Effects of fluidity on the ensemble structure of a membrane embedded α‐helical peptide

Eagleburger, Michael K.; Cooley, Jason W.; JiJi, Renee D.

doi:10.1002/bip.22472

Cited by 5 publications

(4 citation statements)

References 55 publications

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“…Despite the less intense features, this spectrum is still indicative of an α-helical secondary structure, but one that is elongated or stretched. 27 As the two lipid vesicles fuse it can be imagined that there is a mixing of the different length lipids throughout the newly formed vesicle. Indeed, we propose that the PLA7 is stretched in the presence of this new, longer lipid chain to maintain an anchor point at the lipid-aqueous interface.…”

Section: Discussionmentioning

confidence: 99%

Lipid vesicle fusion as a vehicle to study transmembrane protein interactions

Lada¹

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Alzheimer's disease, among other neurologically degenerative diseases, has been linked to protein-enzyme interactions that originate within the transmembrane domain of a cell. The lipid environment that houses these interactions lends difficulty to studying intramembrane interactions, often making for time consuming data analysis. Lengthy data interpretation on top of the rate in which protein-enzyme interactions take place creates a need for a method to overcome these obstacles. The use of lipid vesicle fusion to apply a margin of control over the time frame of interaction combined with deep probing spectroscopic techniques can minimize the interference of the lipid environment. Deep ultraviolent resonance Raman (dUVRR) is a vibrational spectroscopic technique that probes along the protein backbone that allows for removal of lipid environmental interference through background subtraction. Lipid vesicle fusion has been demonstrated by mixing lipid vesicles comprised of oppositely charged head groups (cationic 1,2-diaurroyl-sn-glycero-3-phospho-(1-rac-glycerol) (DLPG)) and anionic 1,2-dilauroyl-sn-glycero-3-ethylphocholine (12:0 EPC) or 1,2-dimyristoyl-sn-glycero-3-ethylphocholine (14:0 EPC)) of equivalent or varying aliphatic tail length, up to a 2-carbon difference. The fluorescent dye, 8-aminonapthalene 1,3,6-trisulfonic acid (ANTS), paired with the quencher, p-xylene-bis-pyridiumbromide (DPX), are separately encased in either DLPG or 12:0 EPC/14:0 EPC, respectively, in aqueous solution, and evidence of lipid vesicle fusion is provided by monitoring fluorescence intensity of ANTS as the two solutions are mixed, resulting in the closing proximity of ANTS and DPX observed as a decrease in fluorescence intensity. Additional evidence is provided by dynamic light scattering (DLS) measurements of both independent vesicle solutions and their mixture showing an increase in hydrodynamic radius (Rh). In addition, cohesion of similarly sized lipids is demonstrated, as DLPG (12-carbon chain) fails to fuse with cationic lipids of chain length 16 carbons or longer. Circular dichroism (CD) is a spectroscopic technique that uses left and right-handed polarized light to obtain the overall secondary structure of proteins. Shown is the use of CD to probe the secondary structure and the changes incurred on PolyLA7 (PLA7), a model ?-helical peptide when placed in a transmembrane or hydrophobic environment, through a change in the lipid environment. PLA7 was inserted in DLPG lipid vesicles and then mixed in solution with lipid vesicles comprised of 14:0 EPC. CD spectra were obtained pre and post vesicle fusion, demonstrating the use of lipid fusion as a means to combine membrane embedded proteins of interest while still being able to observe changes that take place. Finally, we propose an on-demand lipid fusion system in which two separate lipid vesicles could be co-suspended in solution and then chemically or photonically induced to fuse. A titration was performed to obtain the pKa of a synthesized pH inducible cationic lipid (pHiCL). The pHiCL is a dipicolylamine with an attached 12-carbon aliphatic tail. The pHiCL was titrated while suspended in an aqueous environment and while inserted into a lipid vesicle comprised of DLPC, a net neutral lipid also with a 12-carbon length aliphatic tail. The pHiCL will be the first component of a two-part system in which a photoacid (PA) will be used to protonate the pHiCL in solution giving rise to cationic and anionic surfaced lipid vesicles causing vesicle fusion to occur.

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Section: Discussionmentioning

confidence: 99%

Lipid vesicle fusion as a vehicle to study transmembrane protein interactions

Lada¹

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“…Deep Ultraviolet Resonance Raman (dUVRR) spectroscopy has traditionally been applied to secondary structure determination in aqueous proteins. Work with this technique in membrane systems is a relatively new endeavor [1][2][3][4] . Membrane proteins are a vital part of our cellular biology but are not as well studied as their aqueous soluble counterparts due to the experimental challenges of working with lipids for traditional techniques like nuclear magnetic resonance (NMR) or X-ray crystallography.…”

Section: Introductionmentioning

confidence: 99%

“…Deep Ultraviolet Resonance Raman (DUVRR) Spectroscopy is a vibrational technique that has been used to monitor both soluble and membrane-associated proteins 2,24,28,52,58,66 . The utility of dUVRR spectroscopy in protein studies is derived from the resonance enhancement of the structurally informative peptide backbone modes over side chain or background solvent modes, which can dominate the spectra with traditional Raman techniques.…”

Section: Introductionmentioning

confidence: 99%

“…Deep Ultraviolet Resonance Raman (dUVRR) Spectroscopy is a vibrational technique that has been used previously in the determination of secondary structure for both soluble and membrane-associated proteins 2,77,[96][97] . Resonance enhancement of the peptide backbone vibrational modes provides rapid structural insights with little sample preparation.…”

Section: Introductionmentioning

confidence: 99%

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Deep ultraviolet resonance Raman spectroscopy as a tool for biomolecule analysis

Eagleburger¹

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Cholesterol is a major component of membranes, reducing their fluidity at higher concentrations. The UV Raman vibrational mode of the carbon-carbon double bond in cholesterol occurs at 1675 cm-1 is strongly resonance enhanced in deep ultraviolet resonance Raman (dUVRR) spectra of natural and model membranes at an excitation wavelength of 197 nm. DUVRR is a vibrational spectroscopic technique that is used to gather information on protein secondary structure and in this work is extended to study systems in the presence of the membrane component cholesterol and the cholesterol analog 5[alpha]-cholestan-3[beta]-o, which lacks the carbon-carbon double bond. DUVRR studies of melittin indicated that little loss of helical structure occurred with increasing temperature as was suggested by circular dichroism (CD), another structurally sensitive spectroscopic technique. A cholesterol analog was introduced to decrease the fluidity of membrane in a similar manner to cholesterol, while minimizing spectroscopic interference from the resonant enhanced carbon-carbon double bond vibration. With the introduction of the cholesterol analog a loss in helical structure of the melittin was seen in both the dUVRR and CD. Lipoproteins transport cholesterol through the blood stream. Studies have shown that an individual's lipoprotein levels are correlated with their risk of cardiovascular disease. The strongly resonantly enhanced dUVRR feature of the carbon-carbon double bond was used to quantify cholesterol in lipoprotein particles isolated from whole blood. Like cholesterol, unsaturated lipids give rise to a strongly enhanced vibrational mode in dUVRR spectra, which is slightly downshifted from that of cholesterol. The vibrational mode of unsaturated lipids occurs in the region of 1655-1661 cm-1, depending on the type of lipid. The feature arising from the unsaturated lipids was found to be sufficiently resolved from cholesterol's feature to allow quantitation of cholesterol in model membranes and lipoproteins. Isolation and characterization of lipoproteins is an emerging field as studies show that cardiovascular disease risk is a complex function of not just cholesterol and lipoprotein levels but also their size distribution. Spectroscopic methods are readily coupled with chromatographic techniques. Size exclusion chromatography (SEC) was employed to separate lipoproteins based on their size from a series of blood serum samples. Each volume fraction was spectroscopically characterized via dUVRR spectroscopy producing a two-way data matrix for each sample, which could then be analyzed using higher order multivariate methods. Parallel factor (PARAFAC) analysis and multivariate curve resolution-alternating least squares (MCR-ALS) were employed to resolve the underlying spectroscopic and chromatographic profiles. The resolved profiles were attributed to lipid/cholesterol, protein and buffer components.

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