Structure of Fully Hydrated Fluid Phase DMPC and DLPC Lipid BilayersUsing X-Ray Scattering from Oriented Multilamellar Arrays and fromUnilamellar VesiclesNorbert Kucˇerka,* Yufeng Liu,* Nanjun Chu,* Horia I. Petrache,zStephanie Tristram-Nagle,yandJohn F. Nagle*y*Physics andyBiological Sciences Departments, Carnegie Mel lon University, Pittsburgh, Pennsylvania; andzLaboratory ofPhysical and Structural Biology, The National Institute of Child Health and Human Development, National Institutes of Health,Bethesda, Marylan dABSTRACT Quantitative structures of the fully hydrated fluid phases of dimyristoylphosphatidylcholine (DMPC) anddilauroylphosphatidylcholine (DLPC) were obtained at 30°C. Data for the relative form factors F(qz) for DMPC were obtainedusing a combination of four methods. 1), Volumetric data provided F(0). 2), Diffuse x-ray scattering from oriented stacks ofbilayers provided relative form factors jF(qz)j for high qz, 0.22 , qz, 0.8 A˚1. 3), X-ray scattering from extruded unilamellarvesicles with diameter 600 A˚provided jF(qz)j for low qz, 0.1 , qz, 0.3 A˚1. 4), Previous measurements using a liquidcrystallographic x-ray method provided jF(2p h/D)j for h ¼ 1 and 2 for a range of nearly fully hydrated D-spacings. The data frommethod 4 overlap and validate the new unilamellar vesicles data for DMPC, so method 4 is not required for DLPC or futurestudies. We used hybrid electron density models to obtain structural results from these form factors. Comparison of the modelelectron density profiles with that of gel phase DMPC provides areas per lipid A, 60.6 6 0.5 A˚2for DMPC and 63.2 6 0.5 A˚2forDLPC. Constraints on the model provided by volume measurements and component volumes obtained from simulations put theelectron density profiles r(z) and the corresponding form factors F(qz) on absolute scales. Various thicknesses, such as thehydrophobic thickness and the steric thickness, are obtained and compared to literature values.INTRODUCTIONThe phospholipid bilayer is the structural foundation ofbiomembranes and structural information about lipid bilayersis widely used as basic information to help model biomem-brane structure and the functions that take place therein.Structural information is not easy to obtain, however, for themost biologically relevant states of lipid bilayers, namelyfully hydrated, fluid (liquid-disordered Laor even liquid-ordered) phases. The fluidity precludes an atomic level struc-ture within single bilayers. Additionally, traditional arrays ofbilayers that provide strong diffraction signals form liquidcrystals, not crystals. Therefore, conventional crystallographicanalysis breaks down when ample water enters between thebilayers, as occurs for fully hydrated phosphatidyl choline(PC) lipids (Na gle and Tristram-Nagle, 2000) and chargedlipids (Petrache et al., 2004). Partially dehydrating fluidsamples introduces interbilayer forces that distort the struc-ture in ways that become unpredictably nonlinear when theequivalent osmotic pressure exceeds 100 atm (93% relativehumidity (RH)). It is therefore preferable to obtain bilayerstructure for the fully hydrated state in which the bilayers arefar enough apart that interbilayer interacti ons only negligiblymodify the structure compared to isolated bilayers. The abilityto obtain structure from fully hydrated samples leads to thepossibility that peptides and other additives can be studiedwith less concern that there is insufficient water to allow anaqueous alternative for parts or all of the additives.An alternative approach to the structural problem,pioneered by Wilkins et al. (1971), uses unilamellar vesicles(ULV) instead of liquid crystalline arrays. Then the primaryx-ray scattering data straightforwardly provide the continuousform factor F(qz), which is the Fourier transform of theelectron density profile r(z), provided that the vesicles aresufficiently dilute that the interference factor, often called thestructure factor, S(q), is a constant as a function of the scat-tering vector q. This requires that the concentration of lipid be,2% by weight when the ULV diameter is ;600 A˚(Kiselevet al., 2003), which predicates that the scattering is relativelyweak compared to diffraction from bilayer arrays. However,ample scattering above background can be obtained for smallvalues of qz(Kucˇerka et al., 2004b). In this article we have, forthe first time, combined ULV x-ray data with our primaryx-ray data from oriented liquid crystalline arrays of bilayers ina unified analysis.In our earlier modified Caille´ theory (MCT ) method forobtaining fully hydrated structure, the samples were multi-lamellar vesicles (MLV), which are essentially unorientedstacks of bilayers, and the previous results have been re-viewed (Nagle and Tristram-Nagle, 2000). The MCT methodfit the line shapes of the diffraction peaks using liquid crystalscattering theory (Caille´, 1972) to extrapolate the total in-tensity in each diffraction order. These intensities were thenanalyzed, essentially using a crystallographic approach.These earlier MCT results obtained for DMPC (PetracheSubmitted November 19, 2004, and accepted for publication January 3, 2005.Address reprint requests to John F. Nagle, E-mail: [email protected].Ó 2005 by the Biophysical Society0006-3495/05/04/2626/12 $2.00doi: 10.1529/biophysj.104.0566062626 Biophysical Journal Volume 88 April 2005 2626–2637et al., 1998b) confirm our new results from ULV, so only datafrom ULV were obtained for DLPC.Our newer method uses oriented stacks of fully hydratedbilayer arrays. This method defocuses from the peaks, theirshapes, and their overall integrated intensity, and insteadfocuses on the diffuse scattering away from the diffractionpeaks. Because diffuse scattering is relatively weak, this re-quires great er x-ray flux which, in practice, limits instrumen-tal resolution and therefore peak shapes are not resolved.Instead of discrete form factors F(2ph/D) for integral ordersh, this method obtains continuous form factors F(qz). Diffusescattering was previously used for unoriented MLV samplesand applied to POPC by Pabst et al. (2000); form factors wereobtained for qzup to 0.5 A˚1. Our laboratory has developedthe use of diffuse scattering for oriented samples, and we haveobtained continuous form factors for fully hydrated DOPC at30°C for qzup to 0.8 A˚1. This development provides betterspatial resolution of bilayer structure than all previousmethods (Lyatskaya et al.,