Vibrational Spectroscopic and Imaging Studies of Biomembranes

Zachary D. Schultz^a,b, Stephan J. Stranick^a, and Ira W. Levin^b

^a Surface and Microanalysis Science Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899

^b Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892

            The behavior and function of lipids in biological systems play an important part in cell behavior.  The relationship between lipids and proteins is important in cellular processes such as signaling and trafficking, but remain poorly understood.  The existence and characterization of lipid raft formation remains a subject of considerable interest and controversy within the context of biomembrane lipid microdomain organization.  Studies of these systems are complicated by interactions of exogenous probes.  Vibrational spectroscopic techniques (infrared and Raman) provide a label free methods of probing these systems. 

            Model bilayer systems provide useful heterogeneous assemblies for investigating specific component interactions giving rise to lipid microdomains.  An important question concerns the sizes of lipid rafts in membranes, for which various estimates arise from differing measurement techniques.  We present here infrared vibrational spectroscopic measurements as a method for determining the size of raft domains in model systems.  Since raft microdomains are commonly enriched in glycosphingolipids and cholesterol, we use a perdeuterated saturated chain galactocerebroside as a raft marker.  Correlation field splitting is observed in the CH₂ (CD₂) scissor and rocking modes when identical saturated acyl chains interact in a orthorhombic subcell configuration.  The number of interacting chains is determined from the magnitude of the splitting, permitting estimation of domain size.  This splitting is measured in the presence of various biomembrane components such as cholesterol, various saturated and unsaturated lipids, and peptides to assess the effect on domain size and overall bilayer heterogeneity. 

            Intact cells can also be characterized using vibrational microscopy.  The rod photoreceptor cell in the eye provides an interesting example.  The rod outer segment (ROS) contains a series of stacked membrane disks that contain rhodopsin, the initial photoreceptor protein in the vision cascade.  Indirect methods suggest that rhodopsin may be regulated by lipid gradients with the ROS.  Vibrational imaging allows us to probe the spatial distribution of lipids composition of intact cells.  Preliminary results indicate spectroscopic marker bands that can be correlated to the lipid composition and distribution.  

            This work represents important progress using the label free detection of vibrational spectroscopy toward the direct observation of lipid protein interactions in viable cells. 

Your name: Zachary D. Schultz

Mentor’s name: Stephan J. Stranick

Division: Surface and Microanalysis Science Division (837)

Laboratory: Chemical Science and Technology Laboratory

Room and Building address: B217 Bldg. 224

Mail Stop: 372

Telephone #: (301) 975-6969

FAX #: (301) 926-6689

Email: zachary.schultz@nist.gov

Sigma Xi member: No

Category: Biology