Quantitative methods for structural characterization of proteins based on deep UV resonance Raman spectroscopy

Area of Science:

• Biophysical Chemistry
• Spectroscopy
• Structural Biology

Background:

• Protein aggregation is implicated in various diseases.
• Accurate characterization of protein structural elements is crucial for understanding function and dysfunction.
• Deep UV resonance Raman (DUVRR) spectroscopy offers unique insights into protein secondary structures.

Purpose of the Study:

• To develop novel quantitative approaches for protein structural characterization using DUVRR spectroscopy.
• To introduce a new method combining hydrogen-deuterium (HD) exchange and Bayesian source separation for analyzing aggregated proteins.
• To extract DUVRR signatures of specific structural elements, including cross-beta cores and unordered regions of amyloid fibrils.

Main Methods:

• Application of deep UV resonance Raman (DUVRR) spectroscopy.
• Integration of hydrogen-deuterium (HD) exchange with Bayesian source separation.
• Utilizing prior information on concentration matrices and component spectral features to resolve ill-conditioned problems.
• Employing partial least squares (PLS) and least squares support vector machines (LS-SVMs) for initial secondary structure fraction estimation.

Main Results:

• Successful extraction of DUVRR signatures for various structural elements in aggregated proteins.
• Demonstration of the method's efficacy using hen egg white lysozyme undergoing HD exchange.
• Quantification of secondary structure fractions in aggregated proteins.
• Comparison of the proposed method with traditional techniques like classical least squares calibration (CLSC).

Conclusions:

• The combined HD exchange and Bayesian source separation method provides a robust approach for protein structural characterization via DUVRR spectroscopy.
• This technique enables detailed analysis of structural components within aggregated proteins, such as amyloid fibrils.
• The developed method offers advantages over existing techniques for analyzing complex protein structures and aggregation states.