Proteins decoupling

Figure 3. Protein decoupled 13C NMR spectrum at 75 MHz of 17,500 MW polystyrene at 20% w/v in CDCU/CHCU (lower trace). One percent cross-linked polystyrene (Biobeads SX-1) in the same solvent (upper trace).

The 140-residue protein AS is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson s disease. Full-length 13C/15N-labelled AS fibrils and AS reverse-labelled for two of the most abundant amino acids, K and V, were examined by homonuclear and heteronuclear 2D and 3D NMR.147 Two different types of fibrils display chemical shift differences of up to 13 ppm in the l5N dimension and up to 5 ppm for the backbone and side-chain 13C chemical shifts. Selection of regions with different mobility indicates the existence of monomers in the sample and allows the identification of mobile segments of the protein within the fibril in the presence of monomeric protein. At least 35 C-terminal residues are mobile and lack a defined secondary structure, whereas the N terminus is rigid starting from residue 22. In addition, temperature-dependent sensitivity enhancement is also noted for the AS fibrils due to both the CP efficiency and motional interference with proton decoupling.148... 

The 15N spectral peaks of fully hydrated [15N]Gly-bR, obtained via cross-polarization, are suppressed at 293 K due to interference with the proton decoupling frequency, and also because of short values of T2 in the loops.208 The motion of the TM a-helices in bR is strongly affected by the freezing of excess water at low temperatures. It is shown that motions in the 10-j-is correlation regime may be functionally important for the photocycle of bR, and protein-lipid interactions are motionally coupled in this dynamic regime. 

Hamelberg, D. McCammon, J. A., Standard free energy of releasing a localized water molecule from the binding pockets of proteins Double-decoupling method, J. Am. Chem. Soc. 2004,126, 7683-7689. 

Phase incremented double adiabatic decoupling for 13C- and 15N-labelled proteins... 

Fig. 14. SeqHNCA-TROSY experiment for establishing sequential 1HN(i), 15N(i), 13Ca(i— 1) correlations in 13C/15N/2H enriched proteins. Durations of transfer delays A = 1/(4/Hn) 2Ta = 20-27 ms, depending on rotational correlation time of protein 2Tc = 5-7 ms S = gradient + field recovery delay 0 < k < Ta/t2,max- Phase cycling i = y (j>2 = y, — y + States-TPPI 0 = x 0ret. = x, — x. Semi-selective decoupling of 13C spins is attained using a SEDUCE-1 decoupling sequence.95...

When the protein rotational correlation time exceeds 15 ns, the upfield component of the JNH doublet broadens. In such cases the amide JNH coupling can be obtained from the displacement of the 15N chemical shift of the TROSY component (see Chapt. 10) relative to the one from a XH-decoupled HSQC experiment. 

Saridakis, E. and Chayen, N. E. (2000). Improving protein crystal quality by decoupling nucleation and growth in vapor diffusion. Protein Sci. 9, 755-757. 

Figure 5. Proton decoupled 19F-NMR spectrum of pABG5 / -glucosidase inactivated with 2F/ ManF (conditions as described in text). Spectra were recorded on a 270 MHz Bruker/Nicolet instrument using gated proton decoupling (decoupler on during acquisition only) and a 90° pulse angle with a repetition delay of 2s. A spectral width of 40,000 Hz was employed and signal accumulated over 10,000 transients for the native protein and 30,000 transients for the denatured protein in 8M urea, (a) Full spectrum with expansion below it (b) Expansion of spectrum of denatured/dialyzed enzyme.

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