Nuclear Magnetic Resonance (NMR) Spectroscopy Laboratory

Therapeutic Protein Structure by Nuclear Magnetic Resonance Spectroscopy

At the Centre for Vaccine Evaluation, we have developed scientific expertise in various techniques that help provide new tools for evaluating biologics.We use nuclear magnetic resonance (NMR) spectroscopy as a key approach to studying proteins.

 

Why We Study Protein Structure

Proteins consist of polypeptides or long chains of amino acids—the building blocks of life. These chains fold into complex three-dimensional scaffolds or structures (conformations) that directly determine the ability of an active pharmaceutical ingredient (API) or targeted molecule to function properly. As such, proteins are complex biomolecules that form the APIs of biologics such as hormones, antibodies, and toxoid vaccines. These therapeutic proteins target and interact with other proteins in the body.

Various diseases, such as cystic fibrosis, transmissible spongiform encephalopathy (TSE) and Alzheimer’s disease, result from a change of protein conformation. This change can lead to a loss of biological function (for example, cystic fibrosis) or the formation of neurotoxic species (as in TSE and Alzheimer’s disease).

Changes in protein structure can affect how a protein functions. By studying protein structure, we can obtain information on what structural characteristics make an API work well, identify what normal and abnormal proteins look like, and discover how protein function is affected.

 

How We Study Protein Structure Using Nuclear Magnetic Resonance Spectroscopy

At the Centre for Vaccine Evaluation, our team studies protein structure. We focus on the close relationship between biological function and protein conformation. As we obtain a description in structural terms of biologics, proteins or polypeptides, we lay the foundation for building a complete understanding of these complex molecules. We use this knowledge of the relationship between protein structure and function to identify and develop bio-analytical tools for characterizing or assessing biologics.

To study protein structure, our research team has developed expertise in the use of a powerful technique called nuclear magnetic resonance (NMR)spectroscopy. We also use the latest biotechnology methods to produce protein samples necessary for applying NMR techniques. We use recombinant protein and bioprocess technologies to incorporate stable isotopes such as nitrogen-15 and carbon-13 that let us apply multi-dimensional NMR techniques. These techniques greatly increase resolution, so that we can determine and study the structure of proteins. Methods for the analysis of biologics are usually developed and tested with labelled proteins first, before they can be applied to test the proteins in biologic formulations.

Research in this area has applications such as:

  • Characterizing the structure of recombinant protein therapeutics;
  • Developing new methods to assess subsequent entry biologics (SEBs) and other biologics.

 

Concepts and Tools We Use to Study Protein Structure

Nuclear magnetic resonance (NMR) spectroscopy measures the precession frequency of atomic nuclei, such as the hydrogen atom, when subjected to strong magnetic fields. When a molecule (for example, water, protein or drug) is exposed to a very strong magnetic field, the nuclei move like spinning tops that are under the effect of the earth’s gravitational field. This movement, as illustrated in Figure 1, is called precession. The precession frequency of a given nucleus is directly related to the surrounding magnetic field.

Figure 1

Figure 1

1.1: Spinning top
1.2: Nuclei
1.3: Field (Gravitational or Magnetic)

 

© Her Majesty the Queen in Right of Canada, represented by the Minister of Health (2009).

In a protein, the frequencies of nuclei are influenced by their surroundings. Therefore, NMR spectroscopy lets us measure many properties that help determine the structure of a protein at high resolution. Proteins in solution are not static; they possess various degrees of internal motion that can also be studied by NMR spectroscopy.

 

Research Highlight 1: Nuclear Magnetic Resonance Fingerprinting - A New Tool to Analyse Proteins and Identify Biologics

Recombinant protein therapeutics form a class of biologics in which proteins, such as hormones and antibodies, are the key active pharmaceutical ingredients (APIs). These large molecules can vary greatly in shape, composition and structure depending on their environment and how they are produced. The structure of the protein (that is, its conformation) determines its ability to work as a therapeutic agent. A change in conformation may lead to an ineffective therapeutic that may generate unwanted effects.

Delivery of a therapeutic protein into the human body requires that the protein be mixed (formulated) with appropriate additives, called excipients. As added ingredients, excipients allow proper delivery of the APIs, maintain protein conformation, and provide biochemical stability over long periods of time (shelf-life). To assess the quality attributes of these therapeutic products, researchers must develop the ability to analyse the conformation of the protein from formulated products.

The nuclear magnetic resonance(NMR) spectra of a protein measure the frequencies from most nuclei (proton, nitrogen and carbon). These frequencies are directly influenced by the local magnetic field surrounding the nuclei. Therefore, the conformation of a protein will have a unique NMR signature that can be seen as a fingerprint, analogous to the unique fingerprints of a human. Researchers use a protein NMRfingerprint to identify and assess the conformation of the API. They compare it with the matching fingerprint recorded as a reference standard.

Research in this area provides new tools to:

  • assess if changes in formulations of biotechnology derived products affect the structure of the protein and possibly the activity of the biologic;
  • help evaluate the quality of subsequent entry biologics (SEBs) or other recombinant protein therapeutics.


Research Highlight 2: Prions - How Misfolded Proteins Can Cause Diseases

Transmissible spongiform encephalopathy (TSE) diseases are also known as prion diseases. A structural change of the normal cellular form of the protein to a pathogenic form (prion) can spread to other normal proteins.

In animals, prions can cause diseases such as scrapie in sheep and bovine spongiform encephalopathy (BSE) in cows, known as mad cow disease, which in humans is called variant Creutzfeldt-Jakob disease (vCJD). In humans, prions also cause diseases such as Gerstmann-Sträussler-Scheinker (GSS) disease, Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI) and kuru.

The conversion of the normal form to the infectious form can occur sporadically (very rare) or by contact (via ingestion) with infectious material. Genetic mutations have been associated with some forms of TSE diseases. We want to determine how the normal form converts to the infectious form and how mutations contribute to the process. Because there is evidence in the scientific literature that the prion protein interacts with the cellular membrane, we are studying such interactions from a structural perspective.

We are working to:

  • Learn about the structure of prions, which can provide useful information to help understand how prion diseases, such as bovine spongiform encephalopathy (BSE) and fatal familial insomnia , are passed on to people and how the diseases develop.

 

For information about the lead scientist of this laboratory, please visit their Directory of Scientists and Professionals profile.

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