Conformation adsorbed protein

A major area for new research concerns the structural and functional consequences of adsorption of proteins to surfaces (items 3-5 in Table IV). Measurement of conformational change is still in an early stage of development. Most methods for studying adsorbed protein conformation are restricted to comparison of spectral differences induced by adsorption, without knowledge of the actual type or amount of change these differences reflect. Better methodology, especially on quantitative aspects, is sorely needed in this area. The orientation of adsorbed proteins may prove to be readily explored with the monoclonal antibody method and therefore certainly deserves wider application. Finally, the behavior of enzymes and antibodies at interfaces is not... 

It is important to characterize the material adsorbed at the interface, as well as the conformation of the adsorbed proteins, because it determines the properties of the emulsions and gels. However, little is known about the conformational changes of proteins upon adsorption at oil-water interfaces. The main reason for this is the lack of experi-... 

Because membrane filtration is the only currently acceptable method of sterilizing protein pharmaceuticals, the adsorption and inactivation of proteins on membranes is of particular concern during formulation development. Pitt [56] examined nonspecific protein binding of polymeric microporous membranes typically used in sterilization by membrane filtration. Nitrocellulose and nylon membranes had extremely high protein adsorption, followed by polysulfone, cellulose diacetate, and hydrophilic polyvinylidene fluoride membranes. In a subsequent study by Truskey et al. [46], protein conformational changes after filtration were observed by CD spectroscopy, particularly with nylon and polysulfone membrane filters. The conformational changes were related to the tendency of the membrane to adsorb the protein, although the precise mechanism was unclear. 

Thus, cell adhesion is determined by nonspecifically adsorbed serum proteins on the surface. Therefore, it is important to consider the characteristics of adsorbed proteins including the amount, composition, and conformation or orientation. 

The conformation and orientation of adsorbed proteins has been examined with monoclonal antibodies that recognize a specific site in a protein of interest. Keselowsky et al. examined the conformation of Fn adsorbed to SAMs that carried methyl, hydroxyl, carboxyl, and amine groups [79]. They used monoclonal antibodies that recognized the central cell-binding domain of Fn near the RGD motif. Different SAM functionalities differentially modulated the binding affinities of the monoclonal antibodies (OH > COOH = NH2 > CH3). The strength of cell adhesion to these... 

SAMs was correlated to the affinities of the Fn-specific monoclonal antibodies. Although antibody-based measurements could not distinguish between conformational (structural) and orientational changes in the adsorbed proteins, they provided information about the biological activity of adsorbed proteins. 

K.L. Egodage, B.S. de Silva, and G.S. Wilson, Probing the conformation and orientation of adsorbed protein using monoclonal antibodies cytochrome c3 films on a mercury electrode. J. Am. Chem. Soc. 119, 5295-5301 (1997). 

Egodage etal. [103] have described a novel application of monoclonal antibodies for the probing of conformation and orientation of the adsorbed protein using cytochrome c(3) films on mercury electrode. Antibodies were utilized to confirm the presence of three confor-mationally distinct electrochemical forms of cytochrome dependent on the applied potential. 

In order to construct functional microspheres by modification of the surface with adsorbed proteins, e.g., enzymes and antibodies, the conformation and orientation of adsorbed proteins must be controlled to keep them as active as free proteins. If hydrophilic particles are used as a carrier, they hardly suffer nonspecific adsorption, but even antibody cannot be adsorbed. In this case, antibody is immobilized on the particles by chemical reactions such as those mentioned in the previous section (9). 

The other major casein monomer in bovine milk is asi-casein. The SCF theory suggests that a loop-like protein conformation is favoured for adsorbed asi-casein (see Figure 8.1a) (Dickinson et al., 1997 Home, 1998). This implies a reduced hydrodynamic thickness of the adsorbed layer for as]-casein as compared with p-casein. 

The Lundstrom model70 is given in Fig. 13b. He assumes that protein adsorbs with a rate constant ka into State 1. Upon adsorption, some of the adsorbed proteins in State 1 (native) may conformationally change (via rate constant kr) to State 2 (denatured). Letting n, and n2 be the number of molecules per unit area in States 1 and 2, and and a2 be the area fractions occupied in each state, he says (noting that the unoccupied area fraction = (1 — — a2n2) that70) ... 

Beissinger s and Leonard s model (Fig. 13 d) accounts for desorption of both native and denatured adsorbed species (States 1 and 2, respectively). They used the classical Langmuir-Hinshelwood model for catalytic reactions (the surface is the catalyst for conformational change or denaturation of the adsorbed protein), which assumes equilibrium at a steady state between adsorbed and solution molecules. They show ... 

Soderquist and Walton72) added time dependence of the surface reaction to the model. They also allowed for readsorption of desorbed material (Fig. 13e). This model in principle takes into account the time dependence of the conformational change of adsorbed protein. They consider three distinct processes or states ... 

We have already established that the protein adsorption process may result in significant conformational changes. In addition to adsorbed amounts and rates, the orientation and conformation of the adsorbed protein are critical (Fig. 16). Conformation refers to the secondary (a-helix, P-sheet), tertiary, and quaternary structures. 

Many studies of proteins at air-solution interfaces have indirectly established that the adsorbed proteins undergo detectable conformational changes. Similar studies at solid-liquid interfaces are few. We review here only several key studies. 

Fig. 17. At low solution concentration, the protein has no neighbors on the surface and thus can optimally adapt to the surface, maximizing the number of binding interactions. At high solution concentration, any one adsorbed protein is immediately surrounded by neighbors, minimizing the probability that it can conformationally adapt to the interface. This behavior leads to the differences in adsorbed amount and adsorbed protein thickness (determined by ellipsometry), as discussed in the text...

Given sufficient time, adsorbed proteins undergo conformational changes which lead to increased surface interaction. During this process, proteins less optimally adsorbed undergo desorption, hence the overshoot in the time curve. 

Clearly specific antibodies, and particularly monoclonal antibodies, may be very useful in probing the properties of adsorbed proteins. Specific antibodies have been used to probe the structure of antigens in solution 88). Consider the adsorption of a simple protein with a small number of reasonably well-defined epitopes (surface sites with antibody binding activity), as in Fig. 19. Clearly epitopes E and A are not accessible for binding, while B, C, and D would be sterically accessible. One could also envision a conformational change upon adsorption which produces an epitope... 

Clearly a set of monoclonal antibodies may help elucidate the nature of adsorbed protein orientation and conformation. Such studies are in progress by several groups. 

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 ... 

Techniques and methods for the study of protein adsorption have been well reviewed 4). It is now generally recognized that it is not necessarily the type and amount of protein present at the surface which is most important, but rather the orientation and conformational state of those proteins. At present it is virtually impossible to predict the specific conformation of an adsorbed protein at a particular interface. The techniques used in the determination of protein conformation in solution or in the solid state do not usually apply to adsorbed proteins. Hence, the difference between adsorbed and bulk solution protein conformation has to be inferred indirectly. 

In most cases, the adsorbing surface is tacitly assumed to be completely inert and nonresponsive to protein attachment. This assumption may be valid for surfaces of crystalline material but in the case of hairy polymer surfaces, changes in the polymer surface conformation due to the presence of adsorbed protein may be expected. 

For many areas of interest the most valuable information describing the interactions between a protein and a surface is conformational change of the adsorbing protein molecule as it passes from solution to the interface. Low surface area samples in combination with some form of spectroscopic method are generally used in the evaluation of protein conformation. Recent advances in this area warrant a more detailed description of the experimental approaches. 

In conclusion, TIRF promises to be exceedingly useful in the study of protein-substrate interactions. It gives in situ, possibly remote, real-time information about protein adsorption-desorption parameters, conformational changes upon adsorption and hopefully, nanosecond time-resolved fluorescence lifetime information about adsorbed proteins 156). 

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