1.00 Native molecule - Disulfide bonds 0.75 40 Molecules with randomly 110 20 formed disulfides 72 0.50 124 Oxldze Reduce (BME) Denature (Urea) 0.25 Remove urea Remove urea Oxldize 40 m 110 0.0 10 20 30 40 50 +124 Denatured molecule Temperature, °C o Solutlon viscosity o Optical rotatlon at 365 nm A Second denaturatlon A UV absorbance at 287 nm (a) In the classic RNase A refolding experiment of Anfinsen, renaturation before disulfide bond formation yields the active, native conformation, but disulfide bond formation before renaturation yields multiple conformations with little (b) Thermal denaturation of RNase A monitored by various physical methods. Differences between the native and denatured conformations can be detected by several of the spectroscopic methods discussed in Tools of Biochemistry 6A. All three techniques indicate the same fraction unfolding as a function of increasing temperature. Measurements of a second denaturation after cooling (A) recovery of enzymatic activity. produce the same curve, showing that the process is reversible with a melting temperature (T) of 30.0 °C. A FIGURE 6.22 The denaturation and refolding of ribonuclease A. (a) This schematic drawing depicts the classic RNase A refolding experi- ment of Anfinsen. (b) Thermal denaturation of RNase A monitored by various physical methods. This graph shows the fraction of protein that is denatured, as measured by the increase in solution viscosity (O), change in optical rotation at 365 nm (0), or change in UV absorbance at 287 nm (A). The experiments were conducted at pH 2.1, ionic strength 0.019 M. Under physiological conditions, RNase A is much more stable, not dena- turing until about 70–80 °C. Fraction unfolded Three disulfide bonds fomed by side chains of the six cysteines Covalent disulfide bond connecting two Cys residues. Amino acld sequence: RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTC GGA A FIGURE 6.25 Disulfide bonds in bovine pancreatic trypsin inhibitor (BPTI). The main chain of BPTI is shown in cartoon rendering, and the side chains of the six cysteines that form the three disulfide bonds are shown as yellow sticks. The amino acid sequence of BPTI is shown with the strands colored red, the helix cyan, and the cysteines yellow. The covalent disulfide bonds are shown schematically as yellow lines connecting two cysteine residues.