Melting points of lysozyme and ribonuclease A crystals correlated with protein unfolding: a Raman spectroscopic study.
The effects of a temperature increase on monoclinic and tetragonal lysozyme single crystals were investigated by polarizing microscopy, X-ray diffraction and laser Raman spectroscopy. To prevent dissolution, the mother liquor was removed, and the crystals were covered by the oil poly-(chlorotrifluoroethylene). Upon heating, their macroscopic shape was stable beyond 453 K but a change (or loss) of birefringence was observed around 352 and 367 K for the tetragonal and monoclinic crystal forms, respectively, which is associated with tighter packing and higher crystal forces in monoclinic lysozyme. Raman spectral changes in the amide I and amide III regions indicated denaturation of the protein within the crystalline environment at temperature where birefringence changes, and differences in the S-S band suggest that in monoclinic lysozyme, denaturation is accompanied with disruption of some S-S bonds. Comparison with thermal denaturation and gel formation (beta-aggregation) of lysozyme in solution indicates that intermolecular interactions are mainly involved in the stabilization of the denatured lysozyme crystals. The behavior of ribonuclease A is very different. This protein unfolds and refolds reversibly in solution and its crystals melt at the unfolding temperature at 333 K, i.e. loss of structure induces breakdown of crystal lattice and macroscopic shape. Although the crystal lattice of proteins is stabilized by only few intermolecular contacts, its breakdown with increasing temperature is primarily a result of thermal unfolding of the polypeptide chains.
Institut für Kristallographie, Freie Universität Berlin, Takustrasse 6, D-14195 Berlin, Germany.
J
J
Jacob
C
C
Krafft
K
K
Welfle
H
H
Welfle
W
W
Saenger
D002151:Calorimetry; D003460:Crystallization; D009113:Muramidase / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013059:Spectrum Analysis, Raman; D013696:Temperature; D014961:X-Ray Diffraction
D012259:Ribonuclease, Pancreatic; D009113:Muramidase