NMR Assignment Methods for the Aromatic Ring Resonances of Phenylalanine and Tyrosine Residues in Proteins

Abstract

The unambiguous assignment of the aromatic ring resonances in proteins has been severely hampered by the inherently poor sensitivities of the currently available methodologies developed for uniformly 13C/15N-labeled proteins. Especially, the small chemical shift differences between aromatic ring carbons and protons for phenylalanine residues in proteins have prevented the selective observation and unambiguous assignment of each signal. We have solved all of the difficulties due to the tightly coupled spin systems by preparing regio-/stereoselectively 13C/2H/15N-labeled phenylalanine (Phe) and tyrosine (Tyr) to avoid the presence of directly connected 13C-1H pairs in the aromatic rings. The superiority of the new labeling schemes for the assignment of aromatic ring signals is clearly demonstrated for a 17 kDa calcium binding protein, calmodulin.

Synthesis of Isotopically Labeled α‐Amino Acids

“…Asymmetric hydrogenations using chiral rhodium catalysts have also been used for the synthesis of isotopically labeled amino acids [89,90]. For example, Kendall synthesized a multilabeled dehydrotyrosine derivative 75 in five steps from D 6 -phenol 74, which was then hydrogenated in the presence of a chiral rhodium catalyst (Scheme 11.20) [90].…”

Isotope Labelling

“…Another important advantage of the SAIL protein is the improved signal-to-noise ratio, which is mainly derived from the increased T 2 relaxation times, due to the replacement of unneeded 1 H and 13 C with 2 H and 12 C, respectively. In the case of the aromatic groups of Phe, Tyr and Trp, the absence of one-bond carbon-carbon coupling eliminates the need for a constant time scheme, thereby reducing the duration of the pulse scheme [141,142]. To perform this method, 20 special amino acids with a complete stereo-and regio-specific pattern of isotope labelling (SAIL amino acids) have been chemically and enzymatically synthesised (Figure 2.2) [25,[142][143][144].…”

“…In the case of the aromatic groups of Phe, Tyr and Trp, the absence of one-bond carbon-carbon coupling eliminates the need for a constant time scheme, thereby reducing the duration of the pulse scheme [141,142]. To perform this method, 20 special amino acids with a complete stereo-and regio-specific pattern of isotope labelling (SAIL amino acids) have been chemically and enzymatically synthesised (Figure 2.2) [25,[142][143][144]. These SAIL amino acids are commercially available from SAILTechnologies, a company that was established to supply SAIL amino acids to the NMR community (www.sail-technologies.com).…”

Can We Determine a Protein Structure Quickly?

“…There are indeed promising new ways to break this bottleneck. The new methodologies of non-uniform sampling [62][63] , labeling a domain [64][65] , selective labeling [66][67][68] , and more powerful NMR machines will eventually allow us to deal with larger proteins. Computational problems arising along with these new technologies will be very interesting.…”