Nuclear magnetic resonance (NMR) spectroscopy can be used to determine chemical structures and, in some cases, chemical dynamics. By probing the intrinsic spin properties of nuclei, NMR can identify local chemical environments and selective probe atomic species.
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NMR is not just suited to identifying chemically distinct species but can also be used to disentangle different conformers.1 Conformational isomers, or conformers, are molecules with the same atomic composition and connectivity between the atoms. However, they differ in their geometric arrangement. With most conformers, it is possible to interconvert between the different isomeric forms by undergoing some kind of rotational motion.
While conformers may not be distinct in terms of their elemental composition, they can have very different behavior and interactions with other molecular species. Many biological species, including proteins, have a huge number of different conformers. A protein is constructed from building blocks known as amino acids. These amino acid sequences are then linked together with peptide bonds to form the protein structure.
One amino acid, proline, has some unusual properties. When peptide bonds are formed with proline, they can adopt either the cis or trans isomeric forms. While usually the trans form is favored, the interconversion between the cis and trans conformers can play an important role in protein folding.2
Intrinsically Disordered Proteins
Most proteins will have several stable conformers that they can interconvert between but are generally fixed in one secondary structure. However, intrinsically disordered proteins have a much greater degree of conformational flexibility meaning they can bind to multiple targets and may prove a valuable target to drug therapies to aid in the treatment of diseases such as cancer.
Prolines have an important contribution to this conformational flexibility due to their abilities to contribute to cis/trans isomerization in the protein. While this conformational flexibility of intrinsically disordered proteins is interesting from a biomedical perspective for treatments and protein behavior, being able to measure the exact contributions of the amino acids to a given structure or discern the different conformational structures is key to the development of new therapies that make use of these proteins.
Recent work from a collaboration between the Eötvös Loránd University, Hungary, KIT University in Karlsruhe, Germany, and Bruker has involved developing a NMR method that captures these shifting structures for characterization.3
By focusing on 1Hα NMR, the team captured proline-specific signatures for investigation. The challenge they faced was the concentrations of the different isomers could be as low as 5 ppm in some cases. Due to the small energy difference between the two spin states of the atoms of interest, NMR is a relatively insensitive technique. This usually means measurements require large amounts of the sample or long acquisition times.
To capture the dynamical behavior and interconversion between the conformers, long measurement times were not possible as the resulting signal would represent an average of all the different structures the protein had sampled during the measurement.
The new method developed by the team makes use of real-time homodecoupled sequences. Previously, making such measurements on profile to understand the conformational dynamics would have involved making various chemical substitutions on different parts of the protein to investigate the difference between the conformers and try and ‘lock’ certain structures for the duration of the measurement.
With the new methodology, a standard NMR spectrometer can be programmed with the right pulse sequence to make sure of this fast experimental detection times without the need for any additional equipment. This makes NMR a very attractive candidate for structure studies on these types of proteins. NMR is also very attractive as a tool for conformation studies as other structurally sensitive techniques, such as X-ray crystallography or cryo-EM, cannot follow the dynamical processes in the protein. This is because the freezing or crystallization process locks the protein into a particular form.
As well as the ease of use and the lack of need for additional, specialized equipment, another positive aspect of the new NMR methodology is that measurements with this method can be made under physiological conditions. Direct measurements on cells and tissues are often not possible with advanced analytical techniques but given the sensitivity of the protein structure to its environment, it is important that the measurements imitate the ‘real’ environmental conditions for the protein as closely as possible.
The team focused on profiling p53TAD with its new method, which is an example of a disordered p53. p53 is a gene that makes a tumor-suppressor protein and mutations of this gene lead to the formation of cancers. By studying the conformations of p53TAD, it is possible to understand what types of drug structure might be well-suited for targeting genes of this type and help drive better cures for cancers.
References and Further Reading
- Tormena, C. F., Cormanich, R. A., & Rittner, R. (2011). AUREMN NMR Spectroscopy : a Tool for Conformational Analysis. Annual Reviews of Magnetic Resonance, 10(1/2), 1–27. http://www.auremn.org.br/Annals/2011-vol10-num1/AMR2011v10n12p1-27.pdf
- Wedemeyer, W. J., Welker, E., & Scheraga, H. A. (2002). Current Topics Proline Cis - Trans Isomerization and Protein Folding. Biochemistry, 41, 14637–14644. https://doi.org/10.1021/bi020574b
- Sebák, A. F., Ecsédi, P., Bermel, W., & Luy, B. (2021). Selective 1Hα NMR methods to reveal functionally relevant proline cis/trans isomers in IDPs: characterization of minor forms, effects of phosphorylation and occurrence in proteome. Angewandte Chemie - International Edition. https://doi.org/10.1002/anie.202108361