Size limitations in solution-state NMR

X-ray diffraction (crystallography) has been around for much longer than NMR and has been used to determine the precise three-dimensional stmcture of biomolecules as large as viruses (molecular weight in the tens of millions ). This technique requires a high-quality crystal and calculates a three-dimensional map of electron density for the molecule. The 

Thus we cannot escape the depressing reality that 7 2 will get shorter and linewidth will get bigger as we increase the size of the protein studied. The reduced T2 is not only a problem for linewidth, but also causes loss of sensitivity as coherence decays during the defocusing and refocusing delays (1/(2J)) required for INEPT transfer in our 2D experiments. The only ray of hope comes in the form of a new technique called TROSY (transverse relaxation optimized spectroscopy), which takes advantage of the cancellation of dipole-dipole relaxation by CSA relaxation to get an effectively much longer 7 2 value we will briefly discuss TROSY at the end of this chapter. 

Another consideration is the amount of sample (in mg) required for NMR. Because it is the concentration (mM) of molecules that determines the signal strength in NMR, as the molecular size increases the desired concentration of around 1 mM corresponds to larger and larger amounts of protein (Table 12.1). For a small protein (e.g., RNase at 12.6 kD) the sample size is around 6 mg. Talk to a biochemist or molecular biologist if you think this is a small amount of pure protein Now move to chymotrypsin (22.6 kD), which would require 11 mg of pure protein to be soluble and monomeric in 0.5 mL of water. For fatty acid synthase, with 20,000 amino acid residues and 21 polypeptide chains, we would need to dissolve 1.15 g of protein in our 0.5 mL sample volume, clearly an impossibility. 

With so many disadvantages to large-molecule NMR, you might wonder why we all don t trade in our magnets and spectrometers for area detectors and start doing X-ray 

NMR and crystallography should be viewed as complementary rather than competing methods. Different kinds of structural information can be obtained, and information gleaned from one technique can aid in the process for the other. Still, NMR is the younger sister in the family and must meet a higher standard of proof to justify big spending and job security in corporate research and development. 

---

The use of NMR to determine protein structures is a more recent development than X-ray diffraction. It has the advantage that the analysis can be performed in the solution state of the protein which removes any artifacts introduced by crystallization. Its major disadvantage is the size limitation, which restricts most analyses to smaller proteins (<40 kDa), although it is anticipated that improvements in the technology will extend the size limitation. 

The Nuclear magnetic resonance (NMR) image is obtained from the map distribution of spin electrons in a magnetic field and detection of their resonances. The technique can produce detailed information about topography and dynamic movements in either a solution or solid samples. The sensitivity of the technique depends on the amount of magnetic field introduced into the sample and the sample state itself, such as its temperature. NMR technique is limited to the sample size, which has to fit within the magnetic coils (ranges up to 30 cm in diameter) [81]. 

---