Two-dimensional mid-IR and near-IR correlation spectra of ribonuclease A: using overtones and combination modes to monitor changes in secondary structure.
We introduce near-IR spectroscopy as an ancillary tool for monitoring structural changes of proteins in aqueous solution using ribonuclease A (RNase A) as a model protein. The thermal unfolding of RNase A results in clear spectral changes in the near-IR and the mid-IR regions. In the near-IR the most pronounced changes are observed in the spectral region between 4820 and 4940 cm-1. The strong N-H combination band found at 4867 cm-1 in the spectrum of native RNase A shifts to 4878 cm-1 upon thermal unfolding. Hydrogen-deuterium exchange experiments that validate the N-H character of this mode can also be used to estimate the number of unexchanged amide protons after exposure to D2O. The transition profiles and temperatures derived from the temperature dependence of the N-H combination mode were found to be practically identical with those derived from the temperature dependence of the C = O amide I band in the mid-IR region, demonstrating that the near-IR region can be used as a conformation-sensitive monitor for the thermally induced unfolding of proteins in H2O solution. A 2-dimensional correlation analysis was applied to the mid-IR and near-IR spectra of RNase A to establish correlations between IR bands in both regions. The correlation analysis demonstrates that the thermal unfolding of RNase A is not a completely cooperative process; rather it begins with some changes in beta-sheet structure, followed by the loss of alpha-helical structures, and then ending with the unfolding of the remaining beta-sheets.
Schultz
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Institute for Biodiagnostics, National Research Council Canada, Winnipeg, Manitoba, Canada.
Fabian
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Mantsch
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D000577:Amides / Q000737:chemistry; D017666:Deuterium Oxide; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011522:Protons; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013816:Thermodynamics; D014867:Water
D000577:Amides; D011522:Protons; D012996:Solutions; D014867:Water; D012259:Ribonuclease, Pancreatic; D017666:Deuterium Oxide