What is NMR?
Nuclear Magnetic Resonance (NMR) is a spectroscopic technique that can be used to elicit structural and dynamic properties of molecules by exploiting the behavior of certain atoms when placed in very powerful superconducting magnets (180,000 – 360,000 times stronger than the earths magnetic field for magnets at UConn Health). When NMR active nuclei are placed in a strong magnetic field their nuclei align with the field and begin to precess at a frequency dependent on the isotopes gyromagnetic ratio and the strength of the applied magnetic field along with the chemical and physical environment of the atom. The component of the frequency that is dependent on the chemical and physical environment is called the chemical shift. NMR experiments perturb the alignment by applying short pulses of radio frequency (RF) energy to determine the chemical shift of each of the NMR active atoms in the molecule being studied. By using combinations of pulses and delays (what is known as a pulse sequence) additional information may be ascertained such as which atoms are bonded to each other and which atoms are spatially close to each other. By utilizing many different experiments it is possible to determine the three-dimensional structure of molecules, including large bio-molecules such as proteins.
Do I need to understand NMR to obtain useful data?
No. The theory behind NMR is very complicated, however, detailed knowledge of NMR theory is not necessary to collect or interpret NMR data. The NMR facility manager can assist and train users in NMR data collection, processing, and analysis.
What types of biological questions can I answer with NMR?
There are too many uses for NMR of biological samples to list, but here are some of the more common biological questions we routinely address:
- Determining the three-dimensional structure of proteins and protein complexes in solution
- Using NMR relaxation to probe the molecular motions at various timescales
- To determine the site of binding for small molecules, metals, other proteins, nucleic acids, etc.
- To monitor the protein stability for site directed mutants
- For studying conformational exchange in proteins and probing low populated intermediate states
Why is there a size limit for biological samples?
As the molecular size of the sample goes up the NMR signal will decrease and eventually vanish. As the molecular size increases the tumbling rate of the molecule will slow. The slow tumbling will cause the NMR signal to de-phase rapidly causing the signal to broaden and decay quickly. In NMR pulse sequences there are a series of delays. When the relaxation or de-phasing of the NMR signal occurs faster than the delays needed to run the experiment, the experiment will fail.
In general well-behaved proteins below 20 kDa work well. Proteins between 20-30 kDa are more difficult, but can often be studied with conventional techniques. Proteins above 30 kDa are problematic with conventional NMR experiments. However, using high field NMR instruments, such as our 800 MHz instrument, coupled with deuteration of the protein sample, higher molecular weight samples may be studied but with an increased difficulty in interpretation of the data.
Why do I need to 15N and/or 13C label my protein?
The experiments that are used to study proteins rely on observing the 15N and 13C chemical shifts in addition to protons. The natural abundance of 15N and 13C are 0.3% and 1.0% respectively. At such low natural abundance the sensitivity of the experiments is too low to detect. To resolve this issue proteins can be expressed on enriched media allowing full incorporation of these stable isotopes.
How long does it take to run an NMR experiment?
The answer to this question depends on the type of NMR experiment that is being run, the concentration of the sample, and the overall behavior of the system being studied. For a simple 1D spectrum of a small molecule data can typically be collected in 15 minutes. For very insensitive 3D and 4D NMR experiments a single experiment may take a week. To collect an entire set of experiments necessary to calculate a three-dimension structure data collection time can vary from a week to a few months.
Is there training for NMR data processing and/or data analysis?
Yes, the facility manager can help train new users on all steps necessary to utilize NMR in your research. An hourly fee may be charged for such services.
Who can use the facility?
Researchers from the University of Connecticut primarily use the facility. However, access by researchers at other institutions is also available, with a different fee structure.
How do I schedule NMR time?
Contact the director, Dr. Jeff Hoch or the facility manager, Dr. Mark Maciejewski. At this time we are using a first-come first-served approach. If NMR usage for the 800 becomes saturated we will visit some type of priority scheduling. Scheduling calendars can be found at the following links:
What are the costs?
Do I need to be trained to use the facility by myself?
All NMR users should be trained and cleared to use the facility by the NMR facility manager.
How do I obtain keycard (ID badge) access to the facility?
First, you must obtain a current UConn Health ID badge. Internal UConn Health employees should already have a current badge and may update an old badge by going to the public safety office. Non-employees must fill out a background check through public safety. Once the background check has cleared a UConn Health ID badget may be obtained at your next visit. You may download the background check form from the Human Resources website.
There is a $75 background check fee for outside contractors (non students), but the fee is waived for students. The form should be sent to the NMR facility manager who will pass the form on to public safety. Questions can be directed to public safety at 860-679-2511.
Once properly trained the NMR facility manager will submit your ID badge number to public safety for access.
What spectrometer and probes are available in the facility?
400 MHz Instrument
- Broadband probe suitable for 1D proton, 13C, and 31P NMR. This is the default probe in the magnet at most times.
- HCN triple resonance probe with z-axis gradients for biological samples.
500 and 600 MHz Instruments
- HCN cold probe with z-axis gradients suitable for biological samples. The cold probes are in the magnet most of the time.
- HCN room temperature probe with z-axis gradients also suitable for biological samples.
800 MHz Instrument
- HCN salt tolerant cold probe with z-axis gradients suitable for biological samples. The cold probe is in the magnet most of the time.
- HCN room temperature probe with z-axis gradients also suitable for biological samples.
Do I have to run my own experiments?
No, the facility manager can setup experiments for non-experts or for outside users. A user fee will be charged in these cases. See the Services page for more information.
Do you have directions to the NMR Facility?
The Location and Directions page has detailed directions.
Can I walk-up and use an NMR?
Currently the 400 MHz instrument is available for walk-up access. However, on occasion the 400 MHz instrument may be used for long-term experiments. It is thus advisable to check with the calendar before driving to the facility. The 500, 600, and 800 instruments are only available with notification through the facility manager.
Sample Related Questions
Can my protein by studied by NMR?
High-resolution NMR investigations are most feasible for proteins less than approximately 25 kDa in mass and soluble to around 0.5 mM. In certain cases it may be possible to investigate proteins or complexes of larger size or lower solubility. Membrane proteins are difficult to study using high-resolution methods, but new techniques are emerging that may be applicable. Because NMR studies usually require labeling the protein with the stable isotopes 15N, 13C, and sometimes 2H, an expression system suitable for growth in labeled media must be available. Proteins must be purified (typically >95% is required), folded, and at least marginally stable. Preliminary characterization by circular dichroism and thermal or solvent denaturation is recommended.
How can the facility help me?
The facility is mainly used to determine the three-dimensional structures of proteins and protein complexes in solution. Protein structures are frequently helpful for understanding the biological function of a protein. It is not uncommon for structural homology to exist where little or no sequence homology exists, so structure can sometimes reveal function. Structures are also useful for designing biochemical experiments, since they often reveal binding or active sites, epitopes, or exposed residues suitable for labeling. NMR can also be used to studied molecular dynamics or conformational change, and can be used to screen ligand libraries for molecules that bind specifically to a target protein.
How much sample do I need?
This question is difficult to answer, as it is dependent on the questions being asked and the behavior of the sample. Typically 300 ul (with specialized NMR tubes) to 600 ul of sample is needed. Protein concentrations for well-behaved systems should be above 150 uM for structural studies, but lower concentrations may be used for other non-structural studies. While 150 uM is a rough estimate for the lowest concentration to use for structural studies it is advisable to make protein concentrations as high as possible and should be limited by solubility and protein behavior, not the amount of protein prepared. The amount of time running longer experiments to compensate for low concentrations and the increased time to interpret NMR spectra will almost certainly be longer then the time it takes to prepare additional sample.
What buffers can I use?
Traditionally buffers such as sodium phosphate have been popular for NMR studies, as it does not have any detectable NMR signals to interfere. However, for proteins fully labeled with 15N and 13C just about any buffer will work. If buffer concentrations will be very high such that the 1% natural abundance of 13C is detectable the use of deuterated buffers may eliminate some artifacts.
What ionic strengths are appropriate?
The NMR signal strength will drop as ionic strength increases. Thus, it is advisable to use the lowest ionic strength buffer as your sample will tolerate and still be well behaved. While ionic strengths should be kept as low it is possible to collect data with very high ionic strength, but with reduced sensitivity.
How do I label my protein?
Does the facility acquire data for samples other than proteins?
Yes, the 400 MHz instrument is largely dedicated to walk-up access for chemists. The facility is also equipped to collect data for other biological samples such as nucleic acids and carbohydrates, although there is not a lot of expertise in these areas.