Moderate sized labeled proteins

Ligand-detected NMR is a robust method, which is well suited to screening compound mixtures with rapid deconvolution [69], The target protein does not have to be labeled and its size is typically much more than 20 kDa. The protein production requirements are considered to be moderate. If the screen is performed in the presence of a competitor with known interaction mode, active site versus nonactive site binders can be distinguished and binding affinities can be derived. 

The different proteins spinach ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) and thioredoxin m (TRX) and corn NADP-MDH were purified to homogeneity by already published procedures (4,5). EDTA-NaCI washed thylakoids were used to avoid contamination by stromal proteins.The thylakoids were pretreated with high light (4000 pMol quanta.m-2.sec-1) for different lengths of time. All the proteins of the system and NADP-MDH were then added, and MDH activated for a few min under moderate light (300 Mol quanta.m-2.sec-1). The reconstituted system comprised, in 100 pi total volume 100 mM Tris buffer, pH 7.9, thylakoids (20 pg chi), 10 pM Fd, 2 pM FTR, 10 pM TRX and 2.5 pM MDH. MDH activity and the thiol content of the different proteins of the mixture were then measured. Enzyme activity was measured by the decrease in absorbance at 340 nm in the presence of NADPH and oxaioacetate. Protein thiol quantitation was done by derivatization with 14C-labelled iodoacetate or iodoacetamide (6) and either TCA-precipitation of all the proteins, or HPLC separation of each of them. A special separation procedure was devised to recover the proteins from the reaction medium. It was done by molecular size exclusion on a TSK 3000 HPLC column and allowed a quantitative recovery of each of the proteins. The use of iodoacetamide as derivatizing agent and the addition of 300 mM NaCI to the elution buffer were necessary to obtain an efficient separation (7). 

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