Protein segmental labelling

Fig. 1.5 A Natural protein splicing, B Trans splicing with a split intein. The two fragments can be prepared separately and reassembled in vitro to form an active intein domain for protein ligation. C Central segment labeling using two different split inteins.

Xu R, Ayers B, Cowbum D, Muir TW, Chemical ligation of folded recombinant proteins Segmental isotopic labelling of domains for NMR studies, Proc. Natl. Acad. Sci. USA, 96 388-393, 1999. 

Another way of overcoming spectral overlap for proteins is selective isotope labeling of one or several amino acid types, which results in less-crowded H- N spectra with cross-peaks from the labeled residues only. The same is possible for RNA where nucleotides can be selectively isotope enriched. In addition, methods for segmental labeling of parts of the biomolecules have been developed for proteins (48, 49) and for RNA (50). 

The structural analysis on KcsA was performed based on the mobility of each spin labeled side chain in the protein segments under investigation. It is worth recognizing in Pig. 4b that most of the CW RT spectra show multiple spectral components, characterized by different mobility (a few examples are highlighted by arrows). This is a very general property of the R1 side chain in proteins. The components reflect the anisotropy of the spin label reorientational motion, but their appearance could also have other causes. They could arise from a slow equilibrium between two different protein conformations or the presence of asymmetric sites in the protein. The molecular interpretation of different spectral components is cumbersome. Multifrequency EPR [17], temperature analysis of the CW spectra [27], pulse saturation recovery techniques [28], or high pressure EPR [29] can help unravel the possible origins of the spectral components. In the case of KcsA, the spin labels motional information was quantitatively extracted from the inverse central line width (A//q, mobility parameter) and was corroborated by the measure of the accessibility of the spin labeled side chains towards lipids (O2... 

Here, V is the volume of the sphere and // is the viscosity of the solvent As can be seen in Table 1.4, the Tj values of proteins such as bovine serum albumin and trypsin in aqueous solution He in the ns range and become larger with increasing molar mass. The proteins were labeled with fluorescent markers such as l-dimethylamino-5-sulfonyl-naphthalene groups (see Chart 1.10) [38], Segmental motions and molecular flexibiUty have been studied for various polymers, such as polystyrene and the Y-shaped immunoglobulins IgA and IgG. Relaxation times in the range of 10-100 ns were found. In these studies, the... 

Steady state anisotropy measurements described in Section 2.7.5 can sometimes be misleading if a number of sources of depolarization are present. For example, as discussed, it can be difficult to differentiate global molecular rotation from local rotational freedom of the fluorophore when it is attached to a larger molecule [ 1,127]. In these cases time resolved single molecule fluorescence anisotropy measurements can be of use since the contributions to the depolarization may well act on different timescales. For example, for a labelled protein segmental motion of the protein backbone near the label and motion in the linker by which... 

Fig. 1.2 The structure of P450cam (PDB 5CP4) with key helical segments labeled. PDB Protein Data Bank...

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