Protein internal reflectance fluorescence

Protein adsorption has been studied with a variety of techniques such as ellipsome-try [107,108], ESCA [109], surface forces measurements [102], total internal reflection fluorescence (TIRE) [103,110], electron microscopy [111], and electrokinetic measurement of latex particles [112,113] and capillaries [114], The TIRE technique has recently been adapted to observe surface diffusion [106] and orientation [IIS] in adsorbed layers. These experiments point toward the significant influence of the protein-surface interaction on the adsorption characteristics [105,108,110]. A very important interaction is due to the hydrophobic interaction between parts of the protein and polymeric surfaces [18], although often electrostatic interactions are also influential [ 116]. Protein desorption can be affected by altering the pH [117] or by the introduction of a complexing agent [118]. 

S. A. Rockhold, R. D. Quinn, R. A. VanWegenen, J. D. Andrade, and M. Reichert, Total internal reflection fluorescence (TIRF) as a quantitative probe of protein adsorption,... 

D. Horsley, J. Herron, V. Hlady, and J. D. Andrade, Human and hen lysozyme adsorption A comparative study using total internal reflection fluorescence spectroscopy and molecular graphics, in Proteins at Interfaces Physicochemical and Biochemical Studies (J. L. Brash and T. A. Horbett, eds.), ACS Symposium Series No. 343, pp. 290-305, American Chemical Society, Washington, D.C. (1987). 

V. Hlady, D. R. Reinecke, and J. D. Andrade, Fluorescence of adsorbed protein layers Quantitation of total internal reflection fluorescence, / Colloid Interface Sci. Ill, 555-569... 

W. M. Reichert, J. T. Suci, J. T. Ives, and J. D. Andrade, Evanescent detection of adsorbed protein concentration-distance profiles Fit of simple models to variable-angle total internal reflection fluorescence data, Appl. Spectrosc. 41, 503-507 (1987). 

E. Kalb, J. Engel, and L. K. Tamm, Binding proteins to specific target sites in membranes measured by total internal reflection fluorescence microscopy, Biochemistry 29, 1607-1613 (1990). 

The tendency for an adsorbed protein to undergo conformational change is protein-and interface-specific, as well as time-dependent. Total internal reflection fluorescence (TIRF) studies on IgG adsorption and desorption on hydrophobic and hydrophilic surfaces as a function of residence time show clearly both the time-dependence, as well as the surface-dependence, of desorption 92 ... 

To discriminate between surface bound protein molecules and those in bulk solution, total internal reflection fluorescence microscopy (TIRFM)41 55 was employed. TIRFM creates an evanescence wave that decays as a function of distance from the surface as ... 

N. L. Thompson, A. W. Drake, L. Chen, and W. V. Broek, Equilibrium, kinetics, diffusion and selfassociation of proteins at membrane surfaces Measurement by total internal reflection fluorescence microscopy, Photochem. Photobiol. 65, 39-46 (1997). 

A new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labelled solute molecules at surfaces was developed by Thompson et al.74). The technique combines total internal reflection fluorescence with either fluorescence photobleaching recovery or fluorescence correlation spectroscopy with lasers. For example, fluorescent labelled protein was studied in regard to the surface chemistry of blood 75). 

The three-dimensional network of actin stress fiber, which is an association of actin filaments, provides mechanical support for the cell, determines the cell shape, and enables cell movement. Thus the shape change in the cell due to the laser tsunami can be examined by observing the laser-induced dynamics of fibers. The actin stress fiber was visualized by binding it with enhanced green fluorescence protein (EGFP), and monitored by total internal reflection fluorescence (TIRF) imaging [34]. 

Total internal reflection fluorescence spectroscopy has been used to assay the fluorescence of tryptophan in proteins or of fluorescence markers. Morphine has been determined in this manner with a detection limit of 0.2 pmol/l on a quartz support bearing immobilized fluorescein-labeled antihapten (Kronick and Little, 1973). 

As a consequence, researchers from different disciplines of the life sciences ask for efficient and sensitive techniques to characterize protein binding to and release from natural and artificial membranes. Native biological membranes are often substituted by artificial lipid bilayers bearing only a limifed number of components and rendering the experiment more simple, which permits the extraction of real quantitative information from binding experiments. Adsorption and desorption are characterized by rate constants that reflect the interaction potential between the protein and the membrane interface. Rate constants of adsorption and desorption can be quantified by means of sensitive optical techniques such as surface plasmon resonance spectroscopy (SPR), ellipsometry (ELL), reflection interference spectroscopy (RIfS), and total internal reflection fluorescence microscopy (TIRE), as well as acoustic/mechanical devices such as the quartz crystal microbalance (QCM)... 

A variety of other techniques has been used to examine the structure of proteins at surfaces, including electron microscopy (50,51), ellipsometry (52), electrophoretic mobility (53), and total internal reflection fluorescence (TIRF) (54). Several new techniques are being applied at present, including Fourier transform infrared spectroscopy (FTIR) and TIRF (see next section),... 

The application of total internal reflection fluorescence spectroscopy (TIRF) by this laboratory to the study of protein adsorption at solid-liquid interfaces is reviewed. TIRF has been used to determine adsorption isotherms and adsorption rates from single-and multi-component protein solutions. Initial adsorption rates of BSA can be explained qualitatively by the properties of the adsorbing surface. Most recently, a TIRF study using monoclonal antibodies to probe the conformation of adsorbed sperm whale myoglobin (Mb) elucidated two aspects of the Mb adsorption process 1) Mb adsorbs in a non-random manner. 2) Conformational changes of adsorbed Mb, if they occur, are minor and confined to local regions of the molecule. Fluorescence energy transfer and proteolytic enzyme techniques, when coupled with TIRF, can characterize, respectively, the conformation and orientation of adsorbed Mb. 

Current understanding of protein adsorption has been synthesized from investigations employing a number of techniques. One of these techniques, total internal reflection fluorescence (TIRF), has emerged as a versatile tool for studying proteins at surfaces. The investigation of protein adsorption at solid-liquid interfaces in this laboratory using TIRF is reviewed. Several reviews of the TIRF technique have appeared recently (13-15). 

Key words Confocal fluorescence microscopy, Forster resonance energy transfer, Total internal reflection fluorescence microscopy, Single-molecule imaging, GPCR, Heterotrimeric G-proteins, Spatiotemporal dynamics... 

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