Direct observation of structural heterogeneity in a beta-sheet

Area of Science:

• Biophysics
• Structural Biology
• Spectroscopy

Background:

• Proteins often exhibit structural heterogeneity crucial for their function, but conventional detection methods are limited.
• Carbon-deuterium (C-D) bonds serve as sensitive, nonperturbative probes of protein environments due to their unique IR absorption frequencies.

Purpose of the Study:

• To demonstrate that C(alpha)-D bonds can detect and structurally characterize protein heterogeneity.
• To investigate the structural heterogeneity of the N-terminal Src homology 3 (nSH3) domain.

Main Methods:

• Utilized density functional theory (DFT) calculations to predict C(alpha)-D bond sensitivity to local structure.
• Synthesized nSH3 variants with site-specifically incorporated C(alpha)D(2) glycine residues.
• Employed infrared (IR) spectroscopy to analyze C-D bond stretching absorptions.

Main Results:

• DFT predictions confirmed C(alpha)-D bond sensitivity, with red-shifted absorptions for beta-sheet structures compared to turns.
• IR spectroscopy revealed distinct C(alpha)-D bond shifts in nSH3 beta-sheets versus loops.
• Data indicated nSH3 populates at least two distinct beta-sheet core structures, suggesting potential disorder in the folded state and order in the unfolded state.

Conclusions:

• C(alpha)-D bonds are effective probes for detecting and characterizing protein structural heterogeneity.
• The study provides new structural insights into the nSH3 domain, impacting understanding of its folding and function.
• The C-D-based IR technique offers broad applicability for characterizing protein structures in both folded and unfolded states.