Determining Membrane Disrupting Protein Mechanisms Using Polarized FTIR

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The cell membrane serves as a barrier that protects the rich, internal cellular environment from external threats such as pathogens and chemicals. Nevertheless, this barrier is not impenetrable. Pathogens can secrete membrane disrupting toxins which enable them to enter cells, and the advancement of certain diseases, such as Alzheimer’s, has been shown to be connected to membrane disrupting toxins. FT-IR spectroscopy, using polarized filters from Specac, is a crucial tool for understanding the progression of these diseases.

Understanding the processes pathogenic toxins use to disrupt cellular membranes is vital in formulating new methods of treating and preventing pathogenic disease. Moreover, a greater understanding of the role played by toxins in the progression of degenerative diseases will help advance treatment for diseases of the elderly - which are estimated to become more widespread in the future.

Different Types of Membrane Disrupting Proteins

Membrane damaging toxins are typically divided into two distinct categories, channel-forming toxins and enzymatic toxins such as the α-toxin phospholipase of clostridium perfringens, which acts directly on the cell membrane to cause lysis. The channel-forming toxins that form pores in the target cell membrane can be divided into two groups: the RTX toxins and the cholesterol-dependent toxins.

The cholesterol-dependent cytolysins (CDC) are β-barrel pore-forming exotoxins that normally require the presence of cholesterol in the target membrane and the pores developed can be 25-30 nm in diameter. Commonly secreted by gram-positive bacteria, these CDCs are expressed as water-soluble monomers of 50-70 kDa, that bind to the membrane form a circular homo-oligomeric complex – the β-barrel transmembrane structure – which is then inserted into the target cell membrane to form a breach.

RTX toxins are a group of cytotoxins and cytolysins created by bacteria and are typically defined by the presence of a specific tandemly repeated nine-amino acid residue sequence in the protein, and extracellular secretion by the type I secretion systems (T1SS). Haemolysin A (HlyA) of E. coli is a good example. The pores formed are normally cation-selective allowing for an influx of Ca²⁺ into the target cells.

Prions and Amyloids

Apart from bacterial ingress, there are other protein-membrane disruption illnesses. The first and most recognized is the prion-based Creutzfeld-Jakob disease, which is a type of protein that can stimulate normal proteins in the brain to fold abnormally and cause issues in the cell-membrane structure of neurones.

In a similar manner, amyloid plaques are insoluble protein aggregates that are accountable for amyloid diseases such as Huntington's, Alzheimer's, type II diabetes, Parkinson's, and also some cancers. Amyloids are recognized by β-strand repeats running perpendicular to the fiber axis that are the misfolding of this protein structure.

This structure known as cross-β is a unique motif among protein folds and adds to the insolubility and cytotoxic nature of these structures. Although certain amyloids are natural and have a physiological function the mechanism of development is still under research.

Using FT-IR in Membrane Disruption Studies

Several techniques have been used to examine membrane proteins but these are constrained by the restrictions of the quantity of protein material required, the recording time and present insoluble (amphipathic or hydrophobic) aggregates which are in constant flux.

FT-IR provides a unique opportunity to overcome these restrictions and allows aggregates to be characterized during the aggregation process in almost real-time. The recording time is short, preventing time-dependent structural variations and less than 100 ng of protein is needed to assess the secondary structure of these aggregates. For FTIR β-sheet contribution has the maximum absorption coefficient (more than circular dichroism) and this means that FT-IR is mostly well-suited for the investigation of β-sheet-rich proteins such as amyloids. ATR-FTIR overcomes the issue of insolubility in these proteins because the sample is tested as a thin film on the surface of an internal reflection element (IRE).

Using Polarized FT-IR to Determine Protein Orientation

By using polarizing filters and dual beams proteins and molecules in the thin film can be oriented and provide beneficial information about the relative orientation of the matching dipoles and their connection to the orientation of secondary structures. Linearly polarized ATR-FTIR technique is a spectroscopic method frequently used to establish the orientation of functional groups within proteins in very ordered systems such as membranes and β-amyloid plaques.

The technique is based on the observation that the intensity of the IR light absorption is based upon the relative orientation of the transition dipole moment and the excited electric field of the incident light. Absorption is heightened once the transition dipole moment is oriented parallel to the electric field of the incident light. By recording two IR spectra of an oriented sample with light polarized perpendicular to each other the relative orientation can be established. More significantly a different spectrum or dichroic spectrum may be calculated and tested to provide both a quantitative and qualitative assessment of the orientation of dipoles related to particular vibrations.

Polarized FTIR techniques can swiftly be used to confirm cross-β spine conformation in aggregates (secondary structure) as well as the orientation of molecules.

Specac’s Polarizing Infrared Filters

Specac are extremely experienced in FT-IR and offer a full range of infrared polarizers. They provide a variety of high-quality holographic wire grid polarizers fabricated on a range of infrared-transmitting substrates (CaF₂, ZnS, BaF₂, Ge) and available in sizes from 25 mm to 71 mm diameter clear aperture.

All of these filters have an exceptional extinction coefficient characteristic and with grid periodicity of 4000 lines/mm, these polarizers possess heightened transmission throughput characteristics with an optimized performance at short wavelengths for precision applications such as membrane study. The polarizers match with Specac's series of Bench Mount Polarizer rotators.

IR polarizing filters from Specac

All 38 mm clear aperture series Infrared Polarizers can be mounted in the Benchmark™ FTIR Polarizer Rotator for in-compartment polarization experimentation and with Benchmark™ baseplate mounted accessories such as: Golden Gate™ ATR, Silver Gate™ Evolution ATR, and Gateway™ Horizontal ATR systems, and the Cyclone™ and Tornado™ long pathlength gas cells.

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Conclusion

Integrating a polarizer with ATR-FTIR measurements can provide beneficial information about the sample, and determining the polarization delivered is direct. Polarization can improve band intensities and deliver information on oriented functional groups.

Furthermore, FTIR is an excellent method to follow structural variations during the aggregation process, particularly with a small sample size. A direct correlation of molecular conformation with protein local structure is crucial to studies of protein– and peptide–membrane interactions, mainly in the context of membrane-facilitated aggregation, and disordering or disruption. Infrared spectroscopy has been a backbone for defining molecular conformation, following folding dynamics, and characterizing reactions.

References and Further Reading

1. Lally ET, Hill RB, Kieba IR, Korostoff J (1999), "The interaction between RTX toxins and target cells", Trends in Microbiology, 7 (9): 356–361
2. Czajkowsky, D. M.; Hotze, E. M.; Shao, Z.; Tweten, R. K. (2004). "Vertical collapse of a cytolysin prepore moves its transmembrane β-hairpins to the membrane". EMBO J. 23 (16): 3206–3215.
3. Rabia Sarroukh , Erik Goormaghtigh et al., ATR-FTIR: A “rejuvenated” tool to investigate amyloid proteins, Biochimica et Biophysica Acta (BBA) – Biomembranes, Volume 1828, Issue 10, October 2013, Pages 2328–2338
4. Jessica J. Lib, Christopher M. Yip, Super-resolved FT-IR spectroscopy: Strategies, challenges, and opportunities for membrane biophysics, Biochimica et Biophysica Acta (BBA) – Biomembranes, Volume 1828, Issue 10, October 2013, Pages 2272–2282
5. Lisa M. Miller, Megan W. Bourass, Randy J. Smith, FTIR spectroscopic imaging of protein aggregation in living cells, Biochimica et Biophysica Acta (BBA) – Biomembranes, Volume 1828, Issue 10, October 2013, Pages 2339-2346
6. Parsons, Joshua B. et al. “Membrane Disruption by Antimicrobial Fatty Acids Releases Low-Molecular-Weight Proteins from Staphylococcus Aureus.” Journal of Bacteriology 194.19 (2012): 5294–5304. PMC. Web. 19 Oct. 2017.

About Specac Ltd

Specac manufactures an extensive range of FTIR Accessory, IR Polarizer, and Pellet Press Products for Atomic and Molecular Spectroscopy.

These products include ATR Accessories, Specular Reflectance Accessories, Diffuse Reflectance Accessories, Liquid Transmission and Gas Transmission Cells, as well as Infrared and Terahertz Wire Grid Polarizers, Bench-Top Hydraulic Presses, KBr Pellet Presses, XRF Pellet Presses, Thin Film Making Kits, and Evacuable Pellet Dies.

For online optical spectroscopy or FTIR analysis, Specac offers a comprehensive range of NIR Process Cells suitable for liquid and gas/vapour analysis.