Computational and experimental studies of bilayer peptide interactions

This thesis describes the combination of experimental (neutron diffraction) and computational techniques (molecular dynamics simulations) to investigate membrane peptide interactions.The first part deals with a comparison of human and rat form of the amyloid inducing peptide islet amyloid polypeptide (IAPP). Lamellar neutron diffraction was performed and a structural comparison on the differing modes of actions of the rat and human forms of IAPP are reported.A computational model for a di-oleoyl phosphatidylcholine (DOPC) bilayer was then constructed. Once this bilayer had been verified with experimental data (namely area per headgroup, volume per lipid, order parameter of the oleoyl chains and electron density profile) a mixed bilayer of DOPC and di-oleoyl phopshatidylglycerol (DOPG) was then constructed. The mixed bilayer was verified in the same mannerA peptide (adenosine diphosphate ribosylation factor-1 (pARF-1)) was then inserted into the pre-equilibrated mixed bilayer. The orientation of this peptide with respect to the membrane was based on previous neutron diffraction studies, carried out by other group members. Four possible orientations had resulted from analysis of the neutron data. The four orientations of pARF-1 were then subjected to molecular dynamics simulations. The time course of these simulations was 4 ns. The simulation's trajectories were analysed for each of the four models. Particular emphasis was placed upon the positional changes of the phenylalanine label positions that were derived from the neutron data. It was concluded that model A was the most likely orientation of pARF-1 in relation to the bilayer.Having established the technique, and confirmed that the most likely orientation of the peptide was what was originally proposed, another peptide, the fusion peptide of simian immunodeficiency virus (SIV) was placed into a previously equilibrated DOPC bilayer. In this case, only the proposed orientation of the SIV fusion peptide in relation to the bilayer was studied utilizing molecular dynamics simulations. The results are interpreted in relation to the actions of SIV fusion peptide upon the membrane, with particular emphasis on the disruption of oleoyl chain order parameters and secondary structure of the membrane bound fusion peptide

Neutron diffraction reveals sequence-specific membrane insertion of pre-fibrillar islet amyloid polypeptide and inhibition by rifampicin

AbstractHuman islet amyloid polypeptide (hIAPP) forms amyloid deposits in non-insulin-dependent diabetes mellitus (NIDDM). Pre-fibrillar hIAPP oligomers (in contrast to monomeric IAPP or mature fibrils) increase membrane permeability, suggesting an important role in the disease. In the first structural study of membrane-associated hIAPP, lamellar neutron diffraction shows that oligomeric hIAPP inserts into phospholipid bilayers, and extends across the membrane. Rifampicin, which inhibits hIAPP-induced membrane permeabilisation in functional studies, prevents membrane insertion. In contrast, rat IAPP (84% identical to hIAPP, but non-amyloidogenic) does not insert into bilayers. Our findings are consistent with the hypothesis that membrane-active pre-fibrillar hIAPP oligomers insert into beta cell membranes in NIDDM

Conformational effects on the Circular Dichroism of Human Carbonic Anhydrase II: a multilevel computational study

Circular Dichroism (CD) spectroscopy is a powerful method for investigating conformational changes in proteins and therefore has numerous applications in structural and molecular biology. Here a computational investigation of the CD spectrum of the Human Carbonic Anhydrase II (HCAII), with main focus on the near-UV CD spectra of the wild-type enzyme and it seven tryptophan mutant forms, is presented and compared to experimental studies. Multilevel computational methods (Molecular Dynamics, Semiempirical Quantum Mechanics, Time-Dependent Density Functional Theory) were applied in order to gain insight into the mechanisms of interaction between the aromatic chromophores within the protein environment and understand how the conformational flexibility of the protein influences these mechanisms. The analysis suggests that combining CD semi empirical calculations, crystal structures and molecular dynamics (MD) could help in achieving a better agreement between the computed and experimental protein spectra and provide some unique insight into the dynamic nature of the mechanisms of chromophore interactions

Balali-Mood K: Neurotoxic disorders of organophosphorus compounds and their managements

Organophosphorus compounds have been used as pesticides and as chemical warfare nerve agents. The mechanism of toxicity of organophosphorus compounds is the inhibition of acetylcholinesterase, which results in accumulation of acetylcholine and the continued stimulation of acetylcholine receptors. Therefore, they are also called anticholinesterase agents. Organophosphrus pesticides have largely been used worldwide, and poisoning by these agents, particularly in developing countries, is a serious health problem. Organophosphorus nerve agents were used by Iraqi army against Iranian combatants and even civilian population in 1983 -1988. They were also used for chemical terrorism in Japan in 1994 -1995. Their use is still a constant threat to the population. Therefore, medical and health professionals should be aware and learn more about the toxicology and proper management of organophosphorus poisoning. Determination of acetylcholinesterase and butyrylcholinesterase activity in blood remains a mainstay for the fast initial screening of organophosphorus compounds but lacks sensitivity and specificity. Quantitative analysis of organophosphorus compounds and their degradation products in plasma and urine by mass spectrometric methods may prove exposure but is expensive and is limited to specialized laboratories. However, history of exposure to organophosphorous compounds and clinical manifestations of a cholinergic syndrome are sufficient for management of the affected patients. The standard management of poisoning with organophosphorous compounds consists of decontamination, and injection of atropine sulfate with an oxime. Recent advances on treatment of organophosphorus pesticides poisoning revealed that blood alkalinization with sodium bicarbonate and also magnesium sulfate as adjunctive therapies are promising. Patients who receive prompt proper treatment usually recover from acute toxicity but may suffer from neurologic complications

A structural study of the myristoylated N-terminus of ARF1

The effect of myristoylation on the 15-amino-acid peptide from the membrane-binding N-terminus of ADP ribosylation factor 1 (ARF1) was studied using neutron diffraction and circular dichroism. A previous study on the non-acylated form indicated that the peptide lies parallel to the membrane, at a shallow depth and in the vicinity of the phosphorylcholine headgroups. It was suggested that the helix does not extend past residue 12, an important consequence for the linking region of the ARF1 protein. In this paper, we show that the result of myristoylation is to increase the helical content reaching the peptide's C-terminus, resulting in the formation of a new hydrophobic face. This increased helicity may augment the entire protein's membrane-binding affinity, indicating that ARF1 effectively has two interdependent membrane-binding motifs.NRC publication: Ye

Individual contributions of the aromatic chromophores to the near-UV Circular Dichroism in class A β-lactamases: A comparative computational analysis

Class A β-lactamases are enzymes which are responsible for the bacterial resistance against antibiotics and therefore are of great importance in rational inhibitor design. In this paper we comparatively analyze all the individual contributions of the aromatic chromophores in three class A β-lactamases (from Staphylococcus aureus, Streptomyces albus and Bacillus licheniformis) to their near-UV Circular Dichroism. The analysis is performed using recently developed procedure based on established theoretical method. We found that in β-lactamase from S. albus the most significant contributions to the total near-UV CD intensity exhibit Y251 and Y229. In the tryptophan-containing β-lactamases from B. licheniformis and S. albus, W229 and W251 express the strongest individual contributions. A comparative analysis of the individual contributions of conservative chromophores in class A enzymes namely W165, W210, W229, W251, Y97 and Y105 is presented

Cannabidiol lacks the vanilloid VR1-mediated vasorespiratory effects of capsaicin and anandamide in anaesthetised rats

The results of vasorespiratory studies in rats anaesthetised with pentobarbital show that (±) cannabidiol, a cannabinoid that lacks psychotropic actions and is inactive at cannabinoid (CB) receptors, does not affect respiration or blood pressure when injected (1–2000 μg; 3.2–6360 nmol i.a.). Cannabidiol in doses up to 2 mg (6360 nmol) i.a. or i.v. did not affect the fall in mean blood pressure or the increase in ventilation (respiratory minute volume) caused by capsaicin and high doses of anandamide, responses that are mediated by activation of vanilloid VR1 (TRPV1) receptors in this species. Similar results were obtained with (−) cannabidiol (30–100 μg i.a.; 95–318 nmol). It has previously been shown using human embryonic kidney (HEK) cells over-expressing vanilloid human VR1 (hVR1) receptors that cannabidiol is a full agonist at vanilloid VR1 receptors in vitro. However, in the intact rat cannabidiol lacked vanilloid VR1 receptor agonist effects. We conclude that there are substantial functional differences between human and rat vanilloid VR1 receptors with respect to the actions of cannabidiol as an agonist at vanilloid VR1 receptors. Studies in vivo show that cannabidiol lacks any significant effect on mean blood pressure or respiratory minute volume when injected i.a. or i.v., and that this cannabinoid does not modulate the vanilloid VR1 receptor-mediated cardiovascular and ventilatory changes reflexly evoked by capsaicin or anandamide in rats anaesthetised with pentobarbital