Protein sites

Various ligands bind to their protein sites in a diffusive motion. Similarly, the distance between different ends of a folded macromolecule changes in a way which can be described as a diffusive motion in the presence of a constraint potential (that keeps the parts of the molecule near their folded configurations). Brownian-type diffusive motion in the absence of a restrictive potential is characterized by a diffusion constant (Ref. 6)... 

From the various autocorrelation times which characterized macromolecular fluctuations, those associated with the fluctuation of the electrostatic field from the protein on its reacting fragments are probably the most important (see Ref. 8). These autocorrelation times define the dielectric relaxation times for different protein sites and can be used to estimate dynamical effects on biological reactions (see Chapter 9 for more details). 

The energetics of the solute state in the protein site can be expressed as... 

Labelling Na,K-ATPase with ATP analogues provides evidence for contribution from charged residues that are widely separated in the sequence of a subunit of Na,K-ATPase. The first indication came from ATP sensitive covalent insertion of fluorescein-isothiocyanate (FITC) into Lys ° in the a subunit [90], The strong fluorescence signal provides a convenient probe for monitoring conformational transitions in the proteins. Site-directed mutagenesis of Lys reduces the activity of... 

Experiment [80], however, requires that the red-shifted fluorescence comes from molecules that are in an environment quite different from the average, behaving as if in a nonpolar environment (long wavelength, long lifetime). The red-shifted component, therefore, appears to be from chromophores that are embedded more deeply into the membrane. In the case of Aladan, the absorption and fluorescence wavelengths are both longer in the more water-exposed protein sites, but the lifetime is shorter in water [53]. 

Gaertner, H.F., Offord, R.E., Cotton, R., Timms, D., Camble, R., and Rose, K. (1994) Chemo-enzymic backbone engineering of proteins. Site-specific incorporation of synthetic peptides that mimic the 64-74 disulfide loop of granulocyte colony-stimulating factor. J. Biol. Chem. 269, 7224-7230. 

N. Sutin, Brookhaven National Laboratory The 16% decrease in the rate of oxidation of reduced plastocyanin by Co(phen)33 resulting from the attachment of the chromium(III) label seems rather small if the chromium is indeed bound at or near the protein site used for electron transfer to Co(phen)33. ... 

However, modification of the electrode surface is not the only way in which one can facilitate reversible electron transfer between an electrode and a protein site. The indispensable condition is that the electrode surface is electrostatically compatible with the protein surface. 

The ability to biosynthetically incorporate noncoded amino acids into proteins site-specifically has facilitated studies not previously possible. These include studies of protein stability, the initiation of protein translation, electron transfer, protein-protein and protein-membrane interactions, reversal of enzyme substrate specificity, and structure-function relationships, among others. A growing number of research labs have begun to report applications of this technique. A brief look at some recent applications of the suppression mutagenesis technique follows. 

Molecular recognition is one of the keys to life. Scientists are discovering ways both to modify molecular recognition sites and to copy such sites. One approach to modifying molecular recognition sites, namely enzymatic protein sites, is through what is referred to as directed evolution. Arnold and coworkers have employed the combinatorial approach by... 

Figure 11. Extension of the five antigenic sites of Mb. The five antigenic sites (underlined residues) were extended by 6-7 residues each to enhance their T-cell proliferative activity. These peptides were used to prepare T-cell lines and T-cell clones with submolecular specificity to preselected protein sites (73).

By introducing fluorine atoms to the polyenic system of retinal, the geometry, electronic properties, hydrophobicity, and absorption properties of the molecule will be modified. Thus, fluoro derivatives of retinal are useful tools to understand the interactions between retinal and opsin, especially on the level of charge and hydrophobic effects at the protein site. Moreover, fluorine atoms are probes in NMR and allow studies on model molecules of visual pigments Consequently, syntheses of mono-, di-, and trifluoro derivatives of retinal have been the subject of many investigations. 

The fluorescence emission maximum, quantum yield, and lifetime of a fluorophore are very sensitive to its immediate environment. A blue shift in the emission maximum and an increase in the fluorescence quantum yield or lifetime is generally observed when a fluorophore is transferred form a polar solvent to a nonpolar one or when it binds to a hydro-phobic protein site. Furthermore, fluorescence quenching or enhancement may result from interactions of the fluorophore with various structural elements in its vicinity. 

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