FTIR spectroscopy in life sciences: Uses & insights

Infrared spectroscopy, which measures light absorption, provides valuable insights into the structure and conformation of biomolecules. When combined with other analytical techniques, Fourier Transform Infrared (FTIR) spectroscopy can further enhance a molecular biologist’s toolkit.

FTIR is widely used to study conformational changes in proteins. The secondary structure of a protein influences the vibrational modes of the amide groups involved in peptide bonding. This allows FTIR to detect protein folding and aggregation in response to chemical or thermal stress, offering potential insights into the role of protein misfolding in neurodegenerative diseases.

Beyond proteins, FTIR is also valuable for studying large biomolecules such as lipids, phospholipids, and protein-DNA interactions. Regardless of the specific application, the technique can identify structural changes caused by shifts in the molecular environment, helping to reveal their biological effects.

The influence of pH on peptide nanotubes

A self-assembled peptide nanotube, the surfactant like arginine₃–leucine₁₂ (R₃L₁₂), was examined by Castelletto et al.¹ These peptide nanotubes can adopt various structures, including coils and β-sheets, making them promising candidates for biocatalysis and the encapsulation of pharmaceutical molecules for targeted drug release.^3,4

Previous research by the team found that acidic conditions promoted the formation of α-helix structures with cross linkers between R₃L₁₂ nanotubes.⁵ The nanotubes organized themselves into a configuration in which the arginine moiety was situated on the walls of the tubes, both on the inner and outer surfaces, while the leucine fragment was incorporated within the interior of the nanotube.

In this study, the authors were interested in probing the effect of switching the environment to a more basic one, with the pH varied from 9 to 13, at concentrations from 0.04-0.07 wt% of peptide.

To understand the changes undergone by the nanotube, the researchers used transmission electron microscopy (TEM), circular dichroism (CD) spectroscopy, small angle X-ray scattering (SAXS), and FTIR spectroscopy.

As the pH increased, CD spectroscopy revealed the loss of the α-helix structure, while SAXS confirmed the presence of nanotubes at pH 9. At higher pH levels, TEM and SAXS showed that the nanotubes reorganized into globular, micelle-like structures.

However, only FTIR spectroscopy could reveal molecular-level changes, providing key insights into why the nanotubes disassembled at higher pH levels.

While TEM, CD, and SAXS confirmed structural changes, only FTIR spectroscopy provided molecular-level insights, making it essential for understanding why the nanotubes break down at higher pH levels.

At pH 9, FTIR spectra showed the loss of the α-helix structure, indicated by a peak at 1665 cm^-1, which diminished further at pH 12 and 13. However, the amide III band remained consistent across pH levels.

In the CH region, peaks at 2871 and 2959 cm^-1 corresponded to the symmetric and asymmetric stretching modes of CH_₃ terminal groups, while the 2930 cm^-1 peak was attributed to CH/CH₂ stretching modes.

These peaks were linked to the 2-methylpropyl side chain of the leucine peptide, which decreased in intensity as pH increased. This suggests a progressively disordered molecular state, with potential formation of R₃L₁₂ dimers.

Examining bacterial resistance to antimicrobial agents

Another study exploring the use of FTIR was conducted by Schmid et al., which examined the role of ion lipid pairs as a proxy for the chemistry involved in the development of antimicrobial resistance in bacteria.²

The rationale for the study revolved around investigating simple chemical analogs for more complex biological systems, which can then be used to develop novel antimicrobial agents.

The pathogen Staphlyococcus aureus increases the formation of a cationic lipid that limits antimicrobial peptide effects on the bacteria even when formed at low concentrations. The bacterial resistance emerges from the ionic interaction of a negatively charged phosphate functional group in the bacteria’s membrane and a positively charged amine group in the lipid.

In the study, Schmid et al. explored the interaction between an organophospholipid (dipalmitoylglcero-3-phosphoglycerol, DPPG) and a quaternary ammonium compound (dihexadecyldimethylammonium, DHDAB) in an aqueous solution. The DHDAB compound acted as proxy for the bacterial membrane whereas the DPPG compound represented acted as a proxy for the cationic lipid.

The bacterial proxy, DHDAB, was selected specifically to prevent obscuring the PO peaks on the FTIR spectrum of the cationic lipid proxy, DPPG. The FTIR revealed peaks at 1221 and 1201 cm^-1, assigned to PO groups.

When comparing the ratio of the peaks to the concentration, the 1221 cm^-1 peak increased to 1201 cm^-1 as the molar ratio of DPPG:DHDAB decreased, proving the formation of the ion pair.

As the molar ratio changed, FTIR also revealed changes in the C-H stretching region (3000-2800 cm^-1). Specifically, the CH₂ peaks red-shifted with increasing DHDAB concentration, suggesting increased order and packing tightness of the alkyl chains.

At relative molar ratios above 0.5 DHDAB, the trend reversed, with the peaks shifting to higher frequencies (blue shift), indicating decreased molecular order and an increase in the gauche conformer. These changes were attributed to Van der Waals interactions between the alkyl chains of the two ions.

This suggests that interactions in antimicrobial-resistant bacteria may not rely solely on ionic bonding between membrane phosphates and amines. It also helps explain why even a slight increase in amine concentration in bacteria can significantly disrupt the binding between the antimicrobial agent and the bacterial membrane.

This insight could be invaluable in developing novel therapeutics that target this specific resistance mechanism.

Overcoming biomolecular sample challenges

Solutions containing proteins and other large molecules are prone to aggregation and sedimentation, leading to an uneven distribution of target molecules. Depending on the FTIR spectral collection technique used, this can result in low measurement repeatability, as the concentration of the target molecule in the sampled area may vary between measurements.

In Castelletto's study, ion pairs coagulated and formed significant sedimentation when mixed at near 50:50 anion-to-cation ratios.¹ To address this, the researchers used a horizontal transmission cell, ensuring that sediments spread evenly across the window for consistent sampling.

In contrast, a traditional vertical cell arrangement would allow sediments to settle at the bottom, potentially escaping the IR beam passing through the upper portion of the solution.

Another challenge is the high background absorbance of the solvent. Aqueous solutions of proteins and other biomolecules can be difficult to measure because water strongly absorbs infrared energy in many of the same spectral regions as proteins.

There are several ways to address this issue. One approach is careful spectral subtraction using standard water spectra. Another is replacing water with deuterated water (D_₂O), which has lower absorption, though it may alter the structure and stability of protein molecules.⁶

Alternatively, the protein solution can be concentrated into a smaller volume to reduce the relative amount of solvent. However, this also decreases the overall sample quantity. Microvolume ATR analysis offers a solution by enabling measurements of low-concentration samples without requiring large volumes.⁷

Conclusion

FTIR is a valuable tool for obtaining rapid, qualitative insights into the conformations, interactions, and identities of biomolecules. It is sensitive to changes caused by chemical, physical, and thermal perturbations, offering faster comparative analyses than traditional liquid chemistry methods, along with a more straightforward workflow.

FTIR has been applied broadly to detect changes in proteins and lipids, as well as to identify cellular responses to biochemical environments. Its versatility extends across biochemistry, antibacterial coatings, advanced drug formulations, as well as the study of protein aggregation and misfolding in disease processes.

Modern sampling methods, such as ATR-FTIR, also effectively address challenges related to sedimentation and high solvent background absorbance.

References

1. Castelletto, V., et al. (2021). Alpha helical surfactant-like peptides self-assemble into pH-dependent nanostructures. Soft Matter, 17(11), pp.3096–3104. https://doi.org/10.1039/d0sm02095h.
2. Schmid, M., et al. (2018). A combined FTIR and DSC study on the bilayer-stabilising effect of electrostatic interactions in ion paired lipids. Colloids and Surfaces B: Biointerfaces, 169, pp.298–304. https://doi.org/10.1016/j.colsurfb.2018.05.031.
3. Morris, K.L., et al. (2013). The Structure of Cross-β Tapes and Tubes Formed by an Octapeptide, αSβ1. Angewandte Chemie International Edition, 52(8), pp.2279–2283. https://doi.org/10.1002/anie.201207699.
4. PaPandya, M.J., et al. (2000). Sticky-End Assembly of a Designed Peptide Fiber Provides Insight into Protein Fibrillogenesis†. Biochemistry, 39(30), pp.8728–8734. https://doi.org/10.1021/bi000246g.
5. Castelletto, V., et al. (2020). Peptide nanotubes self-assembled from leucine-rich alpha helical surfactant-like peptides. Chemical Communications, 56(80), pp.11977–11980. https://doi.org/10.1039/d0cc04299d.
6. Giulia Giubertoni, Bonn, M. and Sander Woutersen (2023). D₂O as an Imperfect Replacement for H₂O: Problem or Opportunity for Protein Research?. Journal of Physical Chemistry B. https://doi.org/10.1021/acs.jpcb.3c04385.
7. Harrick Scientific Products, Inc. (2007) Analysis of Deuterated Proteins Using the concentratir2tm Atr Accessory, Application Note. No. 21156. Harrick Scientific Products, Inc. https://harricksci.com/content/UQA_App-Notes_Analysis_of_Deuterated_Proteins_21156_Final2.pdf.

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