The satellite band patterns in the near ultraviolet absorption spectra of isotopic compounds of acetaldehyde

The 3700 A - 3000 A absorption spectra of CH3CHO and its isotopic compounds such as CH3CDO, CD3CHO and CD3CDO were studied in the gas phase at room temperature and low temperatures. The low resolution spectra of the compounds were recorded by a 1.5 m Baush and Lomb grating spectrograph. The high resolution spectra were recorded by a Ebert spectrograph with the Echelle grating and the holographic grating separately. The multiple reflection cells were used to achieve the long path length. The pressure-path length used for the absorption spectrum of CH 3CHO was up to 100 mm Hg )( 91 . 43mo The emission spectrum and the excitation spectrum of CH3CHO were also recorded in this research. The calculated satellite band patterns \vhich were ob-tailied by the method of Lewis were used to compare with the observed near UV absorption spectrum of acetaldehyde. These calculated satellite band patterns belonged to two cases: namely, the barriers-in-phase case and the barriers- out-of-phase case. Each of the calculated patterns corresponded to a stable conformation of acetaldehyde in the excited state . The comparisons showed that the patterns in the observed absorption spectra corresponded to the H-H eclipsed conformations of acetaldehyde in the excited state . The least squares fitting analysis showed that the barrier heights in the excited state were higher than in the ground state. Finally, the isotopic shifts for the isotopic compounds of acetaldehyde were compared to the compounds with the similar deuterium substitution.

The effect of lysobisphosphatidic acid (LBPA) on α-tocopherol transfer protein (α-TTP) binding to lipid membranes

The a-tocopherol transfer protein (a-TTP) is responsible for the retention of the atocopherol form of vitamin E in living organisms. The detailed ligand transfer mechanism by a-TTP is still yet to be fully elucidated. To date, studies show that a-TTP transfers a-tocopherol from late endosomes in liver cells to the plasma membrane where it is repackaged into very low density lipoprotein (VLDL) and released into the circulation. Late endosomes have been shown to contain a lipid known as lysobisphosphatidic acid (LBP A) that is unique to this cellular compartment. LBPA plays a role in intracellular trafficking and controlling membrane curvature. Taking these observations into account plus the fact that certain proteins are recruited to membranes based on membrane curvature, the specific aim of this project was to examine the effect of LBP A on a-TTP binding to lipid membranes. To achieve this objective, dual polarization interferometry (DPI) and a vesicle binding assay were employed. Whilst DPI allows protein binding affinity to be measured on a flat lipid surface, the vesicle binding assay determines protein binding affinity to lipid vesicles mimicking curved membranes. DPI analysis revealed that the amount of a-TTP bound to lipid membranes is higher when LBPA is present. Using the vesicle binding assay, a similar result was seen where a greater amount of protein is bound to large unilamellar vesicles (LUV s) containing LBP A. However, the effect of LBP A was attenuated when small unilamellar vesicles (SUVs) were replaced with LUVs. The outcome of this project suggests that aTTP binding to membranes is influenced by membrane curvature, which in turn is induced by the presence of LBP A.

Mechanism of tocopherol transfer by human α-tocopherol transfer protein (α-hTTP)

Vitamin E is a well known fat soluble chain breaking antioxidant. It is a general tenn used to describe a family of eight stereoisomers of tocopherols. Selective retention of a-tocopherol in the human circulation system is regulated by the a -Tocopherol Transfer Protein (a-TIP). Using a fluorescently labelled a-tocopherol (NBD-a-Toc) synthesized in our laboratory, a fluorescence resonance energy transfer (FRET) assay was developed to monitor the kinetics of ligand transfer by a-hTTP in lipid vesicles. Preliminary results implied that NBD-a-Toe simply diffused from 6-His-a-hTTP to acceptor membranes since the kinetics of transfer were not responsive to a variety of conditions tested. After a series of trouble shooting experiments, we identified a minor contaminant, E coli. outer membrane porin F (OmpF) that co-purified with 6-His-a-hTTP from the metal affinity column as the source of the problem. In order to completely avoid OmpF contamination, a GST -a-hTTP fusion protein was purified from a glutathione agarose column followed by an on-column thrombin digestion to remove the GST tag. We then demonstrated that a-hTTP utilizes a collisional mechanism to deliver its ligand. Furthennore, a higher rate of a-tocopherol transfer to small unilamellar vesicles (SUV s) versus large unilamellar vesicles (LUV s) indicated that transfer is sensitive to membrane curvature. These findings suggest that ahTTP mediated a-Toc transfer is dominated by the hydrophobic nature of a-hTTP and the packing density of phospholipid head groups within acceptor membranes. Based on the calculated free energy change (dG) when a protein is transferred from water to the lipid bilayer, a model was generated to predict the orientation of a-hTTP when it interacts with lipid membranes. Guided by this model, several hydrophobic residues expected to penetrate deeply into the bilayer hydrophobic core, were mutated to either aspartate or alanine. Utilizing dual polarization interferometry and size exclusion vesicle binding assays, we identified the key residues for membrane binding to be F 165, F 169 and 1202. In addition, the rates of ligand transfer of the u-TTP mutants were directly correlated to their membrane binding capabilities, indicating that membrane binding was likely the rate limiting step in u-TTP mediated transfer of u-Toc. The propensity of u-TTP for highly curved membrane provides a connection to its colocalization with u-Toc in late endosomes.

The Ligand and Membrane-binding Behaviour of the Phosphatidylinositol Transfer Proteins (PITPa & PITPb)

Human Class I phosphatidylinositol transfer proteins (PITPs) exists in two forms: PITPα and PITPβ. PITPs are believed to be lipid transfer proteins based on their capacity to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments in vitro. In Drosophila, the PITP domain is found to be part of a multi-domain protein named retinal degeneration B (RdgBα). The PITP domain of RdgBα shares 40 % sequence identity with PITPα and has been shown to possess PI and PC binding and transfer activity. The detailed molecular mechanism of ligand transfer by the human PITPs and the Drosophila PITP domain remains to be fully established. Here, we investigated the membrane interactions of these proteins using dual polarization interferometry (DPI). DPI is a technique that measures protein binding affinity to a flat immobilized lipid bilayer. In addition, we also measured how quickly these proteins transfer their ligands to lipid vesicles using a fluorescence resonance energy transfer (FRET)-based assay. DPI investigations suggest that PITPβ had a two-fold higher affinity for membranes compared to PITPα. This was reflected by a four-fold faster ligand transfer rate for PITPβ in comparison to PITPα as determined by the FRET assay. Interestingly, DPI analysis also demonstrated that PI-bound human PITPs have lower membrane affinity compared to PC-bound PITPs. In addition, the FRET studies demonstrated the significance of membrane curvature in the ligand transfer rate of PITPs. The ligand transfer rate was higher when the accepting vesicles were highly curved. Furthermore, when the accepting vesicles contained phosphatidic acid (PA) which have smaller head groups, the transfer rate increased. In contrast, when the accepting vesicles contained phosphoinositides which have larger head groups, the transfer rate was diminished. However, PI, the favorite ligand of PITPs, or the presence of anionic lipids did not appear to influence the ligand transfer rate of PITPs. Both DPI and FRET examinations revealed that the PITP domain of RdgBα was able to bind to membranes. However, the RdgBα PITP domain appears to be a poor binder and transporter of PC.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.