Protein Secondary Structure Estimation Using the Agilent Cary 630 FTIR Spectrometer

Importance of the topic

Proteins fold into defined secondary structures such as α-helices, β-sheets, turns, and random coils. These conformations underlie protein stability and function in biological systems, pharmaceuticals, and industrial applications. Rapid, reliable assessment of secondary structure in solution supports protein engineering, formulation development, quality control, and fundamental research.

Objectives and Overview

This study demonstrates a fast and flexible approach for estimating protein secondary structure in solution using Fourier-transform infrared (FTIR) spectroscopy. The goal is to compare FTIR-derived structure estimates with literature values from X-ray crystallography and to validate the Cary 630 FTIR spectrometer combined with MicroLab Expert software for routine analysis.

Instrumentation

• Agilent Cary 630 FTIR spectrometer with single-reflection diamond ATR module
• MicroLab Expert software (version 1.1.0.1) for data acquisition and curve fitting

Methodology and Instrumentation

Protein samples (BSA, lysozyme, papain, β-casein, myoglobin, monoclonal antibody, IgG) were prepared in aqueous buffer at concentrations between 10 and 25 mg/mL. Spectra were acquired over 4,000–650 cm–1, 4 cm–1 resolution, 140 scans per sample. Water background spectra were subtracted to isolate protein amide bands.

Data processing steps in MicroLab Expert:

• Average of three measurements
• Blank subtraction (water or buffer)
• Merging and smoothing (Savitzky-Golay filter, 2nd derivative for band narrowing)
• Curve fitting of amide I band (1,600–1,700 cm–1) by adding Gaussian/Lorentzian peaks and auto-fitting

Main Results and Discussion

Deconvolution of the amide I band provided relative intensities of α-helix (1,648–1,660 cm–1), β-sheet (1,625–1,640 cm–1), turns (1,660–1,699 cm–1), and random coil (1,640–1,648 cm–1). FTIR-derived percentages broadly agreed with X-ray crystallography references:

• Myoglobin: 72% α-helix (literature 85%)
• Lysozyme: 44% α-helix, 19% β-sheet (literature 45% α-helix, 19% β-sheet)
• IgG: 10% α-helix, 67% β-sheet (literature ~3% α-helix, 67% β-sheet)
• BSA: Lower α-helix (38% vs. crystallographic 74%) due to solution environment effects

Differences in BSA helical content highlight the influence of solvent, pH, temperature, and crystallization state on secondary structure stability. FTIR enables analysis of solution-phase proteins under native-like conditions.

Benefits and Practical Applications

FTIR provides rapid, nondestructive secondary structure estimates with minimal sample preparation and broad concentration tolerance. This technique is suitable for:

• Protein formulation screening and stability studies
• Quality control in biotechnology and pharmaceutical manufacturing
• Monitoring of folding/unfolding and aggregation processes

Future Trends and Potential Applications

Advances may include automated high-throughput FTIR screening, real-time process monitoring in bioreactors, integration with AI-driven deconvolution algorithms, and expanded spectral libraries for diverse protein families.

Conclusion

The Agilent Cary 630 FTIR spectrometer coupled with MicroLab Expert software offers a streamlined method to estimate protein secondary structure in solution. FTIR results show good agreement with X-ray data, demonstrating the method’s utility for rapid structural characterization in research and industrial settings.

Reference

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