Distinct Water Species Confined at the Interface of a Phospholipid Membrane Victor V. Volkov, 1 D. Jason Palmer, 1 and Roberto Righini 1,2 1 European Laboratory for Nonlinear Spectroscopy (LENS), University of Florence, Via Nello Carrara 1, 50019 Sesto Fiorentino, Italy 2 Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy (Received 7 December 2006; published 15 August 2007) The physics of confined water has stimulated extensive research in recent years, in particular, regarding the role of hydrogen bonding as a significant factor in the observed dynamics. In this work, two- dimensional infrared spectroscopy was employed to investigate the response of the OH moiety of water in phospholipid membrane samples. The results show strong evidence for three distinct hydrogen bonding motifs (H 2 O with zero, one, or both OH moieties hydrogen bonded), whose relative proportions at the membrane interface are estimated. DOI: 10.1103/PhysRevLett.99.078302 PACS numbers: 82.53.k, 61.25.f, 78.47.+p Recent studies have identified membrane-mimetic phos- pholipid bilayers as a well-characterized and biologically relevant system for the isolation and study of various water structural motifs [1,2]. The structural stability and biologi- cal activity of a lipid membrane are known to be defined in part by the degree of hydration and the structural dynamics of water molecules at the cellular envelope polar interface [3 – 5]. Therefore, we focus here on the physical insight made available by the segregation of water in the disparate environments present in such samples. It is known from NMR [6 –10] and neutron scattering [3,11] experiments that water molecules occupy the bilayer interface with a steeply decreasing concentration towards the inner hydro- phobic region of the bilayer, an appraisal which has been confirmed by molecular dynamics (MD) simulations [12 – 16]. The majority of the water molecules residing in the polar region of the membrane are H bonded to the phos- phate groups, with a smaller fraction binding the carbonyl groups located deeper in the bilayer [12 –15]. In such an environment, water loses most of its bulk properties. Further, the situation at the membrane interface is appar- ently very different from that of water segregated in reverse micelles [2]. Unique properties of water at the polar inter- face of lipid membranes (see Fig. 1) arise due to the confinement of water molecules; here, interactive engage- ment of water with structural moieties of a phospholipid membrane determine the variability of water molecular forms. In this Letter, we provide spectral and structural identi- fication of the common aqueous structural motifs at the membrane interface using methods of linear and nonlinear infrared spectroscopies. The nonlinear time-resolved re- sponse was detected using a spectrally narrow (45 cm 1 FWHM) pump pulse of 400 fs, and a broadband (300 cm 1 FWHM) probe pulse of 80 fs. The pump frequency was scanned by adjusting a tunable Fabry-Perot etalon, and the transmitted probe beam is spectrally resolved by dispersion in a monochromator and imaging onto a multichannel infrared detector. This is a two-dimensional infrared hole burning technique, wherein the narrow band pump selects a subensemble of vibrational modes within an inhomogene- ous distribution [17]. The time delay between the pump and the probe pulses allows detection of the time evolution of the nonlinear signal of the selected (burned) molecular modes. The black line in Fig. 2(a) shows the steady state linear infrared spectrum of H 2 O stretching modes in dehydrated (lipid:water ratio of 2:1) membrane fragments of 1-palmi- toyl-2-linoleyl phosphatidylcholine (PLPC), whose struc- ture is depicted in Fig. 1. Comparison with the optical response of neat H 2 O (red dashed line) demonstrates that the spectrum of water at the interface is dominated by the hydrogen-bonded water species absorbing on the low fre- quency side of the band. Figures 2(b) and 2(c) represent the nonlinear two-dimensional infrared (2D-IR) spectra of FIG. 1 (color online). PLPC bilayer and phospholipid molecu- lar structures. The large (orange), medium (blue), and small (red) spheres represent phosphorus, nitrogen, and oxygen atoms, respectively. The water molecules are shown with cyan sticks. PRL 99, 078302 (2007) PHYSICAL REVIEW LETTERS week ending 17 AUGUST 2007 0031-9007= 07=99(7)=078302(4) 078302-1  2007 The American Physical Society