Direct affinity of dopamine to lipid membranes investigated by Nuclear Magnetic Resonance spectroscopy

Dopamine, a naturally occurring neurotransmitter, plays an important role in the brain’s reward system and acts on sensory receptors in the brain. Neurotransmitters are contained in lipid membraned vesicles and are released by exocytosis. All neurotransmitters interact with transport and receptor proteins in glial cells, on neuronal dendrites, and at the axonal button, and also must interact with membrane lipids. However, the extent of direct interaction between lipid membranes in the absence of receptors and transport proteins has not been extensively investigated. In this report, we use UV and NMR spectroscopy to determine the affinity and the orientation of dopamine interacting with lipid vesicles made of either phosphatidylcholine (PC) or phosphatidylserine (PS) lipids which are primary lipid components of synaptic vesicles. We quantify the interaction of dopamine's aromatic ring with lipid membranes using our newly developed method that involves reference spectra in hydrophobic environments. Our measurements show that dopamine interacts with lipid membranes primarily through the aromatic side opposite to the hydroxyl groups, with this aromatic side penetrating deeper into the hydrophobic region of the membrane. Since dopamine's activity involves its release into extracellular space, we have used our method to also investigate dopamine's release from lipid vesicles. We find that dopamine trapped inside PC and PS vesicles is released into the external solution despite its affinity to membranes. This result suggests that dopamine's interaction with lipid membranes is complex and involves both binding as well as permeation through lipid bilayers, a combination that could be an effective trigger for apoptosis of dopamine-generating cells

Hydrophobic Effects on Tyrosyl Ring 1H Chemical Shifts in Peptides

poster abstractHydrophobic environmental effects on tyrosine are measurable by 1H NMR spectroscopy and can allow us to detect interactions between peptides and lipid membranes. We first investigated the effects of hydrophobic environments on the 1H chemical shifts of tyrosine ring protons by using varying concentrations of isopropanol to mimic and calibrate the effects of hydrophobicity. Compared with this calibration, we then measured the interaction of tyrosine-containing peptides with sonicated unilamellar vesicles of phosholipids such as phosphatidylcholine and phosphatidylserine that are commonly found in biological membranes

Fluorescence Measurements of Aromatic Amino Acids in the Presence of Lipid Membranes

Amphiphilic peptides are capable of finding their way to, and occasionally through, cellular membranes using a mechanism that includes specific amino acid sequences. Physical measurements of amino acid-lipid interactions are of interest for a quantitative description of peptide affinities to biological membranes. In this study, we investigate small peptide-lipid interactions using the fluorescence of the aromatic amino acids tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe). Reference spectra in deuterated isopropanol solutions are obtained to mimic hydrophobic environments and are used to quantify the interaction of Lys-Tyr-Lys, Trp-Gly, and Gly-Phe with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and palmitoyl-oleoyl phosphatidylserine (POPS) lipid membranes. These fluorescence data complement previously reported UV absorption data and have the advantage of eliminating background and scatter from solution. Together with NMR data, these results can be used to more fully characterize lipid-aromatic amino residue interactions

DOMAIN STRUCTURE OF THE MAJOR ALLERGEN OVOMUCOID BY SOLUTION NMR

poster abstractThe interest in the ovomucoid protein is twofold. First it is a protein of interest for medical studies due to its potent allergen activity. Second, as a special variety of glycosylated protein (Kazal family), it allows us to explore the role of protein glycosylation in protein-membrane interactions for a particular, model case. The nature, location, and orientation of the glycosyl groups are determining factors in proteinmembrane interactions and therefore are critical to biological processes involving glycosylated proteins. We have found that as opposed to other glycosylated proteins, ovomucoid does not induce ionic currents across lipid membranes. This behavior likely has a structural cause, yet very little overall structural data is available. In this study, we use solution NMR spectroscopy to determine the structure of the chicken ovomucoid protein, taking advantage of the division of its structure into three stable domains of 55-65 amino acids each. We present results on the protein purification steps and isolation of separate domains suitable for solution NMR spectroscopy. We then present NMR results acquired on a 500 MHz spectrometer, and we show atomic models of individual domains and of overall protein structure from analysis of NMR spectra

1H NMR of Deep Eutectic Solvents

Deep Eutectic Solvents (DESs) form between a variety of quaternary ammonium or phosphonium salts and hydrogen-bond donors. Over the past decade, DESs have been studied as green solvents with potential applications in industrial processes, chemical extractions, and pharmaceuticals. The recent suggestion that many plants produce natural deep eutectic solvents (NADES) from primary metabolites led to investigation of the potential uses of DESs in biophysics research. This study examined the 1H NMR spectra of the choline chloride:urea 1:2, and choline chloride:ethylene glycol 1:3 molar ratio DES. Spectra of the choline chloride:urea 1:2 with various solutes were acquired to see what effect these solutes had on the DESs NMR spectrum. For both DESs tested, the NMR spectra were a superposition of the spectra of the components. DES-solute spectra showed that interaction between components persisted, indicating the solvent properties of the DESs were not lost upon addition of solutes

Detecting Counterfeit Pharmaceuticals through UV Spectrophotometry

poster abstractAccording to the World Health Organization between 10%-30% of medicines, in Africa, Asia and South America, are counterfeit or sub-standard, affecting the health of millions of people. Currently, there is no effective way to check the quality of a medicine at the point of care, leaving many with treatable diseases at risk. The goal of this study is to identify UV-Vis (240nm - 500nm) absorbance patterns that would indicate if a drug is sub-standard or counterfeit. UV-Vis spectroscopy was selected as the method for testing due to the maturity and availability of the technology. Pure Acetaminophen and Tylenol were used as controls for proof of concept. Samples were prepared by dissolving different combinations of the pure active ingredient and adulterants such as cement, rice flour, vitamin C and lactose in three different types of solvents (H2O, 0.1 M HCl, 0.1 NaOH). Various concentrations (ranging from 0.01mg/ml to 0.04mg/ml) and mixing ratios were analyzed using a UV-Vis Spectrophotometer. It was found that adulterants significantly decrease the absorption of acetaminophen at 245nm by interacting with its benzene ring, while showing a slight increase in other parts of the spectrum. UV-Vis scans show that the amount of change in absorbance at specific wavelengths, coupled with characteristic wavelength shifts produced by different solvents, can be used for detection of counterfeit drugs. The methods presented here could be used for quality control of medicines at or near the point of care in parts of the world at higher risk of encountering defective pharmaceuticals

Finding Active Ingredients in Pharmaceuticals by UV Spectrophotometry

The active ingredient in any pharmaceutical is the chemical that will ultimately deliver the desired effect on a patient. Knowing the quality and the quantity of the active ingredient in a pill right before ingestion is of paramount importance for the patient’s health and the desired results. Unfortunately drugs only undergo quality control testing at the manufacturing plant but not at the point of sale. Moreover, to an untrained eye, one pill may not appear different from another and if the wrong pill or the wrong dose is taken, adverse health effects may arise. Indeed, manufacturers of counterfeit drugs rely on these two points of appearance and testing. In this study we examine whether ultra violet (UV) spectrophotometry absorbance can be used to separate an active ingredient’s UV peaks from the combination of peaks generated by the inactive ingredients of the tablet. A well-understood active ingredient, acetaminophen, was used for this study. Samples were prepared by crushing Tylenol tablets, dissolving the powder in different solvents (0.1M HCl, 0.1M NaOH, and H2O) at various concentrations and mixed by vortex. After preparation, the samples were measured by UV Spectroscopy. Experimental results were compared to standard UV curves for the pure active ingredient to correlate the observed changes in absorbance within the relevant UV wavelength range. We observe that more than one solvent is needed to identify the active ingredient. Development of a simple method to accurately identify the quality of the active ingredient will provide an additional safeguard to consumers, particularly in regions where counterfeit drugs are prevalent

SOLUTION STRUCTURE OF THE TOXIC E. COLI PEPTIDE, TISB

poster abstractAntibiotics act by interfering in bacterial metabolism. Thus, antibiotics are only effective against metabolically active bacteria while dormant cells are highly tolerant to antibiotics. Such persistent bacterial cells may be the main culprits in chronic infectious diseases resistance to antimicrobial thera-py. In Escherichia coli, expression of a toxic peptide, TisB, sends cells into dormancy by decreasing the proton motive force thus decreasing ATP levels. TisB is a 29 amino acid residue peptide with 70% hydrophobic residues. It has a predicted alpha helical transmembrane domain spanning residues 6 - 28. In membrane channel studies, ion transport is observed with TisB and with some TisB mutants. As a preliminary to combining multi-dimensional NMR spectroscopy with circular dichroism to determine the structure of the TisB membrane ion transport complex in lipid micelles, NMR spectroscopy is used to determine the structure of TisB in ethanol