Direct observation of the methionine residues of cytochrome c by 13C nuclear magnetic resonance spectroscopy.

Schejter, Abel; Lanir, Amos; Vig, Ida; Cohen, Jack S.

doi:10.1016/s0021-9258(17)34752-x

Cited by 29 publications

(11 citation statements)

References 11 publications

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“…This is a significant observation, since previous studies have indicated a substantial protein unfolding below pH 2 (Wooten et al, 1981;Drew & Dickerson, 1978; Babul & Stellwagen, 1972). Although state II is commonly assumed to have a random-coil structure, the RR spectra clearly show that this description is inaccurate; likewise, there is 13C NMR evidence (Schejter et al, 1978;Cohen & Hayes, 1974) that the tertiary structure of the protein is maintained to a large degree, below pH 2. At pH 13 (state V), however, the amide I band was observed to broaden markedly, and the peak frequency shifted to 1670 cm'1, consistent with substantial unfolding of the helixes.…”

Section: Resultsmentioning

confidence: 77%

Ultraviolet resonance Raman spectra of cytochrome c conformational states

Copeland¹,

Spiro²

1985

Biochemistry

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Ultraviolet resonance Raman (UV RR) spectra are reported for ferricytochrome c from tuna and horse heart at pH 1.6, 7, 10, and 13, representing distinct conformational states of the protein (states II, III, IV, and V, respectively). The spectra were obtained with pulsed laser excitation at 200 and 218 nm, via H2 Raman shifting the fourth harmonic output of a pulsed YAG laser. At these deep UV wavelengths, strong enhancement is observed for vibrational modes associated with tryptophan, tyrosine, and phenylalanine side chains and with the amide groups of the polypeptide backbone. The amide I peak frequency is consistent with a dominant contribution from alpha-helical regions, although a broad high-frequency tail reflects a variety of unordered conformations. The peak frequency is 12 cm-1 higher for cytochrome c from tuna than from horse, suggesting a less tightly wound structure, which is consistent with the lower denaturation temperature previously reported for the tuna protein. The amide I peak broadens when native protein (state III) is converted to the low- or high-pH forms (states II and IV), reflecting some disordering of the polypeptide chain, but the peak frequencies are unshifted, establishing that the alpha-helical segments are not completely unfolded in these states. Raising the pH to 13 (state V), however, does produce a frequency upshift, reflecting helix unfolding. The amide II and III frequencies are likewise consistent with a dominant alpha-helix contribution in the native proteins; they gain intensity, and amide III is shifted to a lower frequency, in states II and IV, consistent with partial disordering.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 77%

Ultraviolet resonance Raman spectra of cytochrome c conformational states

Copeland¹,

Spiro²

1985

Biochemistry

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“…A monotonic increase in the Met-80 line width occurs with heating above 30 °C until the resonance becomes too broad to observe at ~60 °C. A progressive decrease in the 695-nm absorption band with increasing temperature (Schejter & George, 1964;Kaminsky et al, 1973) indicates that the Met-80 heme iron bond is weakened or disrupted upon heating. This result suggests that the extreme 2H line width observed at elevated temperature results from the rapid interconversion between multiple conformational isomers.…”

Section: Resultsmentioning

confidence: 99%

“…The absence of resolved single-carbon resonances in the 13C NMR spectrum at pH 3 indicates that there are relatively few interactions between amino acid side chains. Nonetheless, no 13C resonance for the Met-80 methyl group in 13C-enriched cytochrome c was observed at pH 2 (Schejter et al, 1978) while an upfield-shifted 2H resonance was observed for in 2H-enriched cytochrome c even at pH 1.8. These results indicate that the Met-80 group remains proximal to the heme although it is likely that there is a great deal of motional flexibility present (vide supra).…”

Section: Mobility Of Methionyl Residuesmentioning

confidence: 88%

“…Usually enrichment also has meant chemical modification, as with carboxymethylation (Eakin et al, 1975) or guanidmation (Stellwagen et al, 1977), but under certain conditions the e methyl of methionyl residues in proteins may be isotopically enriched and yield the native protein (Jones et al, 1976). The preparation of cytochrome c with isotopically enriched methionyl residues (Schejter et al, 1978) is especially fortuitous because of the critical role that Met-80 plays as an axial ligand of the heme iron. It was hoped that enhanced levels of 13C would allow the detection of the paramagnetically shifted Met-80 methyl resonance, but no resonance due to the 13C-enriched methyl group was detected in the native conformation.…”

Section: Discussionmentioning

confidence: 99%

“…The cytochrome c employed for the natural abundance 13C spectra was Sigma Type VI obtained from horse heart. The isotopically enriched ferricytochromes were prepared by established methods (Ando et al, 1966;Jones et al, 1975;Schejter & Aviram, 1972) with only minor modifications. Sigma Type VI protein was treated with either 90% 13C-or 2H-enriched methyl iodide at pH 3.0 and 25 °C for 24 h. At pH 3 the protein is partially unfolded thus allowing greater access to Met-80 which is normally buried within the protein interior.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

pH-Induced conformational transitions of ferricytochrome c: a carbon-13 and deuterium nuclear magnetic resonance study

Wooten¹,

Cohen²,

Vig³

et al. 1981

Biochemistry

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Paramagnetic Tools in Protein NMR

Keizers

Ubbink

2011

Protein NMR Spectroscopy: Practical Techniques and Applications

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Unpaired electrons have a strong magnetic moment and consequently affect nearby nuclear spins. The nuclear resonances can be broadened or shifted and these paramagnetic effects are distance and orientation dependent in a well-understood fashion. Stable unpaired electrons are found on metals and protected radicals, but this does not mean that the observation of paramagnetic effects is limited to metal proteins. It is possible to generate such effects also in other proteins by the introduction of paramagnetic centres, for example by attaching a paramagnetic tag or substitution of a diamagnetic metal, like Ca 2 þ , with a paramagnetic one, like a lanthanide. Paramagnetic effects offer distance restraints up to 60 A , a way to cause partial alignment for the generation of RDCs without the need of external media, the possibility to study protein dynamics and to visualise minor populations. Thus, paramagnetic effects are amazingly powerful and complement more classical restraints, like the NOE.This chapter aims to provide the uninitiated reader sufficient knowledge of paramagnetic NMR tools to enable him/her to select the method of choice for the particular problem at hand. After discussing the type of restraints, choice of metals and the available paramagnetic tags, practical hints are given in a protocol as well as several examples that illustrate the current possibilities. It is not meant as a theoretical description of paramagnetism, for which the reader is referred to other sources [1-3].

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Direct observation of the methionine residues of cytochrome c by 13C nuclear magnetic resonance spectroscopy.

Cited by 29 publications

References 11 publications

Ultraviolet resonance Raman spectra of cytochrome c conformational states

Ultraviolet resonance Raman spectra of cytochrome c conformational states

pH-Induced conformational transitions of ferricytochrome c: a carbon-13 and deuterium nuclear magnetic resonance study

Paramagnetic Tools in Protein NMR

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