Unlike x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy is a unique technique that can determine the structure and dynamics of complex proteins without the requirement of crystals.

Four principle elements were set up by pioneers Wüthrich and his co-workers for protein structure determination.

 

i) The Nuclear Over Hauser effect (NOE) as an experimental NMR parameter in the determination structure and folding dynamics

ii) Sequence specific assignments of NMR peaks  from a protein

iii) Computational tools to  interpret the NMR data

iv) Multi-dimensional NMR  techniques  for  data collection (COSY,SECSY,FOCSY,NOESY, TROSY).

The structural determination of proteins is either carried out in solution state NMR or solid state NMR. In both the techniques the proteins must be labelled with atomic nuclei that are intrinsically magnetic (spin), isotopes displayed this property 13C, 14N, 2H, 19F.

The micelle and bicelles environment is the most-used approach (Lacapère et al.2007). These structures help in the solubility of the proteins into the solution/ solvent.

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These complexes help proteins integrated into NMR solutions 

The spinning of the isotopes cause a magnetic moment when the spin is at ½ it creates a magnetic dipole. When an external magnetic field is applied the magnetic dipoles give rise to spin states called α and β spins. A small difference between nuclear magnetic moments of α and β spins gives rise to small polarization between them causing magnetization and resulting in a NMR signal (Levitt, 2008).

This imbalance is caused by a pulse of electromagnetic radiation also known as a radio-frequency (Rf) pulses, absorption of electromagnetic radiation of appropriate frequency induces a transition from the lower to the upper level (Wider, 2000).

After pulsing the signal undergoes relaxation also known as the free induction decay (FID) is measured. FID measures resonance frequencies of NMR spectra obtained through Fourier transformation techniques. The difference in resonance frequencies is called a chemical shifts (range: 0-9 parts per million) (Sattler, 2004).

The assignment of chemical shifts indicate the chemical group under different conditions like the formation of alpha-helix from a disordered structure in response to change in pH. From these assignments a NOE spectra is formed NOE’s can only be observed when the distance between the spins is 5-6 Å. by using 3-D NOE-spectroscopy . J coupling constants and other parameters like residual dipolar couplings and cross-correlated relaxation effects (CCRs) are measured, to derive structural information as data for computational structure calculation (Wider, 2000).

2                 NMR parameters that contribute to structural determination of proteins

The determination of beta–barrel monomeric/ dimeric structures in transmembrane proteins (TMPs) was carried out by Wüthrich and his coworkers in 2004. Outer membrane protein (OmpX) 16kDa was reconstituted in dihexanoyl phosphatidylcholine (DHPC) micelles.

The use of transverse relaxation-optimised spectroscopy (TROSY) combined with high magnetic fields and backbone resonance assignments of the protons onto specific methyl groups were carried out at a proton frequency 750 MHz (Arora and Tamm, 2001).

The solution state of OmpX elucidated 148 residues long, eight antiparallel beta barrels strands connected with four mobile loops connected by mobile loops. Four protruding beta-sheet were determined (Fernández et .al 2004).

Wüthrich and his co-workers observed that when compared to the X-ray structure they were more complementary than different, both structures showed the protruding beta sheets at the extracellular end and similar b-barrels folding mechanisms (Schulz, 2000). The NMR structure depicted poor loop structures because their beta strands were 2 residues shorter to X-ray structure.

3         The transmembrane β-strands are labelled(β1-β8) where β3, β4 ,β5 and β6 are the protruding β-sheet.  L1-L4 extracellular loops and T1- T3 are periplasmic turns.

Oxenoid et al., 2004 carried out a NMR assignments on diacylglycerol kinase (DAGK) 40kDa with the use of TROSY-based pulse sequences. DAGK elucidated 121 residues and structure is trimetric with each monomer having three transmembrane with one amphipathic helices. Li and her co-workers, 2014 reviewed that solution NMR structure has typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates while the crystal structures show few such perturbations.

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Ribbon structure of DAGK from solution NMR showing poly-topic alpha helixes.

 

Glycophorin A (GpA) is found in human erythrocytes and determines MNs & Ss blood groups. Engelmann et. al, 1997 used solution NMR to solubilize a 40-residue peptide in detergent micelles . GpA is 131 residue long that contains a sequence motif known as the ‘GXXXG’ motif that mediates dimerization to form parallel dimer of helices making NMR solution a viable tool for  helical studies (Arora and Tamm,2001) (Smith et al.2001).

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However studies indicate that solution NMR spectroscopy does not necessarily be method of choice because low spectral dispersions and limited long –range distances from NOE data could impede resonance assignments and tertiary structure determination respectively (Lacapère et al.2007).

Solid state NMR spectroscopy can help overcome these issues it follows similar principles to solution NMR and additionally spins at a magic angle. The random rotational diffusion is simulated by spinning the sample at the ‘magic angle’ of 54.7° using bicelles to carry high-resolution investigation of potassium channels, proton channels, Ca(2+) pumps, G protein-coupled receptors, bacterial outer membrane proteins, and viral fusion proteins to elucidate their mechanisms of action and dynamics (Bechinger et al. 2011)(Kukol,2014) .

The first membrane protein solved by solid state NMR was cation channel gramicidin A, antibiotic that acts as an ion pore. It was used as a study model for other membrane channels because of its cation-selective single-filing pore and acylation of membrane proteins (Seoh and Busath, 1993). The determination on the M2 helix in gramicidin a channel posed as a model study for the nicotinic acetylcholine receptor (Arora and Tamm, 2001). Nicotinic acetylcholine receptor structure was located in the post-synaptic membrane between nerve and muscle cell, transmitted the nerve signal to the muscle  (Celie et al. 2004).The structure compromised of a single alpha helix tilted at 12˚ from which it was understood the ligand-gated cation-selective channel is a pentamer of alpha helices arranged around a central membrane-spanning pore of 290 kDa found abundantly in electric organ of Torpedo ssp (Arora and Tamm, 2001) (Ketchem et al.1993).

 

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Ribbon structure of Gramicidin obtained by solution NMR using DMPC bicelles. Pore channel share structural similarities with the M2 helix of nicotinic acetylcholine receptor .

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Methods using magic-angle spinning solid-state nuclear magnetic resonance (MAS SSNMR) has enabled the investigation of amyloid fibril structures. The study of amyloid fibrils has vastly contributed in understanding Alzheimer, Parkinson and Huntington disease (Müller et al. 2013).

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Depicts the processing of amyloid peptide through the amyloid beta precursor protein. All three domains are in the extracellular matrix.

 

Solid state NMR investigations focused on the β-sheet structures in fibrils formed by fragments of the β-amyloid peptide. It  was observed that the most common structures amongst the fibrils was the  cross- β  structure the  stacking of  β-sheet ribbons made up of  two cross-β units forming glutamine repeats as polar zipper which the main energy barrier for fibril formation and stabilize the structure (Rambaran and Serpell 2008). In β- sheets hydrogen bonding direction runs are parallel to each other and β-strands perpendicular to axis of fibril. Studies of fibrils by SSNMR   have shown that the β-amyloid peptide folds into a β-bend structure that then associates with other molecules to form parallel conformations (Rambaran and Serpell 2008 )(Tycko,2011).

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Ultra-low-temperature MAS and  dynamic nuclear polarization  (DNP) technology has shown success especially in amyloid-forming system Debelouchina et al. have demonstrated the applicability of  DNP to amyloid fibrils, in studies of partially-labelled GNNQQNY peptide fibrils which resulted in studying the conformation of structures at every evolutionary stage.

All these  are just a few examples of  how  NMR Sprectroscopy has helped in creating a better understanding of  pathways and structural determination so as to advance no matter  what the disease or  disorder.  It is also important to appreciate the availability of computational technology that converts the data into desired protein structures, protein docking software’s XPLOR, HADDOCK, ROSETTA, TASSER, ab-initio methods, molecular dynamics simulations and bioinformatics tools and databases that further aids to determine protein structures.

A table to understand  both the technologies in short.

 

         Characteristics  
 

Nuclear Resonance Magnetic  Spectroscopy

 

X-ray crystallography

 

Advantages and  disadvantages

 

Origin of protein

 

 

 

 

Proteins used are usually recombinants protein isotopically labelled.

 

Proteins used can be natural (native conformation) or recombinant proteins. Use of Recombinant proteins in Xray  has an advantage as it  allows the insertion or deletion of fragments  as required .
 Crystals Not required  the  proteins  can be  studied in their  solution or  solid states Crystals are essential for diffraction to take place to determine 3-D structures The disadvantage of using crystal is that some protein  fail to crystallise and becomes a challenge to obtain  crystals whereas  NMR  spectroscopy could dissolve these hard to crystallise proteins in high concentrations and  study them.
 

Protein Size

Molecular weights <40 kDa NMR spectroscopy is method of choice. molecular weights above 50–100 kDa With X-ray crystallography  larger molecules can be studied as long  as they can crystallised unlike in  NMR  spectroscopy large  molecules(>30kDa) were harder to study  till transverse  relaxation optimisation spectroscopy was combined with  2H labelling was used to overcome the size barrier,
Labelling Labelling Isotopes (C13/N15/P31/H1) Uses Selenomethionine (Se-Met) protein labelling and sometimes monoclonal antibodies are used crystallization.

 

 

 

Selenomethionine labelling   into proteins and using  multi-wavelength anomalous diffraction (MAD) incorporation of heavy atoms such as selenium helps solve the phase problem in X-ray crystallography. Whereas the NMR spectroscopy doesn’t face any phase problem as this method depends on the nuclear spins of the isotopes and not the phase itself (Sattler,2004).

 

Catalysts Detergents are used to increase the solubility of the protein/crystal.  NMR spectroscopy use surfactants known as micelles and bicelles. Precipitants are required. Crystallization of protein based on increasing the precipitation of the protein in solution using precipitants such as ammonium sulphate or polyethylene glycol (Drenth, 2007). One of the major advantages with crystallography (Lipid cubic pahses) was that there wouldn’t be any interference of detergents thus resulting in crystal that could  easily give a structure with higher  resolution.

While the disadvantages with NMR its isotopic labelling and deuterated reagents can be expensive, larger proteins can lead to poor spectra.

 

Resolution Å Resolution cannot be  measured Higher resoultions can be obtained

1.5–2.5 Å

Resolution is clearly defined from the diffraction pattern for techniques using crystal

 

There is no way to define resolution for but the quality of the structures might be assessed by the number of long-range NOES.

Initially crystallography also depended on the quality of proteins till  development  of  synchrotron high resolution x-ray diffraction are  obtained

synchrotron

 

Quality of the atomic structure If the root mean square deviation (RMSD) is between 0.5- 0.7 Å indicates high  precision. If the R-factor = 20% or less the structure is considered well defined The % difference between the observe red and calculated diffraction patterns  while root mean square deviation (RMSD) between provide  co-ordinate precision which helps in characterizing NMR structures.
Principles > Nuclear >Overhauser effect  >J- coupling

>Chemical shifts >Dihedral angle        restraints.

>Nuclear magnetic resonance

>Residual Dipolar couplings

>Relaxation and Co-relations

> Fourier transform

> Braggs laws

Fourier transform in X-ray crystallography is a global technique. In contrast the NMR is by definition a local technique (proximity measures) that suffers when distant points are being involved. Constraints are well investigated and applied in X-ray crystallography.
In NMR methods we have to rely on Magnetic Dipole force fields or crude force field approximations.

References you got to have them ! (follow the link below)

https://docs.google.com/document/d/1V_2fs-DnUCGSH5sPLwqbSMl0dhzOO6wYHl4qMZfwWe8/pub

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