Chemical shifts underpin the interpretation of NMR spectra, playing a key role in understanding macromolecules and the active-site chemistry of enzymes, for instance. Now, US researchers are hoping to leverage the chemical shift to improve the level of detail in determining conformation, bonding, and dynamics in important proteins. Their approach relies on measuring not only isotropic chemical shifts but also the full chemical-shift anisotropy (CSA) tensor.

Benjamin Wylie, Lindsay Sperling, Heather Frericks, Gautam Shah, Trent Franks, and Chad Rienstra of the University of Illinois at Urbana-Champaign, point out that until recently, most efforts to measure backbone amide and carbonyl CSAs in enzymes, for instance, use cross-correlated relaxation and residual anisotropic shifts in solution NMR. However, analysing side bands in solid-state NMR has generally only been possible for labelled samples or small peptides.

Now, the team has extended the slow magic-angle spinning (MAS) method of Herzfeld-Berger (first published in 1980, J. Chem. Phys. 1980, 73, 6021-6030) to ¹³C,¹⁵N-enriched solid proteins. The researchers have applied two-dimensional heteronuclear correlation to the microcrystalline protein GB1. The combination of this approach with high magnetic field instrument (750 MHz ¹H frequency) together with pattern labelling of the highly enriched ¹³C sites allowed them to obtain tensors for 42 pairs of amide and carbonyl groups.

The structure of beta1 immunoglobulin binding domain of protein G, commonly known as GB1 was first reported by Gronenborn and co-workers in 1991. "GB1 binds to the Fc region of immunoglobulin G (IgG)," explains Rienstra, "Its function is believed to be a way that Streptococcus helps to evade host defenses by consuming the IgG and alpha1-macroglobulin." He adds that, "It is a commonly used model protein for NMR and protein folding and dynamics studies because it is highly thermostable, easy to prepare in large quantities, and it has both beta sheet and alpha helical domains." Rienstra and his colleagues first reported the solid-state NMR chemical shifts in 2005 (J Am Chem Soc, 127, 12291).

The Urbana-Champaign team has now carried out slow spinning experiments (3-5 kHz) and recorded the N-C' 2D spectra using SPECIFIC CP (spectrally induced filtering in combination with cross polarization) for a single sample of GB1 prepared with ¹⁵N and most ¹³C. Using this approach the team was able to obtain high-quality, slow MAS solid state NMR spectra using a relatively simple experimental setup with diluted-¹³C labelling.

They obtained three data sets, which took one day each to record. These experiments gave them 42 pairs of ¹⁵N and ¹³C' tensors using straightforward fitting procedures. "We envision applications to biological systems by combination with individual or amino acid type-specific labelling," the researchers say, "This approach could have particular value for interrogating active sites of enzymes, where hydrogen bonding and hybridization of intermediate states often report directly on mechanistic details."

Related links:

• J Am Chem Soc, 2007, 129, 5318-5319
• Rienstra Page

Article by David Bradley