Proteins observations

Fig. 8. (a) Structure of the full-length Rieske protein from bovine heart mitochondrial bci complex. The catalytic domain is connected to the transmembrane helix by a flexible linker, (b) Superposition of the three positional states of the catalytic domain of the Rieske protein observed in different crystal forms. The ci state is shown in white, the intermediate state in gray, and the b state in black. Cytochrome b consists of eight transmembrane helices and contains two heme centers, heme and Sh-Cytochrome c i has a water-soluble catalytic domain containing heme c i and is anchored by a C-terminal transmembrane helix. The heme groups are shown as wireframes, the iron atoms as well as the Rieske cluster in the three states as space-filling representations. 

C. L. Voorhees, K. J. Lay, J. O., Jr. Identification of bacterial proteins observed in MALDI TOF mass spectra from whole cells. Anal. Chem. 1999, 71, 3226-3230. 

Haupts U, Maiti S, Schwille P, Webb WW (1998) Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proc Natl Acad Sci USA 95 13573-13578... 

Since protein-observe methods are usually performed at a substance protein ratio of 0.1, they extend the minimum KLl detection limit by roughly two orders of magnitude. The price to be paid is a much higher consumption of both protein and substance to be tested. 

The examples described in this chapter are meant to illustrate the actual application of NMR for the study of protein-ligand interactions in the pharmaceutical industry. In our group, the focus has been mainly on protein observe techniques, as we have been fortunate enough to have a dedicated laboratory for providing both unlabeled and labeled proteins in large quantities. At present, two-dimensional HSQC spectra offer the highest content of information which is not accessible that rapidly by other methods. 2D NMR spectroscopy provides... 

Surprisingly, C. elegans appears to have lost a considerable number of chromatin proteins from the Polycomb group of proteins, observed in Drosophila and in mammals, although other transcription factor genes... 

J. R. Lakowicz and G. Weber, Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies, Biophys. J. 32, 591-601 (1980). 

Deak, J., Chiu, H. L., Lewis, C. M., and Miller, R. J. D. 1998. Ultrafast phase grating studies of heme proteins Observation of the low-frequence modes directing functionally important protein motions,/. Phys. Chem. 102 6621-34. 

The antiapoptotic function of the Bcl-2 protein has been clearly shown experimentally. Its overexpression can prevent initiation of the apoptotic program in various cell types. The oncogenic function of Bcl-2 protein, observed in association with its overex-... 

In E. coli, many proteins are degraded by an ATP-dependent protease called Lon (the name refers to the long form of proteins, observed only when this protease is absent). The protease is activated in the presence of defective proteins or those slated for rapid turnover two ATP molecules are hydrolyzed for every peptide bond cleaved. The precise role of this ATP hydrolysis is not yet clear. Once a protein has been reduced to small inactive peptides, other ATP-independent proteases complete the degradation process. 

Marasmus Marasmus occurs when calorie deprivation is rela tively greater than the reduction in protein. Marasmus usually occurs in children younger than one year of age when the mother s breast milk is supplemented with thin watery gruels of native cereals, which are usually deficient in protein and calories. Typical symptoms include arrested growth, extreme muscle wast ing (emaciation), weakness, and anemia. Victims of marasmus do not show the edema or changes in plasma proteins observed in kwashiorkor. 

Lens aging and opacification can be monitored in situ via structural changes in lens proteins observed in Raman spectroscopy. Ozaki et al. (36) have carried out an extensive study on mouse lens proteins. Figure 6-23 shows the... 

When the operating pH of fused capillaries is reduced to less than 3.0, silanol ionization drops to almost zero and the EOF is drastically reduced. Both peptides and proteins can be separated under these conditions. Note however, that they are partially denatured at extremes of pH range. This may cause dissociation of multimeric proteins into subunits, or multiple conform-ers may be formed. Below pH 3 or above pH 10, only basic or acidic amino acids are ionized, respectively. The reduction in charge diversity of proteins observed at or near physiological pH will diminish selectivity of electrophoretic separations. Polypeptides undergo ion pairing with the acid used to control the pH under acidic conditions. The relative hydrophilicity or hydrophobicity of this acid will have a strong influence on selectivity. 

Figure 10.1 Basic polypeptide geometry. The upper panel shows a short peptide sequence of three amino acids joined by two peptide bonds. A relatively rigid planar structure, indicated by dashed lines, is formed by each peptide bond. The relative positions of two adjacent peptide bond planes is determined by the rotational dihedral angles

In this article, some examples are shown of the application of NMR spectroscopy in chemical biology. We put emphasis on experiments that characterize interactions of proteins and small molecular weight ligands. These interactions can be mapped either by characterizing the target protein (protein-observed experiments) or the ligand (ligand-observed experiments). Finally, the method of structure calculation based on NMR-derived data is briefly introduced. 

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