Ring-current shifted resonances

PMR studies have been performed on a number of other ribosomal proteins isolated by the acetic acid/urea method (Morrison etal., 1977a). The results of these studies have shown that acedc acid/urea-extracted proteins contain little tertiary structure. However, some structure was seen in protein S4 and especially in protein S16 as indicated by the appearance of ring-current shifted resonances in the apolar region of the spectrum (Morrison et al., 1977b). These are due to the interaction of apolar methyl groups with aromatic amino acids in the tertiary structure of the protein. The PMR spectra were recorded either in water or in dilute phosphate buffer at pH 7.0—conditions under which the proteins were soluble. 

The NMR spectra of paramagnetic hemoproteins contain resolved hyperfine-shifted lines in addition to the ring-current shifted resonances (Kowalsky (62) Kurland et al. (64) Wiithrich et al. (112)). Between DSS and ca. +5 ppm the spectra can then contain resolved ring current-shifted... 

In the spectrum of ferricytochrome c (Fig. 19) the resonances between —0.5 and —9 ppm correspond to ca. 650 protons of the polypeptide chain. Essentially all the proton resonances of heme c and the axial ligands were found to be shifted by hyperfine interactions with the unpaired electron (Kowalsky (62) Wuthrich (110)), and some ring current-shifted resonances were also outside of the spectral region from DSS to —10 ppm. From a temperature study similar to that shown for MbCN (Figs. 15 and 16) the temperature dependent hyperfine-shifted resonances in Fig. 20 were identified (110). A tentative assignement of some of these lines... 

Fig. 4. Experimentally measured ring current shifts for lysozyme resonances plotted against ring current shifts calculated from the X-ray structure. The poor agreement for these data, compared to those in Figs. 2 and 3, reflects the theoretical uncertainty in the calculated values (see Ref. 5).

From temperature studies similar to that described for cyanoferri-myoglobin (Figs. 15 and 16) the ring current-shifted high field resonances of ferricytochrome c and cyanoferricytochrome were identified (110,111). The methionyl resonances between 1.9 and 3.7 ppm were not observed for either of the ferric compounds. This was to be expected, since in ferricytochrome c these lines are further shifted by hyperfine interactions, and in cyanoferricytochrome c the methionine is no longer one of the axial ligands. 

Resonances in the region — 5 to — 8 ppm from HDO arise from the ring-current-shifted protons due to those protons located above or below the aromatic amino acid residues and the porphyrins of oxy-Hb A (Fig. 8J). (9) Resonances in the region -6 to -20 ppm... 

K. Deoxy-Hb A Ring-Current Shifted and Hyperfine-Shifted Proton Resonances... 

The potential of the method of using mutant and chemically modified Hbs to assign proton resonances of Hb is greatly enhanced when correlated with both intensity measurements and calculations from the crystal structure of Hb as determined by X-ray diffraction. A representative example of this approach is El 1 Val in HbCO A. Based on intensity measurements on a mixture of HbCO A and ferrocy-tochrome c, Lindstrom etal. (1972b) have shown for the ring-current-shifted proton resonances at —5.86, -6.48, and -6.58 ppm from HDO that each arises from one CH3 per a(3 dimer. In the spectrum of a mixture of HbCO A and HbCO Sydney [(367(E1 l)Val — Ala],... 

Fig. 13. 250-MHz ring-current-shifted proton resonances of HbCO A and HbCO Sydney (p97Val — Ala). [Adapted from Lindstrom et at. (1972b)].

Fig. 30. Effects of alkyl isocyanides (n-series) on the 600-MHz ring-current-shifted proton resonances of isolated a and p chains of Hb A, Hb A, and sperm whale myoglobin (Mb) in 0.2 M phosphate in D20 at pH 6.6 and 21°C CO, carbon monoxide MNC, methyl isocyanide ENC, ethyl isocyanide nPNC, n-propyl isocyanide nBNC, n-butyl isocyanide. [Adapted from Mims el al. (1983a)].

Ring-current-shifted H NMR spectra for these heme proteins containing a series of C-l-substituted (or a-series) ligands, namely, methyl, ethyl, isopropyl, and tert-butyl isocyanide, are given in Fig. 32. The a-chain spectra show a marked downfield shift of the valine peak as methyl groups are added to ethyl isocyanide (Fig. 3IB). For the tert-butyl isocyanide complex, the valine resonance is indistinguishable... 

The NMR spectrum of melittin in the presence of phospholipids was essentially similar to that obtained in phosphate except for the absence of the ring-current shifts of CH3 resonances (Fig. 8). 

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