Relaxation-assisted two-dimensional infrared (RA 2DIR) method: accessing distances over 10 A and measuring bond...

Area of Science:

• Structural biology and biophysics
• Vibrational spectroscopy
• Molecular dynamics and structural analysis

Background:

• Accurate measurement of three-dimensional molecular structures and dynamics is crucial for understanding molecular recognition and catalysis in structural biology.
• Two-dimensional infrared (2DIR) spectroscopy offers a molecular ruler for structural constraints but faces challenges with large molecules, including spectral congestion, limited distance range, and sensitivity issues.

Purpose of the Study:

• To develop advanced 2DIR spectroscopy techniques for overcoming limitations in analyzing larger molecules like proteins.
• To introduce novel vibrational labels and enhanced methods for more sensitive and extended structural analysis.

Main Methods:

• Implementation of dual-frequency 2DIR spectroscopy for enhanced selectivity and sensitivity in measuring vibrational mode interactions.
• Development and application of novel vibrational labels (C≡N, C-D stretching modes) in the water transparency and fingerprint regions for 2DIR.
• Introduction of the relaxation-assisted 2DIR (RA 2DIR) method, utilizing vibrational relaxation and energy transport to amplify cross-peaks.

Main Results:

• The dual-frequency 2DIR approach demonstrated high selectivity and sensitivity for specific vibrational reporter pairs.
• Novel vibrational labels were successfully developed and applied, expanding the utility of 2DIR spectroscopy.
• The RA 2DIR method achieved an 18-fold cross-peak amplification for modes separated by ~11 Å, significantly enhancing sensitivity and enabling longer-range distance measurements.
• Correlation of energy transport time with intermode distance was demonstrated, enabling molecular connectivity mapping similar to NMR methods (TOCSY, HMBC).

Conclusions:

• The developed RA 2DIR method significantly enhances the sensitivity and range of 2DIR spectroscopy for structural analysis of large molecules.
• The ability to measure mode-connectivity patterns offers a powerful new analytical tool for molecular structure determination.
• Further calibration of the RA 2DIR method holds promise for rapid distance and connectivity assessments, potentially reaching analytical method status.