Table 2. Techniques used to monitor protein unfolding
| Technique | Comments | References |
|---|---|---|
| Fluorescence | Reflects the difference in the environment of Trp and Tyr residues in the folded and unfolded states; best for chemical denaturation studies; particularly useful if the protein contains a buried Trp or Tyr that becomes exposed during unfolding; generally requires microgram quantities of protein | RNase T1 (Thomson et al. 1989) Barnase (Pace et al. 1992)Barstar (Khurana and Udgaonkar 1994)trp repressor (Fernando and Royer 1992)Chymotrypsin inhibitor 2 (Jackson et al. 1993) |
| UV absorbance | Similar to fluorescence in that it reflects the difference in the environment of the Trp and Tyr residues in the folded and unfolded states; best for thermal denaturation studies; generally requires milligram quantities of protein | Myoglobin (Pace and Vanderburg 1979) RNase T1 (Thomson et al. 1989) |
| Circular dichroism (CD) | Reflects the difference in secondary structure (α-helix and/or [CD] β-sheet) content in the folded and unfolded states; good for both chemical and thermal denaturation studies; generally requires milligram quantities of protein | Turkey ovomucoid third domain (Swint and Robertson 1993) Actin (Bertazzon et al. 1990)Interleukin 4 (Windsor et al. 1991)RNase T1 (Thomson et al. 1989)HPr (Scholtz 1995) |
| Differential scanning calorimetry (DSC) | Measures the excess heat capacity of a protein solution as a function of temperature, Tm, the calorimetric and van’t Hoff enthalpies, ΔHcal and ΔHvH, and the change in heat capacity, ΔCp, obtained from a single experiment; information about the stability of the protein and about the applicability of a two-state folding mechanism can be obtained; generally requires milligram quantities of protein | RNase T1 (Hu et al. 1992) Transferrin (Lin et al. 1994)Staphylococcal nuclease (Carra et al. 1994)Chymotrypsin inhibitor 2 (Jackson et al. 1993) |
| (Reprinted from Gilleto and Pace 1996 with permission from Academic Press © 1996.) | ||
