Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

The overall goal of this protocol is to study the structures of insoluble and non-crystalline super molecular protein assemblies at atomic resolution by magic angle spinning solid state NMR spectroscopy. We will present the essential methodological steps for studying atomic structures of biomolecular protein assemblies by solid state NMR. Studying atomic structures of these assemblies is extremely challenging because they're inherently insoluble and non-crystalline.

Solid state NMR is an emerging technique able to study molecular structure and dynamics at the atomic resolution without being limited by the size of the assembled object, or by its solubility. We illustrate here the key steps to visualize atomic structures of biomolecular protein assemblies by sold state NMR including the preparation of isotopically labeled samples, the collection and the analysis of structural data from solid state NMR. The first step in a solid state NMR workflow is the production of C13 N15 labeled protein subunits and their in vitro assembly.

Inoculate a 15 mil pre culture of pre-warmed LB medium with one colony of transformed E.coli cells. Incubate at 37 degrees celsius with 200 RPM shaking overnight. To inoculate the main culture, transfer the entire pre culture into one liter of pre warmed N9 medium containing necessary isotopically labeled sources of carbon and nitrogen.

These can include, N15 labeled ammonium chloride, uniformly C13 labeled glucose, selectively C13 labeled glucose, or selectively C13 labeled glycerol Incubate at 37 degrees, and measure the optical density at 600 nanometers as soon as the culture become turbid. When the OD has reached a value of 0.8, induce protein expression with a 0.75 mini molar IPTG for four hours. Note that optimal induction conditions can vary from one protein to another.

Recover the cells by centrifugation for 30 minutes at 6, 000 G and four degrees. After purification of the protein subunits, they are assembled in vitro. Concentrate the protein to around one mini molar in a centrifugal filter unit.

To do so, introduce the sample into the filter unit and centrifuge at 4, 000 G for 30 minutes. Between centrifugation steps, gently mix the solution in the filter unit with a pipette to avoid protein deposition at the filter membrane. Repeat the procedure until the desired concentration is reached.

Transfer the sample into a falcon tube and incubate under agitation for one week at room temperature. Usually, the polymerization of the subunits into filaments is accompanied by the solution becoming turbid. Add 0.02%weight per volume sodium azide to avoid bacterial contamination.

To harvest the protein assembly, centrifuge the sample for one hour at 20, 000 G and four degrees. Aspirate the majority of the supernatant leaving only enough liquid to cover the surface to avoid sample drying, and store the sample at four degrees until measurement. We will present the essential experiments for a structural analysis by solid state NMR.

One dimensional cross polarization, or CP, and inept experiments detected on C13 nuclei are used to detect rigid and flexible protein segments in the assembly, respectively. And to estimate the degree os structural homogeneity and local polymorphism. Here, we use standard experimental values.

Typical parameter ranges are indicated in the protocol. Insert the rota into the NMR magnet and start the magic angle spinning as described in the protocol. Set the desired spinning frequency, and ensure stabilization to within plus or minus 10 hertz.

Record a single pulsed, one dimensional proton spectrum using 16 scans. Set up a 1D proton carbon CP.CP experiments show the signals that arise from residues in a rigid conformation. Initial experiment parameters are taken form a standard optimization procedure on a reference compound.

Pulse calibration and decoupling parameters can be optimized in the sample when the sensitivity is high enough. Here, we record a CP with 16 scans. CP contact time and power levels are chosen based on maximal signal intensity, as demonstrated here for the optimization of the CP contact time.

The maximum intensity in this example is reached at 800 milliseconds. The decoupling parameters should also be readjusted from those originally set form the reference compound calibration. Lastly, carefully observe the localisation of spinning side bands at the given magic angle spinning frequency, shown here at 18 kilohertz, to avoid overlap with the signal.

Record a reference 1D proton carbon CP spectrum that serves as a one dimension spectral fingerprint. Here, we accumulate 128 scans with 800 millisecond CP contact time, 100 kilohertz decoupling strength, a recycle delay of three seconds, and an acquisition time of 20 milliseconds. Process the CP experiment without apathi-zation.

Choose and isolated peak to estimate the C39 within the sample, indicative of structural order and homogeneity. Here, the line width, measured as the full width at half height, is around 60 to 70 hertz, indicative of a well ordered protein structure in the assembly. Set up a one dimensional proton carbon inept experiment to probe for highly mobile parts of the protein assembly.

Record a reference inept experiment to serve as a fingerprint for the mobile protein segments. Typical parameters are 128 scans, and an acquisition time of 25 milliseconds. Process the proton carbon inept experiment.

The number of signals, and their positions, are indicative of the extent of residue mobility and the amino acids composition respectively. Here, only signal from the twist buffer CH2 merit-y is observed as the entire protein is in a rigid regime in the assembly structure. Conformational analysis of the protein structure is based on the solid state NMR resonance assignments for all rigid residues of the assembly.

This is made possible because chemical shifts are highly sensitive probes for local chemical environment, and can be used to predict the protein's secondary structure. A complete 3D structure determination is then based on the collection of structural data such as distance restraints that encode both intra and inter molecular atom proximities up to nine angstrom. Here, we will show how the basic two dimensional experiments are recorded and give an example of a sequential assignment.

We will then give a short demonstration on how to collect distance restraints from experiments recorded on selectively C13 labeled samples. Set up the short mixing time two dimensional carbon carbon PDSD experiment to detect intra residue carbon carbon correlation. Copy the values for the initial proton carbon cross polarization step from the one dimensional CP experiment.

The mixing can be set to 50 milliseconds for uniformly C13 labeled samples. Here, we have chosen 15 and 20 milliseconds acquisition time in the indirect and direct dimensions respectively. To process the PDSD spectrum, we use a QSINE window function with sine-bell shift of 3.5.

To enable residue specific assignment, use the settings of the short mixing time PDSD to record an intermediate mixing time PDSD with a mixing time of 100 to 200 milliseconds. Note that additional spectra, including nitrogen detected experiments, are often required for a complete resonance assignment, and are detailed in the protocol. Choose an NMR analysis program, such as CCPNMR Analysis.

Load the 2D spectra into the software, and create a molecular object with the primary protein sequence. Start with the identification of the amino acid types that are visible in the short mixing time PDSD spectrum. Connecting the carbon atoms of the spin systems allows for residue type specific assignment.

Here, we assign the spin system of a thenium residue. The polarization transfers between the C-alpha, C-beta, and C-gamma nuclei lead to physical cross peaks in the PDSD spectrum. Try to identify as many residues as possible with this procedure.

Overlay both the short and intermediate mixing time PDSD spectrum. The supplementary peaks visible in the intermediate mixing time PDSD commonly arise from contact between sequential residues. Mark the resonance peaks of a spin system and find correlations in the intermediate mixing time PDSD with resonance frequencies of other spin systems.

We show the sequential assignment with thenium 33 to isolutane 32 in our filamentous protein assembly. The resonance frequencies of the thenium spin system are visible on a carbon frequency of the isolutes in 32, and vice versa. Procedures for assignment with nitrogen detected spectra and for obtaining protein secondary structure from the assignments are described in the protocol.

Following thorough resonance assignment, we can proceed with the identification of long range restraints. Long range restraints are intra or inter molecular carbon carbon proximities between distant residues which define both the tertiary fold of the monomeric subunits and the arrangement of the subunits within the assembly. Here, selectively C13 labeled samples are of particular importance.

Overlay an intermediate mixing time PDSD with a long mixing time carbon carbon PDSD recorded with a mixing time between 400 milliseconds to one second on a selectively C13 labeled sample. If possible, both PDSD should have been recorded on the same selectively labeled sample. Supplemental peaks arise from correlations between more distant C13 atoms.

During resonance assignment, take into account the selective labeling scheme. In this case, 1-3-glycerol. Here, we have highlighted and example of a long range contact peak with and unambiguous assignment into both dimensions.

Thenium 33 C-beta with protein 45 C-alpha. After completing long range distance identification, restraints are classified as unambiguous or ambiguous as well as intra or inter molecular. The restraint lists to be prepared for the structural modeling are listed in the protocol.

Tutorials on structural modeling using different programs can be found online. Solid state NMR data, either by itself or in conjunction with complementary data, can allow for the atomic level structure determination of super molecular assemblies. The advantages of solid state NMR are on the one hand, the ability to provide both secondary structure information and atomic distance restraints from intramolecular interfaces, which can be integrated into the modeling process.

Yet, on the other hand, the unique feature of solid state NMR spectroscopy lies in its ability to collect atomic distance restraints at intermolecular interfaces of intact super molecular assembly. The detection and distinction between intra and inter molecular restraint assignments can however be a tedious process, and typically requires a preparation of selectively C13 labeled samples. However, with new developments in selective labeling strategies, spectrometer hardware design, and solid state NMR methodologies, the technique is increasingly fulfilling its theoretical potential of providing atomic level molecular information without limitations of object size, long range order, or crystallinity.

This method allows us to study the atomic structure of biomolecular assemblies. It's very important to carefully prepare the sample to optimize the NMR condition, to obtain high resolution data. With this protocol, we can obtain atomic resolutions which are information of protein assemblies.

And this technique can be combined with other techniques in structural biology to obtain three dimensional models.