Nonlinear infrared spectroscopy of protein conformational change during thermal unfolding

Femtosecond two-dimensional infrared (2D IR) spectroscopy, dispersed vibrational echo (DVE) spectroscopy, and dispersed pump-probe (DPP) spectroscopy of the amide I vibrations are used to follow the thermal denaturing of ribonuclease A. Each experiment measures a spectrum with features that reflect vibrational couplings between amide I transitions of the protein backbone and is therefore more sensitive to protein conformation than traditional FTIR spectroscopy. As the temperature is raised, 2D IR spectra show the disappearance of cross peaks between two vibrational transitions arising from antiparallel beta-sheet structure as well as the blue shift to a diagonally elongated inhomogeneous spectrum. DVE and DPP spectra can be related to projections of the complex 2D IR correlation spectrum and also show clear signatures of beta-sheet structure in the native state. Singular value decomposition of the nonlinear spectra reveal a pretransition at 47 degreesC that precedes the melting of beta sheets in the primary denaturing transition at 65 degreesC. Residual secondary structure is observed in the thermally denatured state. These experiments suggest that nonlinear infrared spectroscopy is an effective probe of protein conformation that can be used to probe the equilibrium thermodynamics and nonequilibrium kinetics and dynamics of protein folding.