Isotope-labeled proteins

Both absolute quantitation and relative quantitation of species in mixtures is of interest in some circumstances. Quantitation in a 5-minute analysis can be achieved by addition of an internal standard, ideally the target microorganism grown in special media to incorporate heavy isotopes92-95 and determination of the relative peak heights of pairs of proteins from the analyte and the standard. Isotope-labeled proteins or peptides, selected to match proteins or peptides characteristic of target microorganisms, can also serve as internal standards for isotope ratio measurement. The addition of unmatched proteins or peptides is less reliable for either ESI or MALDI measurements because of unpredictable suppression in the variable mixture. 

Limited protein stability often hampers successful structure elucidation by X-ray crystallography and/or NMR spectroscopy. Relaxation properties are usually improved at elevated temperatures, and multidimensional NMR experiments require sample lifetimes to extend over several days to weeks in order to acquire all the necessary data. In addition, the activity of contaminating proteases that are sometimes present in purified samples can be significant at the experimental temperatures. Therefore, the stability of a target protein can be a concern, in particular for expensive isotope-labeled proteins. 

The first experiment to be recorded on isotope-labeled proteins is the [ N/HJ-HSQC experiment. Inspection of the [ N. HJ-correlalion map and simple counting of cross peaks reveals whether multiple conformers exist, whether some parts of the backbone signals are broadened, possibly because of slow conformational exchange, or whether parts of the sequence are not visible at all. As mentioned above, the spectrum will also show whether the protein is well folded or not. 

Isotope-Labeled Proteins from Hydrolyzates of the Green Alga Scenedesmus obliquus... 

Although alternative expression systems have been successfully adapted for the production of isotope-labeled proteins (see Sect. 1.5), heterologous expression in E. coli often remains the method of choice for NMR sample preparation. There is a fundamental difference, however, with respect to the kind of medium in which the cells are cultivated. In a so-called chemically defined or minimal medium only one or a very limited number of carbon sources is provided, e. g. glucose or glycerol. All bacterial metabolites have to be biosynthesized by the cells through the various, sometimes lengthy and energy-de-... 

Preparation of Homogenously Isotope-Labeled Protein by Fermentation on Algal Media... 

Keywords Drug interaction In-cell NMR Isotope labeling Protein conformation... 

As demonstrated, different time-resolved FTIR techniques allow to study the complete photocycle of bacteriorhodopsin in the entire range from picoseconds to several milliseconds. Infrared difference spectra trace reactions which take place in different parts of the protein molecule. Isotopically labeled proteins or proteins with mutations at specific sites... 

MacCoss, M.J., Wu, C.C., Matthews, D.E. and Yates, J.R., 3rd (2005) Measurement of the isotope enrichment of stable isotope-labeled proteins using high-resolution mass spectra of peptides. Anal. Chem. 77,7646-7653. 

Romanelli A, Shekhtman A, Cowburn D, Muir TW. Semisynthesis 46. of a segmental isotopically labeled protein splicing precursor ... 

The molecular mass of the complex hetween a domain of protein G and an Fab or an Fc fragment of IgG is approximately 58 kDa, so that a complete structure determination by nmr is not feasible. However, since the structure of each component of the complex is known, it is possible to obtain a medium-resolution model of the complex if the residues in each component involved in the interaction can be identified, at a simple level by using appropriately isotope-labelled proteins to identify residues whose chemical shifts are affected by complex formation. 

Logan TM, Olejniczak ET, Xu RX, Fesik SW (1992) Side chain and backbone assignments in isotopically labeled proteins from two heteronuclear triple resonance experiments. FEES Lett 314 413 18... 

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