Methods for Detecting Binding Site Similarity

A related approach integrating sequence information (conservation), geometric information (cleft detection), and data on local stability calculated by Poisson-Boltz-mann methods was reported by Ota et al. [54], The method was used for predicting catalytic residues (polar atoms only) in enzymes. A number of putative active sites for a series of hypothetical proteins were found and are discussed in the study. 

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Many of the methods described above may suffer from the modifications of the proteins which are necessary to obtain a detectable readout for the interaction under investigation. As a matter of fact, fluorescent labels are attached in close vicinity to the site of interaction, as otherwise binding to a partner is not likely to be detectable. It is conceivable that such labels invoke altered binding properties. Similar skepticism and arguments hold for immobilizing a protein. Non-invasive labeling is possible with radioactive isotopes but these methods bear other disadvantages. 

Once transfectomas have been generated, they must be screened for the production of the desired fusion. The binding site provided by the expression of the VNP heavy chain in J558L cells and association of the heavy chain with the resident light chain means that the protein fusion can be captured on a solid phase by NP or NIP, and detected using commercially available antibody to mouse X chain and an appropriate enzyme conjugate, in a simple ELISA screening procedure. Similarly, purification can be achieved by affinity adsorption of the fusion onto an NP matrix. An immunoblot method is described here for characterization of selected transfectomas, which allows the mol-wt of the fusion product to be estimated. 

It is possible at present to identify two main levels at which molecular similarity is of importance in proteins. First, detection of large-scale similarities between different protein structures, i.e. similarities in the way that the linear polypeptide sequence is folded up to form a three-dimensional structure. This is the subject of the remainder of Sect. 4. Second, comparative analysis of local aspects of protein structure, for example the examination of specific binding sites, or of the environments of particular sidechains. These methods are described in Sect. 5. 

Very fast electron transfers from P+ to bacteriochlorophyl (Bchl) and from (Bchl)- to QA do not depend on media dynamics and occur via conformationally non-equilibrium states (Fig.3.18). The dual fluorophore-nitroxide molecules (D-A) are also convenient objects for analysing the activity-dynamics relationship. The marked irreversible photoreduction of the nitroxide fragment of the dual probe incorporated into the binding site of HSA only took place when the nanosecond dynamical processes around the probe traced by ESR and fluorescence methods were detected (Rubtsova et al., 1993, Fogel et al, 1994 Likhtenshtein, 1986 Lozinsky et al., 2002). Similar results were reported for another model protein system, i.e. a-chymotrypsin with spin labeled methionin-92 groups (Belonogova et al., 1997). In the latter enzyme, the excited tryptophan group serves as an electron donor. 

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