A ABSTRACT At biological system and its surrounding physio- - PDF document

ARTICLE Spatial Distribution of Lipid Headgroups and Water Molecules at Membrane/Water Interfaces Visualized by Three-Dimensional Scanning Force Microscopy Hitoshi Asakawa, † Shunsuke Yoshioka, ‡ Ken-ichi Nishimura, ‡ and Takeshi Fukuma †,‡, * † Bio-AFM Frontier Research Center and ‡ Division of Electrical and Computer Engineering, Kanazawa University, Kanazawa, Japan t the interface between a biological A ABSTRACT At biological system and its surrounding physio- interfaces, fl exible surface logical solution, water molecules in- teract with biomolecules constituting the structures and mobile water surface. Through the interaction, water mol- interact with each other to ecules have signi fi cant in fl uence on the present non-uniform three- structure and function of biomolecules dimensional (3D) distribu- and their assembly. 1 � 3 Therefore, under- tions. In spite of their impact standing the structure and function of a on biological functions, molecular-scale understanding of such phenomena has remained biological system requires investigations on the behavior of interfacial water. The elusive. Here we show direct visualization of such interfacial structures with subnanometer- surface of a biological membrane mainly scale resolution by 3D scanning force microscopy (3D-SFM). We measured a 3D force consists of hydrophilic lipid headgroups. distribution at an interface between a model biological membrane and bu ff er solution by So far, the membrane/water interface has scanning a sharp tip within the 3D interfacial space. We found that vertical cross sections of the been extensively studied by various techni- ques. 4 � 11 These previous works have shown 3D image taken along a speci fi c lateral direction show characteristic molecular-scale contrasts that the water molecules adjacent to a tilted at 30 � to the membrane surface. Detailed analysis of the 3D image reveals that the tilted membrane strongly interact with the head- contrast corresponds to the time-averaged conformation of fl uctuating lipid headgroups. On groups and have signi fi cant in fl uence on its the basis of the obtained results, we discuss the relationships among the hydration structure, mechanical strength and fl uidity. 12 � 14 How- headgroup fl uctuation, molecular fl uidity, and mechanical strength of the membrane. The ever, the molecular-scale origin for such a results demonstrate that 3D-SFM is capable of visualizing averaged 3D distribution of critical in fl uence has remained elusive. fl uctuating surface structures as well as that of mobile water ( i.e. , hydration structure) at One of the major di ffi cultiesinsuchastudy interfaces between biological systems and water. lies in the measurement of molecular-scale structure of a membrane/water interface. As KEYWORDS: biological interface . lipid headgroup . the lipid headgroups exhibit thermal fl uctua- 3D scanning force microscopy tions, the surface structure of a membrane is inherently ill-de fi ned. In addition, the fl uctu- is scanned in XY directions ( i.e. , parallel to ating lipid headgroups interact with mobile the surface) on a sample to produce a two- water, through which the interfacial water dimensional (2D) image of the surface topo- presents non-uniform density distribution graphy. In contrast, several methods have known as hydration structure. Thus, the un- recently been proposed for imaging 3D dis- derstanding of the whole structure of the * Address correspondence to fukuma@sta ff .kanazawa-u.ac.jp. tribution of forces acting on a tip ( F t ) near the membrane/water interface should require a sample surface. 16 � 21 In these methods, a tip method to visualize three-dimensional (3D) Received for review July 12, 2012 is scanned in Z direction ( i.e. , perpendicular distribution of mobile water as well as fl uc- and accepted September 25, 2012. tuating lipid headgroups. to the surface) as well as in XY directions Published online September 25, 2012 Atomic force microscopy (AFM) 15 has been to image the whole 3D interfacial space. 10.1021/nn303229j widely used as a nanoscale surface imag- Among the proposed methods, 3D scanning force microscopy (3D-SFM) 20 has the fastest ing tool. In a typical AFM setup, a sharp tip C 2012 American Chemical Society ’ NO. 10 ’ 9013 – 9020 ’ 2012 ASAKAWA ET AL. VOL. 6 9013 www.acsnano.org

ARTICLE imaging speed and hence is suitable for liquid envi- exhibit much larger thermal fl uctuation even in the gel ronment applications where nonlinear tip drift is di ffi - phase due to the gap between adjacent headgroups. cult to avoid. We performed 3D-SFM imaging at the interface Previously, 3D-SFM has been used for imaging 3D F t between the DPPC bilayer and HEPES solution. In distribution at a mica/water interface. 20 The obtained 3D 3D-SFM, the tip is laterally scanned on a sample as in image showed subnanometer-scale contrastscorrespond- the case of conventional AFM. During the scan, the ingtothespatialdistributionofahydrationlayerandwater vertical tip position ( z t ) is modulated with a sine wave molecules adsorbed on the surface. This previous work faster than the bandwidth of the tip � sample distance highlighted the unique capability of 3D-SFM to visualize regulation (Figure 1b). The F t values varied by the z t hydration structures. However, the method has not been modulation are recorded in real time to construct a 3D used for investigating a biological system. Thus, it has F t image. In this experiment, z t is modulated at 200 Hz remained unknown how the fl uctuating biomolecules with 1.73 nm p � p amplitude while the tip is laterally and interfacial water are visualized and what informa- scanned at 12.2 nm/s. During the scan, the oscillation tion is obtained regarding their in fl uence on the struc- amplitude of the cantilever ( A ) was kept constant at ture and functions of the biological system. 0.095 nm. The variation of F t was detected as a shift In this study, we investigate the membrane/water ( Δ f ) of the cantilever resonance frequency caused by interface by 3D-SFM. We analyze subnanometer-scale F t , namely, using frequency modulation (FM) detection contrasts of the measured 3D-SFM image and correlate method. In a single z t modulation cycle, approaching them to the 3D distribution of interfacial water and and retracting Δ f curves are obtained. In this study, fl uctuating lipid headgroups. We also discuss the we collected approaching Δ f curves at each XY posi- tion to construct a 3D Δ f image (4 � 4 � 1.73 nm 3 , 64 � in fl uence of hydration phenomena on the structure and dynamics of the lipid membrane. 64 � 192 pixels, 53 s per 3D image). Figure 2a shows a model of the interface between RESULTS AND DISCUSSION the DPPC bilayer and water. The model consists of XY and Z cross sections extracted from the 3D Δ f image. In this study, we have investigated a dipalmitoylpho- Figure 2b shows a Δ f versus distance curve averaged sphatidylcholine (DPPC) bilayer supported by mica. over an XY cross section at each z t . The Δ f curve shows DPPC is one of the major phospholipids constituting a gradual increase with oscillatory peaks. These fea- a biological membrane. Thus, a DPPC bilayer has widely been used as a model biological membrane. 22 � 24 A tures suggest the existence of a repulsive long-range DPPC has a tail group consisting of two acyl chains and force ( F LR ) and an oscillatory short-range force ( F SR ). a headgroup consisting of a zwitterionic phosphatidyl- We converted the Δ f curve to F t versus distance curve using the formula proposed by Sader and Jarvis. 25 The F t choline (PC) group (Figure 1a). In aqueous solution, DPPC molecules form a bilayer with the hydrophobic curve (Figure 2c) shows that the oscillatory F SR is much tail groups separated from water and the hydrophilic smallerthan F LR .However,thein fl uenceof F SR isevidentin headgroups adjacent to water (Figure 1b). At room the Δ f curve.ThisisbecauseFM-AFMhasahighsensitivity temperature, a DPPC bilayer is in the gel phase where to the force component with an interaction length com- parable to A (0.095 nm in this experiment). 26 the acyl chains are closely packed to exhibit relatively small thermal fl uctuation. However, the headgroups In the previous study, Δ f curves measured on a DPPC bilayer in phosphate bu ff ered saline (PBS) solu- tion showed an oscillatory pro fi le without in fl uence of F LR . 11 This is due to the di ff erence in the solution conditions used for the imaging and sample prepara- tion. In fact, we experimentally con fi rmed the systema- tic di ff erence between the Δ f curves measured in HEPES and PBS solutions using di ff erent tips (see Supporting Information, Figure S1). We estimated the F LR component by fi tting an exponential function to the F t curve (dotted line in Figure 2c). We subtracted it from the original curve to obtain the F SR component (Figure 2d). For the experi- ments in vacuum, F LR originates from the electrostatic and van der Waals interactions. Thus, functions propor- � 1 or z t � 2 are often used for the fi tting. 27 In tional to z t Figure 1. (a) Molecular structure of DPPC consisting of liquid, these force components are signi fi cantly sup- a phosphatidylcholine (PC) headgroup and acyl chains. (b) Illustration of 3D-SFM imaging at an interface between pressed, whereas the contribution of the hydration a DPPC bilayer and HEPES bu ff er solution. A tip is scanned in force becomes evident. 28 The distance dependence of Z direction as well as in XY directions to image the whole 3D the hydration force varies depending on the solution space at the interface. ’ NO. 10 ’ 9013 – 9020 ’ 2012 ASAKAWA ET AL. VOL. 6 9014 www.acsnano.org