Bilayer thickness and thermal response of dimyristoylphosphatidylcholine unilamellar vesicles containing cholesterol, ergosterol and lanosterol: A small-angle neutron scattering studyIntroductionMaterials and methodsResultsVesicle size and polydispersityMembrane thicknessThermal area expansion coefficientsDiscussionConclusionsAcknowledgementsReferencesUNCORRECTED PROOFARTICLE IN PRESS12 Bilayer thickness and thermal response of dimyristoylphosphatidylcholine3 unilamellar vesicles containing cholesterol, ergosterol and lanosterol:4 A small-angle neutron scattering study5 Jeremy Pencera,b,c,*, Mu-Ping Nieha, Thad Harrouna,d, Susan Kruegerc,6 Carl Adamsb, John Katsarasa,d,e7aNRC, Canadian Neutron Beam Centre, Chalk River Labs, Chalk River, ON, Canada K0J 1J08bDepartment of Physics, St. Francis Xavier University, Antigonish, NS, Canada9cNIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD, USA10dDepartment of Physics, University of Guelph, Guelph, ON, Canada11eBiophysics Interdepartmental Group and Guelph-Waterloo Institute of Physics, University of Guelph, Guelph, ON, Canada12 Received 19 July 2005; received in revised form 19 October 2005; accepted 31 October 20051314 Abstract15 Small-angle neutron scattering (SANS) measurements are performed on pure dimyristoyl phosphatidylcholine (DMPC) unilamellar vesicles16 (ULV) and those containing either 20 or 47 mol% cholesterol, ergosterol or lanosterol. From the SANS data, we were able to determine the17 influence of these sterols on ULV bilayer thickness and vesicle area expansion coefficients. While these parameters have been determined18 previously for membranes containing cholesterol, to the best of our knowledge, this is the first time such results have been presented for19 membranes containing the structurally related sterols, ergosterol and lanosterol. At both molar concentrations and at temperatures ranging from 1020 to 45 -C, the addition of the different sterols leads to increases in bilayer thickness, relative to pure DMPC. We observe large differences in the21 influence of these sterols on the membrane thermal area expansion coefficient. All three sterols, however, produce very similar changes to22 membrane thickness.23 D 2005 Elsevier B.V. All rights reserved.2425 Keywords: Cholesterol; Ergosterol; Lanosterol; Unilamellar vesicle; Hydrophobic thickness; Small-angle neutron scattering; Dimyrisotylphosphatidylcholine26 (DMPC)2728 1. Introduction29 Membrane hydrophobic thickness has been identified as an30 important modulator for the insertion [1], folding [2] , multi-31 meric assembly [3] and function [4–7] of trans-membrane32 proteins. Since membrane protein reconstitution and activity33 studies often utilize unilamellar vesicles (e.g., [8]), ULV have34 become the focus of a number of studies pertaining to the35 bilayer’s hydrophobic thickness [9–11], and its modulation by36 cholesterol [12], proteins [13], solutes [14] and other additives37 [15]. Both SANS and small-angle X-ray scattering (SAXS)38have proven particularly useful for such studies, since the39scattering data yielded by these techniques are directly related40to vesicle structural parameters (e.g., [10,13]). It has been41hypothesized that the correlation between protein function and42cholesterol levels in membranes may be related to the43modulation of the membrane’s hydrophobic thickness (e.g.,44[7]). However, while cholesterol has been identified as a45modulator of membrane protein f unction ([16,17]), such46modulation could occur via any combination of fact ors47including: hydrophobic mismatch between the membrane and48transmembrane protein segments ([4 – 7]), membrane dynamics49[18], membrane elasticity [7], membrane lateral pre ssure50profile [19] , and membrane lateral organization [20], since51cholesterol affects all of these membrane properties. Thus, the52identification of the particular mechanisms for cholesterol53modulation of protein function clearly requires knowledge of0005-2736/$ - see front matter D 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.bbamem.2005.10.017* Corresponding author. NRC, Canadian Neutron Beam Centre, Chalk RiverLabs, Chalk River, ON, Canada. Tel.: +1 613 584 8811x6475; fax: +1 613 5844040.E-mail address: [email protected] (J. Pencer).Biochimica et Biophysica Acta xx (2005) xxx – xxxhttp://www.elsevier.com/locate/bbaBBAMEM-78951; No. of pages: 8; 4C:+ modelUNCORRECTED PROOFARTICLE IN PRESS54 how cholesterol modulates membrane properties, such as the55 hydrophobic thickness, elasticity, and lateral organization.56 Synthetic and naturally derived sterols that are structurally57 related to cholesterol have been utilized as biomolecular58 probes allowing researchers to identify the specific structural59 features of cholesterol that enable it to aid in the function of a60 variety of membrane proteins (e.g., [21,22] ). Such probes also61 present the opportunity to identify the structural features62 responsible for cholesterol’s abilities to modulate membrane63 permeability [23], elasticity [24,25], lateral organization64 [26,27], and acyl chain order [24,28–30]. As such, sterols65 that are structurally related to cholesterol can help us to66 elucidate cholesterol’s role in modulating the function of67 transmembrane prote ins.68 Ergosterol and lanosterol are two specific sterols that have69 attracted some interest in comparative studies with cholesterol70 [24,29–31]. Ergosterol differs from cholesterol in having a71 double bond at the base of its tail, joining C22 and C23, and an72 additional methyl group attached to C24 (Fig. 1). On the other73 hand, lanosterol has two additional methyl groups on the74 otherwise flat alpha face, attached to C4 and C14 and one75 additional methyl group on its beta face, also attached at the C476 position (Fig. 1). Similar to ergosterol, this sterol also has a77 double bond in its tail, joining C24 and C25. The dimensions78 of ergosterol and lanosterol (total length of the molecule and79 length of the rig id steroid portion) a re comparable to80 cholesterol.81Interest in comparisons of ergosterol and lanosterol to82cholesterol does not only arise from their structural sim ilar-83ities, but also their biosynthetic relationships. Ergosterol is84the final step in the biosynthetic pathway for sterols of a85variety of yeasts, thus presenting a functional evolutionary86alternative to cholesterol [32,33]. Lanosterol, on the