Partitioning of semisynthetic lipidated N-Ras in lipid raft nanodomains determined by FRET to lipid domain markers
Summary (3 min read)
- Separation with the nanometer-scale liquid-ordered phase (aka "lipid rafts" or Lo) surrounded by the liquid-disordered phase (Ld).
- Their cellular counterparts are expected to be much smaller, nanometer-sized, making them only resolvable by electron and atomic force microscopy techniques11-14.
- In a cell, many membrane proteins permanently reside in raft membrane domains, which is essential for their function5, 15-19.
- Ras proteins are represented by three Ras isoforms with a high degree of homology and nearly 90% sequence identity in the N-terminal GTPase domain33.
1. Lipid membrane mimic with nano-scale lipid domains
- To create lipid bilayers that spontaneously forms nanometer-sized raft domains (approx. ranging from 4 to 15 nm), the authors followed Pathak and London50 and utilized a lipid mixture of sphingomyelin (SM), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol in the equimolar ratio (referred to in the following as the raft lipid mixture).
- Confocal fluorescence microscopy of a supported lipid bilayer made of the raft lipid mix confirmed that these bilayers do not form non-physiological micrometer-sized rafts .
- The Ro of 36Å allows for sensitive detection of formation and melting of nanoscopic raft nanodomains50.
- Increase of relative access of Rhod-DOPE acceptors (magenta stars) to the DPH donor molecules (green stars) upon heating.
- The light scattering by LUV is relatively temperature- independent, therefore, the F/Fo variation with temperature reflects the relative change of FRET from DPH to Rhod-DOPE.
2. Preferential localization of N-Ras C-terminal lipopeptide
- The N-methylanthranyl group (mant) was attached to the lipopeptide N-terminus to serve as a donor fluorophore.
- Heating led to increasing F/Fo indicating reduction of the FRET efficiency at higher temperatures, while homogeneous lipid showed relatively constant F/Fo values.
- In an analogous system, Fastenberg et al. explained such increasing pattern by hypothesizing that donor is present in the same disordered phase as the acceptor59.
- Intensity ratio, F/Fo, was calculated using mant emission of F and Fo samples, containing and lacking RhodDOPE, respectively.
- A similar increasing trend in the temperature dependence of F/Fo values was observed confirming their conclusion of the localization of the mant-labeled C-terminal N-Ras lipopeptide outside of lipid rafts—readily accessible by acceptor fluorophores.
3. Test of the raft-boundary localization of the C-terminal N-Ras peptide
- Experiments with mant-lipopeptide revealed that the lipopeptide is accessible to the acceptor fluorophore, Rhod-DOPE, at all times .
- The lineactant facilitates increase of the total length of the boundary thus promoting breaking the existing rafts into smaller ones (destabilization of large rafts).
- Reduction in raft size will be detectable in FRET experiments with DPH and Rhod-DOPE, because DPH will be more effectively quenched by Rhod-DOPE in smaller rafts.
- Reduction in F/Fo values upon heating due to melting of lipid rafts occurs in a similar temperature range both in the absence and the presence of the lipidated N-Ras peptide.
- This observation implies that the raft boundary does not significantly attract the lipopeptide.
4. Determination of the domain localization with time-domain fluorescence measurements
- Analysis of the FRET donor distribution among ordered and disordered lipid domains in the heating/cooling experiments described above relied on a measurement of relative fluorescence intensities in the two samples with and without acceptor (F and Fo), which required exactly matching concentrations of the donor.
- This is easy to accomplish for lipid mixtures that are made by taking accurate aliquots of fluorophore stocks, yet is very hard to achieve for the protein associated with LUV.
- The homogeneous and raft mixtures lacking acceptor (Fo samples; green and black symbols) exhibited relatively invariable lifetimes throughout the full temperature range.
- Containing mixtures in the presence and the absence of acceptor Rhod-DOPE.
- (B) FRET efficiency calculated using Eq. 1 (see Materials and Methods) from lifetimes of DPH in panel A.
5. Preferential localization of N-Ras bound with fluorescent GDP and GTP-mimics
- To mimic a full-length N-Ras with the native posttranslational lipidation pattern (one palmitoyl and one farnesyl chain), the authors prepared a semisynthetic protein following protocols developed by Herbert Waldman group68-70.
- To establish the predominant localization of the N-Ras bound to mant-nucleotides, the authors determined efficiency of FRET between mant group and Rhod-DOPE in homogeneous and raft LUV.
- N-Ras in its activated conformation (bound to GTP-mimic) was localized at the raft boundary or in a disordered lipid phase.
- The full-length gene of the wild-type N-Ras was a gift of Dr. Robert Deschenes, University of South Florida.
- For bacterial expression, N-Ras gene was subcloned into the pET vector (EMD Millipore, Billerica, MA).
- The truncated N-Ras construct ending with the cysteine 181 was prepared by introducing a stop codon in place of the methionine 182 codon.
- The C118S mutation was introduced to avoid possible side reactions between the only exposed cysteine on the G domain and maleimido group of the lipidated peptide93.
- All mutagenesis steps were performed using the QuikChange Site-Directed Mutagenesis Kit (Life Technologies, Grand Island, NY).
- Expression and isolation NRas-C118S-181 was performed as described earlier for a similar construct of H-Ras with little modifications92, 94.
- Final yield was approximately 0.5-2 mg of 95% pure protein from each liter of the expression medium.
Preparation of the lipidated peptides
- Fmoc-protected farnesylated cysteine was prepared as described95.
- The synthesized peptides were purified using the RP-HPLC-C4 column .
- This non-ionic detergent undergoes phase separation in aqueous solutions at temperatures above 30°C.
- The Triton X-114 solution was prepared prior to the reaction to achieve the final concentration of about 30 g/L as described98.
- Figures S5 and S6 show an expected increase in mass of 1315 Da indicating that Ras was successfully conjugates to the lipidated peptide.
- Lipids and their fluorescent derivatives were dissolved in chloroform (with the exception of DPH, which was dissolved in ethanol) and stored at -20°C.
- Lipid unilamellar vesicles, LUV, were prepared by extrusion following published protocols50, 99.
- To make the Fo samples, Rhod-DOPE was substituted by 2% DOPG to remove acceptor fluorophore but maintain the negative charge of the bilayer.
- Donor fluorophores DPH, dansyl-DOPE, and mant-lipopeptide, were added to F and Fo samples to 0.1% mol of total lipid, respectively.
- The temperature of the exturder block was maintained at 75 oC.
Preparation of Ras-LUV samples
- Fluorescent Ras-GDP and Ras-GTP complexes were prepared using the (2'-(or-3')-O-(N- methylanthraniloyl) guanosine 5'-diphosphate, mGDP, and the slowly hydrolysable GTP mimic 2'/3'-O-(N-methyl-anthraniloyl)-guanosine-5'-[(β,γ)-imido] triphosphate, mGppNHp, respectively.
- To prepare N-Ras-mGDP or N-Ras-mGppNHp associated with LUV, the lipidated Ras samples were subject to the nucleotide exchange followed by association with LUV and chromatographic separation as described in the following.
- The reaction mixtures were incubated for 2 hours at room temperature.
- Sizeexclusion elution profiles were monitored by tyrosine and mant fluorescence for protein, and rhodamine fluorescence for LUV.
- Confocal microscopy of supported lipid bilayers Supported lipid bilayers were created using raft LUV and observed with Nikon Perfect Focus Ti-E inverted research microscope using standard laser and filter sets.
- Measurements of steady-state and time-resolved fluorescence were performed using the Photon Technology International QM40 QuantaMaster system equipped with Pico-Master 1 time- correlated single-photon counting unit (HORIBA Scientific, Edison, NJ).
- A four-position Peltier- based Turret 400 (Quantum Northwest, Shoreline, WA) allowed for simultaneous temperature control and observation of up to four replicates for each sample condition.
- Time-domain fluorescence decays were analyzed using DecayFit software (kindly shared by Søren Preus; available from www.fluortools.com).
Lipidated N-Ras protein
- Figure S5. SDS-PAGE of the C118S N-Ras protein before (lanes 2 and 4) and after conjugation with lipidated peptide (lane 3).
- The difference in masses of the major peaks is 1315 Da. Figure S7.
- Two-dimensional excitation-emission spectra of the full-length lipidated C118S N-Ras bound with mGDP (left) and mGppNHp .
Number of peptide molecules per LUV =
- Maximum possible number of rafts per LUV = LUV surface area / Area of raft = 400; Rafts are estimated to occupy approximately 10-40% of the membrane106-108 in the cell and 50% in the lipid mixture that the authors are using50, which results in ca. 200 rafts per LUV.
- Calculation of the protein surface density for Ras-LUV complex.
- The external surface area of LUV per liter of the sample is 6.8 m2.
Did you find this useful? Give us your feedback
"Partitioning of semisynthetic lipid..." refers methods in this paper
...In the following subsections we (1) evaluated the model lipid bilayers to confirm that they form nanometer ordered domains mimicking size of cellular rafts, (2) detected non-raft localization of the C-terminal lipidated peptide of N-Ras, (3) evaluated a hypothesis that the C-terminal peptide may be attracted to the raft boundary, (4) established lifetime-based detection of nanodomain localization, and (5) detected distinct nanodomain preferences of N-Ras in active and inactive states (bound to GTP mimic or GDP)....
Related Papers (5)
Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "Partitioning of semisynthetic lipidated n-ras in lipid raft nanodomains determined by fret to lipid domain markers" ?
CC-BY-NC-ND 4. 0 International license under a not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.