Lesson 14
Magnetic Resonance
In magnetic resonance techniques, a magnetic field is applied to a sample which can result in excitation of electrons or atomic nuclei (see this video). Electron paramagnetic resonance (EPR) involves the excitation of an unpaired electron, while nuclear magnetic resonance (NMR) involves the excitation of atomic nuclei. These principles also apply to magnetic resonance imaging (MRI).
Nuclear Magnetic Resonance (NMR)
NMR can be used to determine high resolution structures of proteins. NMR structure determination utilizes the natural spin of stable isotopes (generally 13C and/or 15N) that are incorporated when expressing the protein. The stable isotopes have spin states that will give rise to a nuclear magnetic resonance which depends on both the frequency of the electric field and the electronic environment that each atom is in. Pulse programs that manipulate the orientation and length of time the magnetic field is applied have been developed which allow for the resonance of only certain atoms to be detected. For example, the first step in solving an NMR protein structure is to assign a heteronuclear single quantum coherence spectra (HSQC). The pulse program used allows for the resonance of only 15NH groups to be detected. Since each amino acid in a protein contains one NH in the backbone, each peak in this spectra corresponds to one amino acid in a unique environment. The next step is to assign each of the peaks in the HSQC to its specific amino acid in the protein. This is completed through the use of 3-dimensional NMR spectra that correlate the 15NH of one amino acid to the 15NH in the proceeding amino acid. Finally, spectra based on the nuclear Overhauser effect (NOE) are used, which utilize cross-talk between different spins through space (rather than through bond as above) to determine the distances between atoms. This allows for the mapping of inter-protein interactions that leads to a 3-dimentional high resolution structure.
Linda's postdoctoral work was using NMR to investigate membrane protein structure. Tracy's undergraduate work was focused on using NMR for soluble protein structure determination.
Left is a picture of the NMR instrument at UVA.
Watch this video from St. Jude Children's hospital for an example of how NMR is used in simplified terms. At the beginning of this video what other techniques to determine protein structure are depicted?
Electron Paramagnetic Resonance (EPR)
Electron paramagnetic resonance (EPR) is the measurement of the absorption of electromagnetic radiation equaling the energy of the splitting of the energy states of an unpaired electron in a magnetic field. EPR requires a paramagnetic center with an unpaired electron. As most proteins do not naturally contain an unpaired election, they must be experimentally added via site-directed spin labeling (SDSL). For SDSL, any reactive native cysteine residues must first be removed from the protein (generally these are replaced with alanine or serine residues) via mutagenesis. Then, the site(s) of interest are mutated to cysteine residues. Then, the spin label is chemically reacted with the thiol group of a protein’s cysteine residue to result in a covalently attached spin label at that specific protein site.
Continuous wave (CW) EPR is an excellent technique to probe local protein properties such as dynamics, local environment, secondary structure, tertiary contacts, and solvent accessibility. To the untrained eye, CW spectra are just a bunch of squiggles that are hard to interpret. However, CW lineshapes of nitroxide spin labels can give important information about the dynamics of the spin label. In the fast motional limit (~0.1 nsec, such as free spin label) the CW spectrum shows three sharp lines of equal height. As motion slows, the lineshape broadens, the amplitudes decrease, and the lineshape becomes asymmetric. Thus, the position of the spin label on the protein can be categorized as mobile, such as on a loop; semi-restricted, such as a surface exposed site containing secondary structure; or immobile, such as buried in the protein core. Linda's PhD thesis was focused on quantifying dynamics from EPR spectra on proteins.
Double electron electron resonance (DEER) spectroscopy (also called pulsed-electron double resonance, PELDOR), can be employed to measure the distance distribution between two spin labels. These spin labels may be within the same protein, aiding in identification of protein structure and conformational dynamics; or, two different proteins can be singly labeled to investigate binding and the complex structure. DEER extracts distance information from pairs of spin labels due to the distance dependence of the energy of the dipolar interaction between the unpaired electrons. Tracy's PhD applied CW and DEER spectroscopy to LspA and will be the topic of your assignment.
Assignment: For this week we will do a journal club looking at a paper (in preparation) examining LspA structure and dynamics using EPR as well as molecular dynamics (computation). I will email you the paper!
Congratulations!
We have come to the end of our tutorial! It has been a pleasure working with each of you! I hope you have learned a little bit about what biophysics is and what it looks like to be a biophysicist. I have enjoyed watching you grow as scientists even in this short time. You have learned how to think and ask questions like a true scientist and I hope you continue to ask questions about the world around you!