No Label Required: Protein Binding at Membrane Interfaces Visualized through Colloid Phase Transitions Claudia Steinem* [a] and Andreas Janshoff [b] In 1972, Singer and Nicolson proposed the fluid mosaic membrane model that is instructive for our current understand- ing of the structure and function of the biological membrane. The model pre- dicts lateral and rotational freedom and random distribution of molecular com- ponents in the membrane: ™a two di- mensional oriented solution of integral proteinsº.in the viscous phospholipid bilayer∫. [1] Based on this model, the plasma membrane of a living cell has been accepted as a semipermeable, ho- mogeneous cover separating the cell's interior from its environment. Thirty years later, this picture has been rein- vented by the idea that the biological membrane is not a homogeneous two- dimensional fluid in which proteins are embedded, but a structured system with nonrandom distribution of proteins and lipids. The term ™rafts∫ was created. [2] This idea has brought back lipid mem- branes and their functions into the focus of current research. One approach to elucidate the func- tion of particular components of a bio- logical membrane is to use artificial membranes. Succeeded by the pioneer- ing work of Brian and McConnell, [3] tech- niques to immobilize membranes on a solid support, such as glass, mica, metals, and polymers were developed and these solid-supported membranes (SSMs) in combination with surface-sen- sitive- and electrochemical methods turned out to be an invaluable tool to investigate particular functions of biolog- ical membranes. Besides various advan- tages such as long-term and mechanical stability, the major drawback of SSMs is, however, the direct contact of one leaflet of the lipid bilayer with the solid sup- port, which complicates the insertion of transmembrane proteins and may reduce the lateral mobility of the lipids. For the investigation of reactions occur- ring at the outer leaflet of the immobi- lized lipid membrane, these restrictions, however, do not hold. Thus, SSMs have been widely used to investigate pro- tein±receptor interactions occurring at the membrane's interface. Several tech- niques were developed to scrutiny li- gand±receptor couples. Some of them are highlighted here: If a fluorescence label is attached to the ligand or recep- tor, fluorescence-based techniques, such as total internal reflection fluorescence [4] and confocal laser scanning microscopy [5] can be used. Although fluorescence- based techniques are very sensitive, the question always arises of how to control the attachment of the fluorescence label and whether the label alters the kinetics and binding affinity of the ligand to the surface-bound receptors. This is not the case if label-free detection schemes are used. In this respect, surface plasmon resonance (SPR) spectroscopy, the quartz crystal microbalance (QCM), ellipsometry (ELL), and reflectometric interference spectroscopy (RIfS) are well-established techniques. [6] All these methods allow online monitoring of binding of a certain ligand to its receptor, which is embed- ded in a lipid bilayer attached to the solid substrate; in the case of SPR, ELL, and QCM this is a gold surface and in the case of RIfS it is a thin polymer layer (Figure 1a). From the time course of the transducer's response, which is depend- ent on the ligand concentration in solu- tion, binding kinetics, that is, the corre- sponding rate constants of adsorption and desorption as well as the equilibri- um binding constants can be extracted. Very recently, Groves and co-workers reported on a new label-free technique to detect and quantify binding of pro- tein ligands to immobilized membrane- confined receptors. They exploited phase transitions occurring in colloidal systems caused by changes in the pair interaction potential. [7] Colloidal particles self-assemble into a variety of ordered phases and they display intriguing phase transitions between gas, liquid, liquid- crystalline, and solid phases. In general, phase diagrams of colloidal systems are determined by their interaction forces: while simple systems of hard spheres ex- hibit merely fluid and crystal phases, the introduction of long-range attractive forces results in three-phase equilibria between gas, liquid and crystal phases. In the case of shorter-range attraction, equilibrium between gas and crystal phase is found, but the liquid±liquid transition becomes metastable. Though phase transitions have been studied for decades, the prediction of phase dia- grams is not always realized as recently reviewed by Anderson and Lekkerkerk- er. [8] Colloidal systems are often trapped in gel-like states, or become under- cooled or supersaturated. In many cases the end products strongly depend on the starting position in the phase dia- gram and this renders the prediction of a phase difficult or even impossible. Since the phase behavior of a colloidal system is dominated by the interaction potential between particles, it is desira- ble to find ways to tune it. Yethiraj and van Blaaderen [9] recently presented a col- loidal system in which both the repulsive range and the anisotropy of the pair in- [a] Prof. Dr. C. Steinem Institut f¸r Analytische Chemie, Chemo- und Biosensorik, Universit‰t Regensburg 93040 Regensburg (Germany) Fax: (+ 49)941-943-4491 E-mail: claudia.steinem@chemie.uni-regensburg.de [b] Prof. Dr. A. Janshoff Institut f¸r Physikalische Chemie Johannes-Gutenberg-Universit‰t Jakob-Welder-Weg 11, 55128 Mainz (Germany) ChemPhysChem 2004, 5, 1121±1124 DOI: 10.1002/cphc.200400092 ¹ 2004 Wiley-VCH Verlag GmbH&Co. 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