Site specific affinity

A careful study by McDermott and co-workers of the high-K+ and low-K+ states of the KcsA channel has focused exclusively on the selectivity filter.101 They discovered that low K+ also induces the "non-con-ductive" or "collapsed" structure of the selectivity filter at neutral pH, but only if the sample remains well hydrated this state is lost if the bulk buffer is removed from the NMR rotor after spinning. Comparison of the measured chemical shifts with predictions by the SHIFTX and SPARTA programs identified the crystal structures that are most consistent with the selectivity filter conformations in the high-K+ and low-K+ proteolipo-some samples. Titrations of the chemical shift changes were used to measure site-specific affinities for K+. Based on the slow exchange rate between these conformations (<500 s 1), the authors suggest that the low-K+ conformation is relevant to channel inactivation rather than to conduction.101... 

Site-specific affinity binding of proteins to self-assembled molecular architectures... 

To target a specific site. Connecting specialized functional groups that have site-specific affinity (peptide, antibody, etc.) can allow the parent drug to be delivered to the targeted area of the body to produce site specific therapeutic action. 

A system which is depending on adsorption onto sites of the type described in Section IV, B, 2, b is open to competitive inhibition by solutes, generally of analogous structure, who show for the same site specific affinities which often depend on very small structural differences (Le Fevre, 1961). 

There is good agreement that the two high-affinity Ca sites are within = 10 A of each other (Table II) [132,390,404-409], Their localization within the bilayer is supported by the observation [130,131] that site-specific mutagenesis of several amino acids within the putative transmembrane helices interferes with Ca binding and with the Ca -dependent phosphorylation of the enzyme by ATP, but has no effect on the Ca -independent phosphorylation by inorganic phosphate. 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

Lee, S., Young, N.L., Whetstone, P.A., Cheal, S.M., Benner, W.H., Lebrilla, C.B., and Meares, C.F. (2006) A method to site-specifically identify and quantitate carbonyl end products of protein oxidation using oxidation-dependent element coded affinity tags (O-ECAT) and nanoLiquid chromatography Fourier transform mass spectrometry./. Proteome Res. 5(3), 539-547. 

Rhode, B.M., Hartmuth, K., Urlaub, H., and Liihrmann, R. (2003) Analysis of site-specific protein-RNA cross-links in isolated RNP complexes, combining affinity selection and mass spectrometry. RNA 9, 1542. 

Schepartz, A., and Cuenoud, B. (1990) Site-specific cleavage of the protein calmodulin using a trifluoperazine-based affinity reagent./. Am. Chem. Soc. 112, 3247-3249. 

High affinity, site specific DNA binding. DNA-dependent protein/protein interaction... 

The success of treating tumours, especially solid tumours, by systemic therapy depends on various characteristics of the tumour. Besides the importance of intrinsic drug activity and the potential targets within the tumour cells, drug pharmacokinetics and whole body distribution, site of delivery and the ability of site-specific targeting (affinity) are important features. 

Colman has also produced a definitive account of site-specific modification of enzymes, and her chapter is particularly instructive about the range and utility of reaction types that can be gainfully exploited in affinity labeling experiments. 

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