Chapter III: Materials and Methods
12 STRUCTURE DETERMINATION BY SOLUTION NMR
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122..11 NNMMRRddaattaaaaccqquuiissiittiioonn
NMR spectra were acquired at 292 K on Bruker DRX500, DRX600, or DRX900 spectrometers with cryogenic triple resonance probes. Spectra were processed with NMRPipe (Delaglio et al., 1995) and analyzed using NMRVIEW (Johnson and Blevins, 1994).
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122..22 BBaacckkbboonneeaassssiiggnnmmeennttooffcchheemmiiccaallsshhiiffttss
A combined set of heteronuclear multidimensional NMR experiments were recorded for the assignment of the 1H, 13C, and 15N chemical shifts of uniformly labelled protein. The resonance assignment strategy involved the concerted use of four 3D triple-resonance experiments [HN(CO)CA, HNCA, CBCA(CO)NH and 13C-resolved three-dimensional NOESY]. The central feature of this policy was the concurrent assignment of both backbone and side-chain aliphatic atoms, which was critical for overcoming ambiguities in the assignment process. The combination of the 3D experiments HNCA and HN(CO)CA was used to establish backbone sequential connectivities by connecting the resonance frequencies of spins with those of preceding residues. The CBCA(CO)NH experiment was used to extend the connectivities from the backbone to Cβ as the chemical shifts of the side-chain carbons are characteristic for the amino acid type. This information can be utilized to position sequentially connected fragments within the amino acid sequence (Sattler et al., 1999).
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122..33 SSttrruuccttuurreeccaallccuullaattiioonnaannddddeetteerrmmiinnaattiioonn
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and T2) were performed. 15N relaxation (T1, T2) and heteronuclear 1H-15N NOE was measured on a 15N-labelled protein sample at 292 K (Farrow et al., 1994). The experimentally determined distance and dihedral restraints (Table 4, Figure 26 C) were applied in a simulated-annealing protocol using ARIA (Linge et al., 2001) and CNS (Bruenger et al., 1998). NOEs were manually assigned and distance calibrations were performed by ARIA. The final ensemble of NMR structures was refined in a shell of water molecules (Linge et al., 2003). Structural quality was analyzed with PROCHECK (Laskowski et al., 1996).
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122..44 NNMMRRttiittrraattiioonneexxppeerriimmeenntt –11HH--1155NNHHSSQQCC
The 1H-15N HSQC (heteronuclear single quantum coherence) NMR experiment is a two dimensional experiment in which each amino acid residue (except for proline) in a given protein is described by one peak in the spectrum. The chemical shift of a peak at (ω1, ω2), where ω1 and ω2 are the amide 15N and 1H shifts, respectively, depends from the chemical surrounding of the amino acid amide group in the protein. In addition, the side chain amides of glutamine and asparagine are represented in the spectrum. A folded protein structure will generally produce a 1H- 15N HSQC spectrum with a broad distribution of well separated signals. Changes in the environment of a spin due to binding of a ligand give rise to chemical shift changes in the NMR spectrum. These changes are expected to be largest near the binding site and the interface of a protein with a ligand can be easily mapped.
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122..55 IIssoottooppeeffiilltteerriinnggeexxppeerriimmeennttss
To understand the function of biological macromolecules, it is important to illuminate the molecular crosstalk between these molecules. For studying the structures of molecular complexes by NMR spectroscopy, it is essential to distinguish between intra- and intermolecular NOEs. This task can be achieved by heteronuclear filtered NOE experiments performed on a sample of a complex consisting of differentially labeled molecules. In the binary complex consisting of 13C, 15N-labeled SRI domain (A) and unlabeled peptide (B), four kinds of NOE cross peaks can be observed (Table 12).
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Table 12: NOE cross peaks observed in complexes
intramolecular: intermolecular:
A – A: 1H-(13C,15N) – 1H-(13C,15N) A – B: 1H-(13C,15N) – 1H-(12C,14N) B – B: 1H-(12C,14N) – 1H-(12C,14N) B – A: 1H-(12C,14N) – 1H-(13C,15N)
The connectivity of protons to the heteronuclei 13C and 15N can therefore be used to separate intra- and intermolecular NOEs and the intramolecular NOEs of A and B (Sattler et al., 1999). For that purpose, a 2D ω2-13C,15N-filtered NOESY was acquired to examine especially the intermolecular NOEs between peptide and SRI domain.
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122..66 TTOOCCSSYYeexxppeerriimmeennttss
In general, the HCCH-TOCSY (Total correlation spectroscopy) experiment correlates all aliphatic 1H and 13C spins within residues, and is used to assign aliphatic 1H and 13C resonances and connect the side-chain chemicals shifts with the backbone assignment (Teng, 2005). Here, the scalar couplings observed in different TOCSY experiments (mixing time 30 or 60 ms) were used to correlate the spins within the spin system of the 'free' and unlabeled peptide. The measurements were either performed in the same buffer conditions as for the NMR-titration experiment (20 mM sodium phosphate pH 6.5, 200 mM NaCl, 2 mM DTT/2 mM peptide concentration) or in 100 % D2O (0.4 mM peptide concentration).
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122..77 RROOEESSYYeexxppeerriimmeennttss
ROSY (Rotating-frame Overhauser effect spectroscopy) is an experiment in which homonuclear NOE effects are measured under spin-locked conditions. Both ROESY
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TOCSY although the cross-peaks of a ROESY spectrum have an opposite phase to those in the TOCSY spectrum (Teng, 2005).