Raman spectroscopy is a powerful technique for analyzing the molecular structure and interactions of various substances, but it has been challenging to apply it to live proteins without damaging them. A new method developed by researchers at Texas A&M University overcomes this limitation and opens up new possibilities for biomedical applications.

What is Raman Spectroscopy and Why is it Useful?

Raman spectroscopy is a type of spectroscopy that uses monochromatic light, usually from a laser, to illuminate a sample and measure the scattered light that emerges. The scattered light can have different wavelengths than the incident light, depending on how the molecules in the sample vibrate, rotate or undergo other excitations. This phenomenon is called Raman scattering, and it provides a unique fingerprint of the molecular structure and composition of the sample.

Raman spectroscopy is useful for identifying and characterizing different chemical species, such as organic molecules, minerals, semiconductors and biological macromolecules. It can also reveal information about the molecular interactions, such as protein-ligand binding, that are crucial for many biological processes and drug development. Raman spectroscopy is non-destructive, requires minimal or no sample preparation, and can be performed in various conditions, such as in solution, in solid state or in vivo.

What are the Challenges of Raman Spectroscopy of Live Proteins?

Despite its advantages, Raman spectroscopy has some limitations when applied to live proteins. One of the main challenges is that the laser light used to excite the sample can also generate heat, which can damage or denature the proteins and affect their function and stability. This leads to poor reproducibility and reliability of the measurements, especially for low-concentration and low-dose samples. Moreover, the Raman signal from proteins is often weak and obscured by background noise or fluorescence from other molecules in the sample.

For more than 50 years, biomedical researchers have been frustrated by these difficulties and have tried various methods to overcome them, such as using different wavelengths of light, cooling the sample or adding labels or enhancers to increase the signal. However, these methods have their own drawbacks, such as reduced sensitivity, specificity or compatibility with physiological conditions.

How Does the New Technique Solve These Problems?

A group of researchers with the Institute for Quantum Sciences and Engineering at Texas A&M University and the Texas A&M Engineering Experiment Station (TEES) have developed a novel technique that solves these problems and enables low-concentration and low-dose screenings of protein-to-ligand interactions in physiologically relevant conditions.

The technique is called thermostable-Raman-interaction-profiling (TRIP), and it is based on cooling down the surface or substrate where the proteins are immobilized before exposing them to the laser light. This way, the proteins are protected from heat damage and can maintain their native structure and function during the optical measurements. The researchers also optimized the experimental parameters, such as laser power, exposure time and detection system, to achieve high signal-to-noise ratio and reproducibility. The researchers published their findings in the Proceedings of the National Academy of Sciences.

What are Some Examples of Protein-Ligand Interactions?

Protein-ligand interactions are ubiquitous in biology and play important roles in many cellular functions and pathways. Some of the protein-ligand interactions are well known, such as the interaction between an enzyme with its substrate, the interaction of an antibody with its antigen, the interaction of protease with its inhibitor, interaction of ATPases with ATP, the interaction of GTPases with GTP, and interaction of transcription factors with DNA motifs.

Other protein-ligand interactions are less obvious but equally important, such as the interaction of hormones with their receptors, the interaction of growth factors with their receptors, the interaction of cytokines with their receptors, the interaction of neurotransmitters with their receptors, the interaction of drugs with their targets, and the interaction of toxins with their targets⁴

Protein-ligand interactions can be classified into different types based on their strength, specificity, reversibility and dynamics. For example, some protein-ligand interactions are strong and specific but reversible and dynamic (e.g., enzyme-substrate), while others are weak and nonspecific but irreversible and static (e.g., covalent modification).

Some protein-ligand interactions are mediated by a single binding site on each molecule (e.g., antibody-antigen), while others involve multiple binding sites on each molecule (e.g., avidin-biotin). Some protein-ligand interactions are cooperative, meaning that the binding of one ligand affects the binding of another ligand (e.g., hemoglobin-oxygen), while others are independent, meaning that the binding of one ligand does not affect the binding of another ligand (e.g., albumin-drugs)

How Can the TRIP Technique Detect and Characterize Protein-Ligand Interactions?

The TRIP technique can detect and characterize protein-ligand interactions by measuring the changes in the Raman spectra of the proteins before and after binding to the ligands. The Raman spectra reflect the vibrational modes of the molecules, which are sensitive to the molecular structure and environment.

When a protein binds to a ligand, some of its vibrational modes may change due to conformational changes, charge transfer, hydrogen bonding, or other effects. These changes can be observed as shifts, intensities, widths, or shapes of the Raman peaks in the spectra. By comparing the Raman spectra of the free and bound proteins, one can infer information about the binding site, affinity, kinetics and thermodynamics of the protein-ligand interaction.

The TRIP technique can also monitor the protein-ligand interactions in real time and under different conditions, such as temperature, pH, salt concentration, or presence of other molecules. This can provide insights into the dynamics and regulation of the protein-ligand interactions and their response to external stimuli or perturbations. The TRIP technique can also detect multiple protein-ligand interactions simultaneously by using different wavelengths of light or different detection channels. This can enable high-throughput screening and analysis of protein-ligand interactions in complex biological systems.

What are the Implications and Applications of This Technique?

The TRIP technique is a paradigm-shifting answer to a long-standing problem that provides label-free, highly reproducible Raman spectroscopy measurements of live proteins. The technique can detect protein-ligand interactions in real time and reveal their binding kinetics and thermodynamics. This can be useful for studying various biological processes, such as signal transduction, immune responses and gene regulation, that depend on these interactions.

Moreover, the technique can be used for screening potential drugs or vaccines that target specific proteins or modulate their activity. The technique can also be applied to clinical diagnostics, as it can identify pathogens or biomarkers based on their protein signatures in a fast and accurate way.

The TRIP technique has several advantages over existing methods for Raman spectroscopy of live proteins. It requires smaller sample size and lower protein concentration, which makes it more cost-effective and less invasive. It also does not require any labels or enhancers, which avoids any interference or alteration of the protein function or structure. It also works under physiological conditions, which preserves the relevance and validity of the results. Finally, it is compatible with various types of substrates and surfaces, which increases its versatility and applicability.

Conclusion

In this article, we have introduced a novel technique for Raman spectroscopy of live proteins, called thermostable-Raman-interaction-profiling (TRIP), that overcomes the limitations of conventional methods and enables low-concentration and low-dose screenings of protein-ligand interactions in physiologically relevant conditions. We have explained the principles and advantages of Raman spectroscopy and the challenges of applying it to live proteins.

We have described how the TRIP technique solves these problems by cooling down the surface or substrate where the proteins are immobilized before exposing them to the laser light. We have also given some examples of protein-ligand interactions and how the TRIP technique can detect and characterize them by measuring the changes in the Raman spectra of the proteins. Finally, we have discussed the implications and applications of this technique for biomedical research, drug development and clinical diagnostics.

The TRIP technique is a breakthrough in the field of Raman spectroscopy and protein-ligand interactions, as it provides label-free, highly reproducible and reliable measurements of live proteins without damaging them.

The technique can reveal information about the binding site, affinity, kinetics and thermodynamics of protein-ligand interactions, as well as their dynamics and regulation under different conditions. The technique can also be used for screening potential drugs or vaccines that target specific proteins or modulate their activity, as well as for identifying pathogens or biomarkers based on their protein signatures. The technique is cost-effective, versatile and compatible with various types of substrates and surfaces.

The TRIP technique opens up new possibilities for studying various biological processes and pathways that depend on protein-ligand interactions, such as signal transduction, immune responses and gene regulation.

It can also facilitate the discovery and development of new drugs or vaccines that can modulate these interactions for therapeutic purposes. Moreover, it can improve the diagnosis and treatment of various diseases that are caused by or involve protein-ligand interactions, such as infections, cancers, autoimmune disorders and neurodegenerative diseases.

The TRIP technique is a powerful tool for advancing our understanding of the molecular mechanisms and functions of proteins and their interactions with other molecules. It is also a promising tool for translating this knowledge into practical applications for improving human health and well-being. We hope that this article has provided a comprehensive overview of this technique and its potential benefits for biomedical science and engineering.

FAQ

Q: What is Raman spectroscopy? A: Raman spectroscopy is a type of spectroscopy that uses monochromatic light, usually from a laser, to illuminate a sample and measure the scattered light that emerges. The scattered light can have different wavelengths than the incident light, depending on how the molecules in the sample vibrate, rotate or undergo other excitations. This phenomenon is called Raman scattering, and it provides a unique fingerprint of the molecular structure and composition of the sample.

Q: What are protein-ligand interactions? A: Protein-ligand interactions are interactions between proteins and other molecules, such as small molecules, peptides or nucleic acids, that form a complex through non-covalent interactions. Protein-ligand interactions are important for many biological processes and drug development.

Q: What are the challenges of Raman spectroscopy of live proteins? A: The challenges of Raman spectroscopy of live proteins are that the laser light used to excite the sample can also generate heat, which can damage or denature the proteins and affect their function and stability. This leads to poor reproducibility and reliability of the measurements, especially for low-concentration and low-dose samples. Moreover, the Raman signal from proteins is often weak and obscured by background noise or fluorescence from other molecules in the sample.

Q: What is the TRIP technique? A: The TRIP technique is a novel technique for Raman spectroscopy of live proteins, developed by researchers at Texas A&M University. It is based on cooling down the surface or substrate where the proteins are immobilized before exposing them to the laser light. This way, the proteins are protected from heat damage and can maintain their native structure and function during the optical measurements.

Q: What are the advantages of the TRIP technique? A: The advantages of the TRIP technique are that it provides label-free, highly reproducible and reliable measurements of live proteins without damaging them. It also requires smaller sample size and lower protein concentration, which makes it more cost-effective and less invasive. It also does not require any labels or enhancers, which avoids any interference or alteration of the protein function or structure. It also works under physiological conditions, which preserves the relevance and validity of the results. Finally, it is compatible with various types of substrates and surfaces, which increases its versatility and applicability.

Q: How does the TRIP technique detect and characterize protein-ligand interactions? A: The TRIP technique detects and characterizes protein-ligand interactions by measuring the changes in the Raman spectra of the proteins before and after binding to the ligands.

The Raman spectra reflect the vibrational modes of the molecules, which are sensitive to the molecular structure and environment. When a protein binds to a ligand, some of its vibrational modes may change due to conformational changes, charge transfer, hydrogen bonding, or other effects. These changes can be observed as shifts, intensities, widths, or shapes of the Raman peaks in the spectra. By comparing the Raman spectra of the free and bound proteins, one can infer information about the binding site, affinity, kinetics and thermodynamics of the protein-ligand interaction.

Q: What are some examples of protein-ligand interactions? A: Some examples of protein-ligand interactions are:

• The interaction between an enzyme with its substrate
• The interaction of an antibody with its antigen
• The interaction of protease with its inhibitor
• Interaction of ATPases with ATP
• The interaction of GTPases with GTP
• Interaction of transcription factors with DNA motifs
• The interaction of hormones with their receptors
• The interaction of growth factors with their receptors
• The interaction of cytokines with their receptors
• The interaction of neurotransmitters with their receptors
• The interaction of drugs with their targets
• The interaction of toxins with their targets

Q: What are some applications of this technique for biomedical research, drug development and clinical diagnostics? A: Some applications of this technique for biomedical research, drug development and clinical diagnostics are:

• Studying various biological processes and pathways that depend on protein-ligand interactions, such as signal transduction, immune responses and gene regulation
• Screening potential drugs or vaccines that target specific proteins or modulate their activity
• Identifying pathogens or biomarkers based on their protein signatures in a fast and accurate way

Q: How can I learn more about this technique and its potential benefits for biomedical science and engineering? A: You can learn more about this technique and its potential benefits for biomedical science and engineering by reading the original article published in the Proceedings of the National Academy of Sciences , or by contacting the authors at Texas A&M University.

Q: Where can I find more resources on Raman spectroscopy and protein-ligand interactions? A: You can find more resources on Raman spectroscopy and protein-ligand interactions by visiting the following websites

References

Altangerel, N., Wang, Y., Li, X., & Yakovlev, V. V. (2021). Thermostable-Raman-interaction-profiling enables low-concentration and low-dose screenings of protein-to-ligand interactions in physiologically relevant conditions. Proceedings of the National Academy of Sciences, 118(37), e2107042118. https://doi.org/10.1073/pnas.2107042118

Williams, M. A. (2013). Protein–Ligand Interactions: Fundamentals. In D. G. Hardie (Ed.), Protein-Ligand Interactions (pp. 1–26). Humana Press. https://doi.org/10.1007/978-1-62703-398-5_1

Bruker. (n.d.). What is Raman Spectroscopy? Retrieved October 14, 2021, from https://www.bruker.com/en/products-and-solutions/infrared-and-raman/raman-spectrometers/what-is-raman-spectroscopy.html

Wikipedia. (2021, September 28). Protein–ligand complex. Retrieved October 14, 2021, from https://en.wikipedia.org/wiki/Protein%E2%80%93ligand_complex