Noncovalent Binding

It is well known that some chemicals have a preferential location inside specific pockets of the 3D protein structures. When fluorescent, they can be used for reporting conformational changes in and around these specific subdomains, for most of the fluorescence properties are sensitive to changes in the microchemical environment. That is specifically the case when the preferential location results from a noncovalent binding to a specific amino acid. Then, changes in protein conformation may influence fluorescent properties through either changes in the microchemical environment, a change in binding conditions, or even both at the same time. Sometimes, the binding of the exogenous fluorophore may induce some energy transfer from a neighboring tryp-tophan, which makes it possible to probe the interaction through FRET techniques.

Some years ago, it was demonstrated that Mag-indo 1, a fluorescent chemical initially used for probing intracellular calcium concentration, was able to interact with some proteins through a noncovalent binding with some histidine residue(s). Such a binding induces a specific shift in the fluorescence spectrum of Mag-indo 1, making it possible to quantify this binding using specific data treatments (Fig. 4) (25,26,39). The interaction of Mag-indo 1 with turkey egg white lysozyme (Tlys) in the absence of calcium has been studied using the synchronous fluorescence technique (40,41). This technique, in which both excitation and emission wavelengths are scanned simultaneously with a constant 5X, compresses the spectral information to a relatively narrow domain of wavelengths. This generally reduces the spectral overlap from various fluorescent components. In this study, it allows the simultaneous recording of synchronous fluorescence spectra both of free Mag-indo 1 and protein-bound Mag-indo 1 and of the protein itself. Moreover, a potential energy transfer from one of the protein tryptophans cannot affect the fluorescence intensity of the synchronous fluorescence spectrum of the protein-bound Mag-indo 1. As a consequence, the value of the dissociation constant characteristic of the interaction can be easily obtained. On the contrary, changes in the intensity of the protein fluorescence spectrum may reflect both a change in the chemical microenvironment of a tryptophan close to the interaction site and some energy transfer from this tryptophan to the protein-bound Mag-indo 1. The energy transfer resulting from the interaction can be monitored through changes in the intensity of the Mag-indo 1 excitation spectrum when excited in the range of

TLys BSA

Fig. 7. Respective 3D conformations of TLys (left) and BSA (right). Chemical structures of tryptophan and histidine residues appears as balls and sticks.

TLys BSA

Fig. 7. Respective 3D conformations of TLys (left) and BSA (right). Chemical structures of tryptophan and histidine residues appears as balls and sticks.

wavelengths of tryptophan absorption (42). Furthermore, the amino acid sequence of Tlys has only two histidine residues, and only one of them, His 121, is located in the vicinity of one tryptophan in the stable 3D conformation. Thus, the existence of some energy transfer allows easy determination of the protein subdomain accessible through this interaction (Fig. 7).

Fluorescence techniques were also used to monitor conformational changes resulting from the binding of fluorescent substrates (4-methlyumbelliferyl chitobiosides or chitotriosides) to turkey or hen egg white lysozyme. In both cases, a quenching of the protein fluorescence was observed when the substrates were engulfed in the enzyme cleft, but no energy transfer was evidenced. This finding suggests that the quenching results only from a change in the chemical environment of one tryptophan, probably Trp 108, associated with the presence of the substrate inside the cleft (unpublished results).

Interactions of bovine serum albumin (BSA) and human serum albumin (HSA) with Mag-indo 1 have also been studied (42). These homologous proteins present two hydrophobic pockets but differ in their number of tryptophan residues: BSA has two tryptophans, one in each hydrophobic pocket, but HSA has only one, lacking the Trp 134 present in BSA. It was demonstrated that both proteins interact with Mag-indo 1 but that interaction induces a quenching of the protein fluorescence only for BSA. This quenching was associated with an energy transfer from BSA to Mag-indo 1 that parallels the protein-probe

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Eosin concentration (^iM)

Fig. 8. Influence of increasing eosin concentrations on fluorescence intensity of BSA trytophans (□) and on fluorescence intensity of BSA-bound eosin (■). Fluorescence intensities on the y-axis are in arbitrary units. BSA concentration was 1.8 |iM.

binding. These results suggest that interaction takes place in the vicinity of Trp 134 and can be used for monitoring unfolding and refolding processes of this BSA subdomain in the presence of guanidine hydrochloride (43).

Recently, interactions of eosin with, respectively, BSA and HSA have been reconsidered from the conformational change point of view (J. M. Salmon and L. Bilia, personal communication 2001). The interaction of eosin with these proteins induces both an increase in intensity and a red shift in the eosin fluorescence spectrum that allows quantifying the interaction. The stoichiometry of the interaction is 1/1 for HSA and 1/2 for BSA. Moreover, this interaction induces a quenching of the fluorescence of both tryptophans of BSA (Fig. 8). Comparison with results obtained with HSA confirmed that the eosin interaction occurs with amino acid(s) located in the two hydrophobic pockets of BSA.

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