Multi-mode two-dimensional infrared spectroscopy of peptides and proteins

Abstract

In this thesis, a methodology for understanding structural stability of proteins through multi-mode two-dimensional infrared (2D IR) spectroscopy is developed. The experimental framework for generation of broadband infrared lasers and robust new approaches to 2D IR spectroscopy are demonstrated. Long-term phase stability is achieved through the development of a passively stabilized diffractive optic and wedge interferometer. A new approach for acquisition of 2D IR spectra in the pump-probe geometry reduces overall experimental complexity. These technological advances extend the capabilities of 2D IR to further resolve inter- and intramolecular couplings, relaxation pathways and structural kinetics in complex systems. Characterization of multi-mode spectra is first performed on model protein systems to reveal detailed information on the effects of solvation and structure on the amide vibrations. Differences in vibrational coupling, transition dipole angles and the anharmonic potential of the amide vibrations of isotopologues of N-methylacetamide arise from significant change in the local mode composition of the amide II band due to isotopic substitution of the peptide group. Extension of multi-mode 2D IR to study the amide I'-II' spectra of an ideal protein system, poly-L-lysine, provides direct evidence for the structural sensitivity of the amide II' vibration, particularly to the !-helix moiety. This structural sensitivity arises from through bond coupling and structure induced symmetry and orientation of adjacent residues. Integration of these tools with hydrogen exchange techniques allows for the protein structural kinetics and stability to be observed through protein-solvent interactions with enhanced structural sensitivity relative to amide I spectroscopy alone.

The amide II' diagonal provides a measure of the degree of exchange and the cross peaks between the structurally sensitive amide I/I' vibration and the solvent exposure sensitive amide II and II' modes reveal the location of exchange. Partial exchange of the secondary structure of ubiquitin is revealed by correlation of the different amide signatures through analysis of cross peak line shapes, positions and amplitudes. Results provide direct evidence for a highly stable helix and labile "-sheet structure.