Hole burning spectroscopy of ribonuclease A.

We present pressure tuning hole burning experiments with the enzyme ribonuclease A using the UV-absorbing amino acid tyrosine as a probe. We show that, at 2 K, the protein is intact, and that at least four different regions which we associate with different tyrosine sites can be distinguished through their specific response to pressure. For one site we could determine the compressibility to 0.15 GPa(-1). Upon denaturing the protein with guanidine hydrochloride, one of the tyrosine sites is preserved to a large extent. Reducing the sulfur bonds has a more drastic effect: the tyrosine sites lose most of their individual features and their compressibilities come close to that of tyrosine in solution.

Local compressibilities in insulin as determined from pressure tuning hole burning experiments and MD simulations.

We present a hole burning study on insulin in a glycerol-water solvent by using the intrinsic amino acid tyrosine as a photochemical probe. The focus of the experiments is on the comparative pressure response of the spectral holes for insulin in its native state, in its chemically denatured state and for tyrosine in the glycerol-water solvent. From an analysis of the color effect of the pressure response, we can identify two different spectral ranges characterized by a markedly different sensitivity to pressure. We conclude that at least two tyrosines (or two groups of tyrosines) out of the eight in the insulin dimer are photochemically labeled, and that they are characterized by markedly different compressibilities, namely 0.08 and 0.13 GPa(-1), respectively. An interesting observation concerns the compressibility in the unfolded state: It is significantly lower and closer to the value measured for the pure tyrosine molecule in a glycerol-water solvent. In contrast to the native state, the response of the various tyrosines in the unfolded state to pressure variations is quite uniform. The experiments are compared with MD simulations of monomeric insulin at ambient temperature. The computational results show that the local compressibilities around the different tyrosines vary significantly and that they strongly depend on whether just the first shell of molecules or the first two shells are included in the local volume.