D [15n1htrosy

For large proteins, 2D [15N,1H]-TROSY provides fingerprints with improved resolution and sensitivity compared with conventional experiments. Ms is illustrated in Fig. 5.4, panel A, which shows a [15N,1H]-TROSY spectrum of the integral membrane protein OmpX in dihexanoylphosphatidylcholine (DHPC) micelles with an overall molecular mass of 60 kDa. 2D [15N,1H] correlation spectra constitute a many-parameter NMR probe with high sensitivity to changes in the protein structure and environment (Hajduk et al. 1999; Pellecchia et al. 2002; Meyer and Peters 2003); therefore, they are widely used to study intermolecular interactions, either with low molecular mass ligands or with other biomolecules, such as peptides, proteins, and nucleic acids. For example, chemical shift mapping of the amide groups is used in NMR screening to detect binding of compounds that can be optimized to high-affinity ligands by a strategy named "SAR-by-NMR" (Shuker et al. 1996). 2D [15N,1H] correlation spectra are also very useful in providing information about the quality of a protein preparation with regard to purity, folding state, stability, and aggregation properties (Woestenenk et al. 2003). In this way, NMR can be efficiently applied during the process of sample optimization at the onset of NMR studies and even X-ray crystallography studies, provided that 15N-labeled and possibly deuter-ated protein can be produced. Moreover, 2D [15N,1H] correlation spectra are the basis for studies of the dynamic properties of biomolecules based on spin relaxation experiments (Kay et al. 1989), and for the extraction of experimental data that yield structural information, e.g., based on amide proton exchange rates (Wüthrich 1986), coupling constants (Griesinger et al. 1999), or residual dipolar couplings (RDCs) (Prestegard et al. 2000). tte introduction of the TROSY technique extends the applications based on 2D [15N,1H] correlation experiments to much larger structures.

A) 2D [15N,1H]-TROSY


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Fig.5.4. Impact of TROSY on NMR spectra. The spectra were measured at a 1H resonance frequency of 750 MHz with a sample of the uniformly 2H,13C,15N-labeled Integral membrane protein OmpX In dlhexanoylphosphatldylchollne (DHPC) micelles, a 60-kDa complex. A, B 15N-H correlation spectra that were recorded and processed Identically, except that TROSY was used In A only (experimental scheme In Fig. 5.3a). The inserts show cross sections that were taken parallel to the w2(1H)-axls at the position Indicated by the horizontal broken lines. C Strips along the 13C-dlmenslon from a 3D [15N,1H]-TROSY-HNCA spectrum (experimental scheme In Fig. 5.3b). The strips were taken at the 15N chemical shifts (Indicated at the bottom of the strips) of the amino acid residues 12-16, and are centered on the corresponding amide proton chemical shifts, w3(1HN). Horizontal and vertical broken lines demonstrate the connectivities that can be obtained, leading to complete resonance assignments of backbone 1HN, 15N and 13Ca nuclei. D Corresponding strips to those In C extracted from a conventional 3D HNCA spectrum. (Reproduced from Fernandezand Wider 2003 with permission)

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