Labeling techniques chains

It is clear that, having the same chain length, these macromolecules are in reality experimentally indistinguishable. One could however think of a labelling technique to make Nf different from Nj, for example by using a chromophore-bound radical scavenger which added selectively to the macroradicals issued from the chain scission. Equation (87) can be split into a system of two equations (100) and (101)... 

Organometallic compounds appear to be a serious alternative to state-of-the-art labeling techniques as shown in the ongoing preclinical and clinical studies with organometallic-labeled tumor affine peptides and single-chain antibody fragments. ... 

It now remains to determine the dimensions of a single Ionomer chain within an associated aggregate. To this end, the mixed-labeling technique described In reference 7 was used. As demonstrated (17) this technique can be used to Isolate single-chain Information without additional corrections for the second vlrlal coefficient. 

Abstract EPR spectroscopy of site-directed spin labeled membrane proteins is at present a common and valuable biophysical tool to study structural details and conformational transitions under conditions relevant to function. EPR is considered a complementary approach to X-ray crystallography and NMR because it provides detailed information on (1) side chain dynamics with an exquisite sensitivity for flexible regions, (2) polarity and water accessibility profiles across the membrane bilayer, and (3) distances between two spin labeled side chains during protein functioning. Despite the drawback of requiring site-directed mutagenesis for each new piece of information to be collected, EPR can be applied to any complex membrane protein system, independently of its size. This chapter describes the state of the art in the application of site-directed spin labeling (SDSL) EPR to membrane proteins, with specific focus on the different types of information which can be obtained with continuous wave and pulsed techniques. 

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... 

---