Doubly-labeled proteins

Most recently, Peti et al.225 have demonstrated the potential of pCoil for the chemical shift assignment of aromatic side-chain protons of proteins. Using the conserved hypothetical protein TM0979 from Thermotoga maritima, the authors demonstrated the sequence-specific backbone resonance assignments with < 500 pg of 13C15N-doubly labeled protein. 

Doubly labeled protein is obtained in a similar manner to N-labeled protein. The bacterial cell line expressing the protein are simply grown in minimal media containing N ammonia and glucose. The cost of this media is considerably more than the N-labeled media, approximately 1000 per liter. It is often advantageous to have a separate sample that is labeled with alone because the coupling increases the line width of the resonances, decreasing the resolution of the separated NOESY and TOCSY spectra. Thus both a N-labeled and a C- N-labeled sample are desirable. 

In most cases, EPR distance measurements are performed to determine a distance within a nanoobject, e.g., the spin-spin distance in a doubly labeled protein. It is desirable to consider an isolated pair of spin labels therefore dipolar interactions to spins of neighboring objects, e.g., intermolecular interactions,... 

From early studies with doubly labelled transferrin it became clear that whereas iron is rapidly cleared from the circulation, the protein recycles many times - in... 

The nitrogen In secondary compounds also appears to be readily mobile and metabolizable. When doubly-labeled nicotine ( C - N) was fed to tobacco (j. rustics), carbon was recovered In alkaloids, free amino acids, pigments, free organic acids and free sugars and nitrogen was recovered In proteins, alkaloids, free amino acids and pigments (17). 

Double-quantum coherence (DQC) was used to measure interspin distances in eight doubly labelled derivatives of T4 lysozyme.25 Distances between 20 and 47 A with distance distributions of 1.0 to 2.8 A were measured. For some variants two conformations were detected with distances that differed by 5 to 5.4 A. It was shown that a few measurements of large distances provided significant constraints on protein structure. 

The doubly labeled water technique measures CO production, and not O production. Because the heal created per mole of expired CO differs between the three major fuels (carbohydrate, fat, protein), the calculation of energy expenditure requires a knowledge of the Lntemal fuel being oxidized. Thus, the doubly labeled water technique, coupled with infomnatior from RQ measurements, may be used instead of the method of indirect calorimetry (using an insulated chamber to measure heat production) to determine total energy production over the course of several days. 

Doubly labelled ( H C) protein hydrolysates strongly acidic cation exchanger... 

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