ATR FT-IR: A New Vision on Protein Structure and Aggregation

3D render of a protein structure © VRAYVENUS - stock.adobe.com

The intricate world of proteins—molecules central to life processes and industrial applications—demands precise structural characterization. While traditional methods such as X-ray crystallography nuclear magnetic resonance (NMR) spectroscopy have been foundational, they present significant limitations, including high costs, laborious sample preparation, and technical constraints. A team of scientists from the University of Belgrade’s Department of Biochemistry has delved into the underutilized potential of attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectroscopy, a technique with unique advantages in protein structural analysis (1,2).

Breaking Down ATR FT-IR Spectroscopy
At its core, ATR FT-IR employs infrared light to examine the vibrations of chemical bonds within molecules, particularly focusing on the amide I region (1700–1600 cm⁻¹). This spectral range is highly sensitive to secondary structures such as α-helices, β-sheets, and unordered conformations, enabling the detection of subtle changes in hydrogen bonding patterns. Unlike traditional infrared spectroscopy, ATR FT-IR analyzes samples in a dry state, making it particularly adept at handling aggregated protein forms that might challenge other methods, like circular dichroism (CD) (1).

Advantages Over Traditional Techniques
The study emphasizes the comparative benefits of ATR FT-IR. X-ray crystallography, for instance, requires painstakingly prepared protein crystals and does not always indicate the natural state of proteins in solution. NMR spectroscopy, while retaining proteins in their natural environments, struggles with larger molecules and requires isotopically labeled samples. ATR FT-IR circumvents these issues, requiring minimal sample preparation and offering rapid analysis. Moreover, ATR FT-IR can distinguish between native and aggregation-prone β-sheets—key markers in studying amyloid formation and protein misfolding disorders (1,2).

Insights Into Protein Structure and Aggregation
The authors, Đorđo Tintor, Katarina Ninković, Jelica Milošević, and Natalija Đ. Polović, from the University of Belgrade, underscore ATR FT-IR’s capabilities in monitoring structural transitions and aggregation. By deconvoluting the amide I region, researchers can quantitatively estimate secondary structure changes. For instance, shifts in spectral peaks corresponding to α-helices (1654 cm⁻¹) and β-sheets (1620 cm⁻¹) reveal critical insights into processes like amyloid fibrillation, a hallmark of neurodegenerative diseases such as Alzheimer’s. ATR FT-IR 's ability to normalize and compare spectral intensities further bolsters its quantitative utility, extending applications to biopharmaceutical stability studies and food protein research (1,2).

Applications Beyond Basic Research
ATR FT-IR spectroscopy's utility extends beyond laboratory investigations, with implications for pharmaceuticals, biotechnology, and food industries. For example, monitoring the stability of commercial proteases and biopharmaceuticals like antibodies highlights its role in ensuring product efficacy and shelf life. Additionally, the ability to analyze collagen and gelatin transitions underscores its versatility in materials science (1).

Future Challenges and Opportunities
Despite its strengths, ATR FT-IR is not without limitations. Issues such as slower scanning processes and limited temperature control during analysis present challenges, particularly for unstable intermediates or rapid conformational transitions. However, ongoing advancements in spectral analysis software and instrument design promise to address these hurdles, further solidifying ATR FT-IR’s position as a cornerstone of protein structural analysis (1,2).

This comprehensive research review underscores ATR FT-IR spectroscopy as a powerful, accessible alternative for protein structure analysis. With its ability to provide qualitative and quantitative insights into secondary structures and aggregation, ATR FT-IR emerges as a pivotal tool in both academic research and industrial applications. As technology advances, the method’s limitations are likely to diminish, making a way for broader adoption in the study of proteins and other complex molecules.

References

(1) Tintor, Đ.; Ninković, K.; Milošević, J.; Polović, N. Đ., Gaining Insight into Protein Structure via ATR-FTIR Spectroscopy. Vib. Spectrosc. 2024, 134, 103726. DOI: 10.1016/j.vibspec.2024.103726.

(2) Maguire, S., Scheibe, C., Eisgruber, T., Mosesso, N., Isono, E. and Hauser, K., 2024. Membrane-specific and calcium-dependent binding of the Arabidopsis C2 domain protein CaLB revealed by ATR-FTIR spectroscopy. Spectrochim. Acta A: Spectrochim. Acta - A: Mol. Biomol. Spectrosc. 2024, 307, 123629. DOI: 10.1016/j.saa.2023.123629