glycerol dibiphytanyl glycerol tetraether (GDGT) Archaeal lipids has been linked to variation in environmental temperature. However, recent work with GDGT‐making archaea highlight the roles of other factors, such as pH or energy availability, in influencing the degree of GDGT cyclization. To better understand the role of multiple variables in a consistent experimental framework and organism, we cultivated the model Crenarchaeon
Sulfolobus acidocaldarius DSM639 at different combinations of temperature, pH, oxygen flux, or agitation speed. We quantified responses in growth rate, biomass yield, and core lipid compositions, specifically the degree of core GDGT cyclization. The degree of GDGT cyclization correlated with growth rate under most conditions. The results suggest the degree of cyclization in archaeal lipids records a universal response to energy availability at the cellular level, both in thermoacidophiles, and in other recent findings in the mesoneutrophilic Thaumarchaea. Although we isolated the effects of key individual parameters, there remains a need for multi‐factor experiments (e.g., pH + temperature + redox) in order to more robustly establish a framework to better understand homeostatic membrane responses. -
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Summary Microorganisms regulate the composition of their membranes in response to environmental cues. Many Archaea maintain the fluidity and permeability of their membranes by adjusting the number of cyclic moieties within the cores of their glycerol dibiphytanyl glycerol tetraether (GDGT) lipids. Cyclized GDGTs increase membrane packing and stability, which has been shown to help cells survive shifts in temperature and pH. However, the extent of this cyclization also varies with growth phase and electron acceptor or donor limitation. These observations indicate a relationship between energy metabolism and membrane composition. Here we show that the average degree of GDGT cyclization increases with doubling time in continuous cultures of the thermoacidophile
Sulfolobus acidocaldarius (DSM 639). This is consistent with the behavior of a mesoneutrophile,Nitrosopumilus maritimus SCM1. Together, these results demonstrate that archaeal GDGT distributions can shift in response to electron donor flux and energy availability, independent of pH or temperature. Paleoenvironmental reconstructions based on GDGTs thus capture the energy available to microbes, which encompasses fluctuations in temperature and pH, as well as electron donor and acceptor availability. The ability of Archaea to adjust membrane composition and packing may be an important strategy that enables survival during episodes of energy stress. -
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Abstract Archaeal glycerol dibiphytanyl glycerol tetraethers (GDGT) are some of the most unusual membrane lipids identified in nature. These amphiphiles are the major constituents of the membranes of numerous Archaea, some of which are extremophilic organisms. Due to their unique structures, there has been significant interest in studying both the biophysical properties and the biosynthesis of these molecules. However, these studies have thus far been hampered by limited access to chemically pure samples. Herein, we report a concise and stereoselective synthesis of the archaeal tetraether lipid parallel GDGT‐0 and the synthesis and self‐assembly of derivatives bearing different polar groups.
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Abstract Archaeal glycerol dibiphytanyl glycerol tetraethers (GDGT) are some of the most unusual membrane lipids identified in nature. These amphiphiles are the major constituents of the membranes of numerous Archaea, some of which are extremophilic organisms. Due to their unique structures, there has been significant interest in studying both the biophysical properties and the biosynthesis of these molecules. However, these studies have thus far been hampered by limited access to chemically pure samples. Herein, we report a concise and stereoselective synthesis of the archaeal tetraether lipid parallel GDGT‐0 and the synthesis and self‐assembly of derivatives bearing different polar groups.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/ijms20215308
