Welcome to part two of “What Can NMR Do For You?”, a three-part series in which we see how you can use simple NMR experiments in your research. In part one, we went over some key points to keep in mind when doing NMR on proteins and DNA, such as sample preparation, and saw how NMR can be used to assess protein folding. In this article, we will see how NMR can be used to determine whether a protein is a dimer in solution. All the key points from part one still apply, and, because we are keeping this simple, we will be focusing on 1D experiments that do not require isotopic labeling of your sample.
First, why use NMR to look at protein dimerization? NMR has two main advantages over other techniques. First, NMR can detect weak dimers, with mM range affinity. Depending on your system, that may or may not be an advantage, since the biological relevancy of such interactions is questionable. However, this approach may be useful if you’re working with protein domains, which may have weak affinity in isolation and a stronger tendency to dimerize in the context of the whole protein. Second, generating your protein sample and collecting NMR spectra are relatively quick. You don’t have to make two differently tagged proteins like you do for a pull-down, which may interfere with binding, and the spectra themselves take minutes to collect.
To understand how you can measure protein dimerization with NMR, we need to go into a little bit of how NMR works. NMR is like any other type of spectroscopy. You hit your sample with a certain frequency of radiation, the sample absorbs that radiation and produces a signal, and the signal decays over time. In NMR, the bigger the molecule, the quicker the signal decays. This is why it’s difficult to study molecules above a certain size, though the size limit of NMR is continuously increasing. If you can measure the rate at which the signal decays, you can get an idea of the size of your molecule. Luckily, you can!
Let’s take another look at a 1D NMR spectrum of a protein:
Let’s ignore all the complexity for now, and just look at the area underneath all of the peaks in the amide region (dotted blue line): we’ll call that the signal intensity. Now, what if you waited a short time (we’re talking milliseconds here) between irradiating the sample and collecting the spectrum? Well, you would see the same spectrum, but, because the signal is decaying over time, the signal intensity would be a little bit weaker. If you collect several spectra as a function of different waiting times (also known as delay times), you can fit those data to an exponential curve and graph the rate of decay. It might look something like this:
(Note: These are not real data. No real data ever look this good.)
In NMR, there are two main rates of interest: T1 and T2. They give you slightly different information, but the basic idea is the same: hit your sample with a certain frequency of radiation, wait a little bit, and then collect a spectrum.
So, what does all this have to do with dimerization? Well, if you know T1 and T2, you can calculate what’s known as the rotational correlation coefficient of your protein, which (drumroll please) is inversely proportional to the size of the protein. So, if you know you have an 8 KD protein, and the rotational correlation coefficient is more consistent with that of a 16 KD protein, you probably have a dimer. I’m not going to bore you with the equations, but here’s a nice article that goes through the details of monitoring dimerization by 1D NMR. If you’re feeling especially industrious, you could even generate the rotational correlation time as a function of protein concentration and get a sense of how weak or strong the dimerization is.
So, next time you’re thinking of measuring dimerization, consider NMR. Also, if you’ve got an NMR enthusiast nearby, ask him or her what extra information you could get from isotopically labeling your protein. You may just be able to determine the dimerization interface, test conditions that disrupt or promote dimerization, or see if other molecules bind to your protein.
What do you think? How do you monitor protein dimerization? Leave your ideas in the comments below.
Although his name could fit in easily to the early 1980’s Hip-Hop Scene, Jerzy Nomarski (or ‘George’) was actually a Polish physicist with an interest in optical theory. Born in 1919, he eventually became a member of the Polish Resistance fighting in the Second World War. He was captured by enemy forces and held as […]
It’s great to have you in the Bitesize Bio family! We’ve sent you an email to confirm your registration. Please click on the link in the email or paste it into your browser to finalize your registration.
For more information on how to use Bitesize Bio, take a look at the following image (click it, for a larger version)
An error occured while registering you, please reload the page and try again