Aromatic Side Chain Assignment Management

10.1.2

HETERONUCLEAR RESONANCE ASSIGNMENTS FOR ISOTOPICALLY LABELED PROTEINS

The conventional sequential or main chain directed assignment strategies also are used to obtain sequence-specific assignments from 3D heteronuclear NMR spectra. The principal advantage of using 3D heteronuclear-edited NOESY and TOCSY (Section 7.2), rather than homonuclear 2D experiments, for resonance assignments is the significant reduction in cross-peak overlap. The 3D 1H-15N NOESY-HSQC experiment is used to identify sequential through-space dαN, dβN, and dNN connectivities. Amino acid spin systems are identified by observation of direct and relayed through-bond connectivities between the 1HN spins and the 1Hα and aliphatic side chain protons using the 3D 1H-15N TOCSY-HSQC experiment (Section 7.2.2). Alternatively, complete side chain assignments also can be obtained from HCCH-COSY and HCCH-TOCSY experiments (Section 7.3). In this case, the side chain spin systems are connected with the backbone 1HN and 15N resonances via 1HN-15N-1Hα and other correlations observed in an 1H-15N TOCSY-HSQC spectrum, or by using correlations observed in one or two of several triple-resonance experiments (Section 7.4) [e.g., HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HBHA(CBCA)NH, or HBHA(CBCACO)NH]. The heteronuclear-edited NOE-based sequential assignment method has been successfully applied to a number of proteins, with molecular masses up to ∼20 kDa (40–46).

The triple-resonance experiments introduced in Section 7.4 offer an alternative to the NOE-based strategy for sequentially assigning 1HN, 15N, 13CO, 1Hα, 13Cα, 1Hβ, and 13Cβ resonances. Using these experiments, sequential correlations are established via the relatively uniform and well-resolved heteronuclear one-bond and two-bond couplings, without any prior knowledge of spin system types. Side chain assignments are completed using the TOCSY-HSQC, HCCH-COSY, and HCCH-TOCSY experiments. Potential errors that arise from misassignment of sequential and long-range connectivities in the NOE-based procedures are avoided because assignments are based solely on predictable through-bond scalar correlations. Triple-resonance approaches were developed by Bax and co-workers to overcome difficulties in obtaining resonance assignments for calmodulin by homonuclear techniques (47). Calmodulin (16.7 kDa) is largely α-helical and has very narrow chemical shift distributions for both 1HN and 1Hα spins.

For example, the combined use of the HNCA (Section 7.4.1) experiment, which provides intraresidue (together with some sequential) correlations between 1HN, 15N, and 13Cα resonances, and the HN(CO)CA experiment (Section 7.4.2), which gives solely interresidue correlations between the 1HN and 15N resonances of one residue and the 13Cα resonance of the preceding residue, provides an obvious route to sequential assignment. Each 13Cα resonance is linked to both its intraresidue and sequential 1HN and 15N resonances. Ambiguities caused by chemical shift degeneracy are solved by using additional experiments that provide alternative correlations. For instance, the HNCO (Section 7.4.4.1) and HN(CA)CO (Section 7.4.4.2) experiments correlate the 1HN and 15N resonances with both the intraresidue and the sequential 13CO signals (rather than the 13Cα spins). To “align” the backbone sequential assignments with the protein amino acid sequence, side chain amino acid spin systems are identified from HCCH-COSY, HCCH-TOCSY, and 1H-15N TOCSY-HSQC experiments.

A different set of experiments, CBCA(CO)NH [or HN(CO)CACB] and HNCACB (or CBCANH), together with the closely related HBHA (CBCACO)NH and HBHA(CBCA)NH experiments (Section 7.4.5), offers an alternative sequential assignment strategy for proteins with 13Cα and 1Hα chemical shift degeneracy. Using these experiments, the 1HN and 15N resonances are correlated with the intraresidue and sequential 13Cα and 13Cβ (or 1Hα and 1Hβ) resonances. Information regarding amino acid type can be obtained from the 13Cα and 13Cβ chemical shifts (see later). The HCC(CO)NH-TOCSY (48–52) and CC(CO)NH-TOCSY (53) experiments, discussed in Sections 9.1.6 and 9.1.7, also provide sequential and spin system assignment information. These experiments are similar in principle to the CBCA(CO)NH and HNCACB experiments, except that 13C isotropic mixing is used instead of COSY-type transfer to relay aliphatic side chain magnetization to the 13Cα nucleus. Sequential and intraresidue correlations are obtained between backbone 1HN and 15N resonances and the side chain (either 13C or 1H) resonances. Finally, banks of experiments have been designed to achieve resonance assignments for particular side chain topologies, such as aromatic amino acids (54, 55), side chain 1H–15N groups (56), arginine amino acids (57), and proline amino acids (58, 59).

Increasingly, resonance assignments of proteins with molecular masses of > 20 kDa are obtained using 2H/13C/15N triply labeled proteins to reduce losses due to efficient 1H-13C dipole-dipole relaxation, as discussed in Section 9.1. Triple-resonance experiments for backbone resonance assignments discussed in Section 9.1.5 are based on out-and-back transfers from the 1HN spin. Assignments are obtained primarily from three pairs of three-dimensional experiments: HNCA and HN(CO)CA, HNCACB and HN(CO)CACB, and HNCO and HN(CA)CO experiments. These experiments also have four-dimensional variants, such as HNCOCA (60). Assignments of aliphatic and aromatic side chains using combined analysis of 13C/15N doubly labeled and 2H/13C/15N triply labeled proteins, random fractionally deuterated 2H/13C/15N proteins, or selectively protonated proteins have been described in Sections 9.1.6, 9.1.7, and 9.1.9. Nearly complete backbone and methyl resonance assignments of malate synthase G, with 723 amino acid residues and a molecular mass of 81.4 kDa, have been achieved by Kay and co-workers (61, 62).

Information on amino acid type is obtained from 13C chemical shift data. Random coil 13C chemical shifts have been determined for unstructured peptides (1, 2, 63). The dependence of 13C chemical shifts on protein secondary structure is discussed elsewhere (11, 64–66). The distributions of aliphatic and aromatic 13C chemical shifts for different amino acid residues compiled in a database at BioMagResBank (12) are plotted in Fig. 10.7. The characteristic 13Cα and 13Cβ chemical shifts of alanine, threonine, serine, and glycine residues (Fig. 10.7) allow ready identification of these amino acid types. Clearly, knowledge of other aliphatic 13C chemical shifts can also be used to assign a given spin system to a unique or limited number of possible amino acid types. This information, coupled with the alignment of sequentially connected spin systems with the known amino acid sequence, leads to unambiguous assignment.

The relative simplicity and predictability of triple-resonance 3D and 4D spectra used for protein assignment purposes makes these experiments particularly amenable to automated or semiautomated analysis. Current efforts at automated assignment generally begin with automatic peak picking. The reduced resonance overlap in 3D and 4D triple-resonance spectra, relative to 2D homonuclear spectra, increases the reliability of this process; however, the final peak lists usually must be edited (by the spectroscopist) to distinguish “real” resonance peaks from spectral artifacts. The peak lists are searched automatically to find expected intraresidue and interresidue correlations, and the spin systems are identified and sequentially ordered according to these correlations. Information regarding spin system type, which may be obtained from 13Cα and 13Cβ chemical shifts values, for instance, is incorporated and the ordered spin systems are aligned with the known amino acid sequence. Current state-of-the-art automatic assignment methods have been reviewed (33–36, 67).

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