Wednesday 10 July 2013, Sidlaw Room, 11:00-13:00
Biological structure and function
(invited) Single-crystal neutron structural studies of carbonic anhydrase: Elucidating enzyme mechanism and aiding drug design
R McKenna 1 , Z Fisher 2 , M Aggarwal 1 and D N Silverman 1
1 University of Florida, USA, 2 Los Alamos National Laboratory, USA
The carbonic anhydrases (CA) comprise of a group of well-studied and distinct gene families (a,b and γ ) of mostly zinc-metalloenzymes that catalyze the hydration of carbon dioxide. These ubiquitous enzymes, present throughout virtually all living organisms, including animals, plants, algae, bacteria, and archaebacteria, are involved in a vast number of physiological processes and therefore are a uniquely broad and interesting family of enzymes, since the reaction they catalyze is linked to respiration, acid-base homeostasis, photosynthesis, and other biosynthetic pathways. The mammalian a-class of CA is comprised of 14 expressed isozymes (CA I – XIV) with varying tissue distributions and catalytic activity. Human CA II is the most extensively studied of these isozymes, as it has widespread tissue distribution and because it is the most efficient of the HCAs with a catalytic turnover rate of 10 6 s -1 .
From a basic science stand-point, HCA II is an excellent model system for the study of an enzyme mechanism and proton transfer, the rate limiting step in CA catalyzes. Recent studies, combining high-resolution x-ray and medium resolution neutron crystallography, with molecular dynamics and kinetic studies have yielded a new level of understanding of the proton transfer mechanism.
In addition, from an applied science application, the broad involvement of CA in physiological processes makes it an attractive drug target in the treatment of human diseases such as glaucoma and cancer, and in industrial uses for carbon sequestration.
Both current basic science and application studies of CA will be discussed and the possible future directions of CA studies will be considered.
(invited) Planar lipid bilayers structural charaterization: Moving towards complexity
G Fragneto
Institut Laue-Langevin, France
Neutron and X-ray reflectometry are now established techniques for the study of bio-interfaces. The importance and progress of their complementary aspects will be outlined [1].
One of the recent advances in the field of membrane biophysics is the discovery of the existence of coexisting micro-domains within a single membrane, important for regulating some signaling pathways. Many important properties of these domains remain poorly characterized.
coupled to deuterium labeling provide the possibility to highlight signal from these domains.
Recent results on the structural characterization of complex biomimetic systems via neutron reflectometry will be presented. These will include the structural characterization of the effect of cholesterol on the distribution of the ganglioside GM1 in model membranes [2] as well as on the action of a Feline Immunodeficiency Virus peptide [3].
The importance of using an appropriate model system will be pointed out [4] as well as the usefulness of model systems in the study of the effect of nanoparticles on membranes [5].
Current efforts towards the structural characterization of different reconstituted membranes extracted from fully deuterated
cells and their interaction with the antifungal amphotericin B (AmB) by means of neutron
ICNS 2013 International Conference on Neutron Scattering

reflectivity. It has been shown that AmB interacts with ergosterol –the main sterol in fungal cell- by forming channel which leads to the cell death. In this study results from three different membrane systems reconstituted and brought in contact with AmB will be presented. The first membrane contained only polar lipids, i.e. the glycerophosphospholipids and sphingolipids. The second one contained polar lipids and sterols. The third one contains polar and nonpolar lipids (di- and tri-glycerides, fatty acids, cholesteryl ester) without sterols [6].
[1] A. Hemmerle, L. Malaquin, T. Charitat, S. Lecuyer, G. Fragneto, J. Daillant, PNAS (2012)
[2] V. Rondelli, G. Fragneto , S. Motta, E. Del Favero, P. Brocca, S. Sonnino, L. Cantù, BBA 1818 (2012) 2860
[3] G. Vitiello et al., Soft Matter 2013, accepted
[4] Y. Gerelli , L. Porcar, G. Fragneto, Langmuir 28 (2012) 15922
[5] M. Maccarini et al., Langmuir 2013, accepted
[6] A. deGhellinck et al., in preparation
The interaction of amyloid-ß with membranes in Alzheimer’s disease
T Hauß 1 , H Karastaneva 1 , A Buchsteiner 2 and N A Dencher 3
1 Helmholtz-Zentrum Berlin für Materialien und Energie, Germany, 2 Forschungszentrum Jülich, Germany, 3 Technische
Universität Darmstadt, Germany
Alzheimer’s disease is the most common cause of dementia with symptoms of gradually progressing loss of memory and finally leads to death. In the case of sporadic Alzheimer’s disease the symptoms appear at an age of about 65 and the prevalence is increasing rapidly with age. Last estimates by the WHO number the cases worldwide to 115 millions in 2050, more than double the number of today. As of today, no treatment to delay or halt the progression of Alzheimer's disease is available. Amyloid-ß peptides (Aß) are the main constituents of senile plaques and are considered as pathological hallmark in Alzheimer's disease. Recently, attention has become focussed on prefibrillar monomeric Aß and small oligomers of Aß  as a trigger of Alzheimer's disease. Unlike in senile plaques,
Aß  monomers and oligomers are water-soluble and membrane active. We are engaged in the study of the interaction of monomeric or small oligomeric Aß with biological model membranes to shed light on this particular aspect of a probable molecular origin of Alzheimer’s disease. In a series of neutron diffraction and quasi-elastic neutron scattering experiments in combination with specific deuteration we were able to demonstrate changes in membrane structure and dynamics caused by Aß. In this contribution we will present new findings on the dynamical and structural nature of membranes in Alzheimer’s disease.
Adsorption and activity of Thermomyces Lanuginosus Lipase (TLL) at the oil-water interface using neutron reflectometry
T Nylander 1 , H Wacklin 2 , A Svendsen 3 , M Skoda 4 , J Webster 4 and A Zarbakhsh 5
1 Lund University, Sweden, 2 European Spallation Source ESS AB, Sweden,
UK, 5 Queen Mary University of London, UK
3 Novozymes A/S, Denmark, 4 ISIS,
This study aims to provide a better understanding of the lipid-aqueous interface structure due to the action of lipases on the lipid, e.g. triglycerides. Due to the lipolysis products (e.g. di- and mono-glycerides and fatty acids) formed from triglycerides, the interfacial lipid layer undergo sequential changes with respect to the liquid crystalline structure also affected by, e.g. pH and ionic strength. So far most studies of such processes only concern the interfacial reactions, using monolayers that cannot be formed from triglycerides. It is also challenging to get detail information on oil-water interfacial changes from studies of emulsion system. We used the recently developed spinfreeze-thaw technique to form stable, uniform and ~2 μm thick triglyceride films. Since the triglyceride it self is the natural substrate for the wild type lipase, we were able to study lipolysis at the triglyceride oil-water interface, under conditions that could mimic those in the gastro intestinal tract as well as in laundry applications. The use of htriolein and d-triolein with contrast matching allowed us to capture the structural changes occurring at the lipid
ICNS 2013 International Conference on Neutron Scattering

aqueous interface. Wild type and inactive mutant of TLL made it possible to separate the effect of enzyme adsorption from changes due to activity. We found that the inactive mutant shows enzyme monolayer adsorption only, while the wild type activity lead to the formation of a liquid crystalline phase that depends on solution pH.
Structure and function of potassium ion-channel membrane proteins vectorially-oriented in lipid bilayer membranes at solid/liquid interface via x-ray and neutro
S Gupta
Drexel University, USA
Voltage–gated ion channels (VGIC) constitute a superfamily of integral membrane proteins forming selective pore for potassium, sodium, or calcium ions stimulated by transmembrane electric potentials. They underlie various physiological processes, most notably, the generation and propagation of action potentials during neurotransmission. Among VGICs, the potassium (Kv-) channels are prototypical examples. The molecular architecture of
KvAP (from Aeropyrum pernix) channel consists of a homo-tetramer with 6 transmembrane helices in the monomeric unit, in which the S1-S4 helices form the voltage-sensor domain (VSD) conferirng voltage sensitivity to
KvAP switching it from a resting to an activated state in response to membrane depolarization. The S5-S6 helices contribute to form the pore domain (PD) in the tetrameric protein. The cooperative mechanism of electromechanical coupling that leads to the switching is highly dependent on the properties of the host lipid bilayer. Vectoriallyoriented self-assembled monolayers of isolated VSD and full-length KvAP channels are prepared and reconstituted into a single phospholipid bilayer studied via neutron and X-ray reflectivity at solid/liquid interface providing 1-D scattering length density profiles elucidating the spatial extent and uni-directionality of the transmebrane helices in retention of protein’s 3-D tertiary structure [Gupta et al. Phys. Rev E. 2011, 84; Gupta et al. Langmuir 2012, 28].
Tthe distribution of water solvating the proteins and exchangeable hydrogens throughout the profile structure are also determined. Financially supported as a research fellowship from the NIH/NINDS Program Project P01
GM086685 awarded to Prof. J. K. Blasie (University of Pennsylvania).
ICNS 2013 International Conference on Neutron Scattering