Abstract: The presence of disulfide bonds can be detected unambiguously only by X-ray crystallography,  and otherwise must be inferred by chemical methods. In this study we demonstrate that ¹³C NMR chemical shifts are diagnostic of disulfide bond formation, and can discriminate between cysteine in the reduced (free) and oxidized (disulfide bonded) state. A database of cysteine ¹³C C^a and C^b chemical shifts was constructed from the BMRB and Sheffield databases, and  published journals. Statistical analysis indicated that the C^b shift is extremely sensitive to the redox state, and can predict the disulfide-bonded state. Further, chemical shifts in both states occupy distinct clusters as a function of secondary structure in the C^a/C^b chemical shift map.  On the basis of these results, we provide simple ground rules for predicting the redox state of cysteines; these rules could be used effectively in NMR structure determination, predicting new folds, and in protein folding studies.

Biomol NMR 2000 Oct;18(2):165-71  [PubMed PMID: 11101221]

Ground Rules for deducing the redox state of cysteines in an unknown protein:

Redox state of cysteines in all proteins except those that are paramagnetic can be determined using the following ground rules. To ensure that the redox states are correctly assigned, we suggest that all of the steps are used

Step 1: Ensure that the C^a and C^b assignments of cysteine are correct.

Step 2: If the C^b shift is less than 32.0 ppm or greater than 35.0 ppm, the redox state is assigned to reduced or oxidized, respectively. If the shift lies in the overlap region (32.0 �V 35.0 ppm), the redox state can be assigned using step 3.

Step 3: For proteins with a single cysteine showing a C^b shift in the overlap region, the cysteine must be reduced. For proteins with multiple cysteines, of which one of the C^b shifts lies in overlap region, the oxidation state of this cysteine is established from the C^b shifts of the other cysteines in the protein using Figure 1 and further confirmed from Figure 2. For proteins with multiple cysteines of which more than one lie in the overlap region, the redox state is assigned using Figure 2. In this case, Figure 1 alone cannot unambiguously assign the redox state.

Figures from the article:
Figure one
Figure two
Figure three
Figure four
Figure five

Tabular Data:
Table one
Table two
Table three
 

Also available is a Microsoft Excel spreadsheet listing data pertaining to the proteins used in this study.

Protein List
this list includes links to the BioMagResBank database and PDB files.