Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study

Abstract

Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial N-, C -, and C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves N, 1- C, 2- C, fully N/ C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide H and N resonances need to be obtained, even in cases where sensitivity is the limiting factor.

Selective isotope labeling strategy and computational interpretation of spectra for protein NMR analyses

“…To reduce the required number of labeled samples based on this point-of-view, the information content (entropy) per labeled sample should be increased. Therefore we introduced fractional stable isotope labeling to achieve ternary labeling levels; namely, 50%, 75%, or 100% for 15 N and 0%, 50%, or 100% for 13 C, while the other combinatorial labeling methods used binary labeling levels. An example of the labeling pattern is shown in figure 1b.…”

“…The protein sample is usually stable-isotope labeled, to obtain rich information about the targets using the NMR-detectable nuclei, 1 H, 13 C, and 15 N, and to perform various measurements that rely on stable isotope enrichment. Two-dimensional 1 H- 15 N spectroscopy is one of the basic methods for protein analyses. It uses amide moieties as probes to obtain residuespecific information about the target protein.…”

“…It uses amide moieties as probes to obtain residuespecific information about the target protein. For example, it includes chemical shift perturbation, transferred cross saturation, 15 N relaxation, and paramagnetic effects. To perform such analyses, the assignment of the amide signals is required beforehand.…”

“…For single selective labeling, the amino-acid typing of the residue can be performed by acquiring the standard 1 H-15 N correlation spectrum, such as 15 N-HSQC, with 19 labeled samples corresponding to all amino acids except for proline. For dual selective labeling, each sample is both 15 N-labeled with a specific amino acid and 13 C-labeled with another specific amino acid [4]. With the two-dimensional HN(CO) measurement, which is the most sensitive method among the triple resonance experiments, one can perform amino-acid typing of the preceding residue (residue i-1) as well as the residue including the amide (residue i), since the HN(CO) signal is only observed if both the carbonyl carbon at residue i-1 and the amide nitrogen at residue i are isotopically labeled.…”

“…The methods were designed for various objectives, including the discrimination of 16 amino acids with only 5 samples [7]; the discrimination of amino acids that have close chemical shifts at the alpha and beta carbons [8,9]; the design of a labeling pattern specialized for a specific amino-acid sequence to achieve maximal unique assignments [10][11][12][13][14]; the use of the alpha carbon as well as the carbonyl carbon to discriminate amino acids with various combinations of triple resonance experiments [15,16]; the avoidance of signal overlap considering amino-acid frequency [17][18][19]; the labeling of a specific amino-acid set for the assignment of the transmembrane region of membrane proteins [16,20,21]; and the selective inverse labeling, or selective unlabeling, which is compatible with various protein production methods, including in vivo expression [9,22].…”