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

Wooten, Jan B.; Cohen, Jack S.; Vig, Ida; Schejter, Abel

doi:10.1021/bi00522a007

Cited by 29 publications

(24 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The pKa value of cytochrome c in D20 at 27°C was calculated as 9.5 + 0.1 from the forward and reverse rate constants, kf and kr, using the expression: pK~ = pH* + log(kr/kf); the same value was derived from the integrated areas of NMR peaks for the native and basic forms. This calculated pKa value agrees with that measured by others [1][2][3][4][5][6][7][8][13][14][15][16][17]22]. To calculate activation parameters for this process, rate constants were measured at six different temperatures ranging from 27 to 55°C.…”

Section: Resultssupporting

confidence: 87%

“…This work extends previous studies of the NMR spectrum of cytochrome c as a function of pH [13][14][15][16][17]. We report two new findings.…”

Section: Introductionsupporting

confidence: 88%

“…Details of this technique may be found elsewhere [20]. It has been noted previously that the alkaline isomerization is not completely reversible [16]. However, we observed no changes in the kinetics of a given sample over the course of many days.…”

Section: Methodsmentioning

confidence: 54%

See 2 more Smart Citations

NMR study of the alkaline isomerization of ferricytochrome c

Hong

Dixon

1989

FEBS Letters

View full text Add to dashboard Cite

The pH-induced isomerization of horse heart cytochrome c has been studied by tH NMR. We find that the transition occurring in D20 with a pKa measured as 9.5+0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0+0.6 s -~ at 27°C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are zlH~ = 12.8 +0.8 keal.moP, z/S~ = -12.9 __+ 2.0 e.u. for the forward reaction and ,Jilt = 6.0_+0.3 kcal.mo1-1, AS~= -35.1 + 1.3 e.u. for the reverse reaction (pH* =9.28).ztH ° and z/S ° for the isomerization are 6.7 + 0.6 kcal.mol -~ and 21.9 + 1.0 e.u., respectively.

show abstract

Section: Resultssupporting

confidence: 87%

“…This work extends previous studies of the NMR spectrum of cytochrome c as a function of pH [13][14][15][16][17]. We report two new findings.…”

Section: Introductionsupporting

confidence: 88%

See 1 more Smart Citation

NMR study of the alkaline isomerization of ferricytochrome c

Hong

Dixon

1989

FEBS Letters

View full text Add to dashboard Cite

show abstract

“…pH-induced denaturation has been observed in many other systems and by many techniques, including 'H, 2H, and`3C NMR (30)(31)(32). In a general sense, our results are similar to those of Epstein et al (30), who monitored H+-induced denaturation of staphylococcal nuclease by using 'H NMR.…”

Section: Methodssupporting

confidence: 88%

pH-induced changes in Rhodospirillum rubrum cytochrome c2 and subsequent renaturation: an 15N NMR study.

Smith

1988

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The 15N-enriched ferrocytochrome c2 from Rhodospirillum rubrum was studied by '5N NMR at different solvent pH values. The m bility and chemical shift of the N-terminal glutamic acid (35.i.4 ppm at pH 5.1) were found to depend on pH. It was least mobile between pH 8 and 9.0, which is explained in terms of pH-dependent conformational changes and formation of salt linkages and/or hydrogen bonds. The resonances of the lysine side chains are centered around 341.7 ppm at low pH and move upfield with pH by about 8.4 ppm with pK. values of 10.8. The exchange rates of the eNH protons are lowest near their pK. values. The protein is very stable in the pH range between 4.9 and 10.0 but unfolds abruptly at pH 10.5-11. Denaturation was verified by the measurement of several parameters by NMR. The renaturation of the protein demonstrates that the folding begins with reformation of heme coordination and establishment of a hydrophobic core, followed by positioing of side chains and peptide backbones linking the nucleation centers. The repositioning processes had time scales of minutes to hours in contrast to the reported values of seconds in some studies.The cytochromes c2 are a class of electron-transfer proteins found in the non-sulfur purple photosynthetic bacteria. One characteristic of the cytochromes is their redox potential, which, for cytochrome c2, has been found to be very pH dependent (1,2). Ionizable groups in the protein include the ligand histidine and a non-ligand histidine, a single invariant tryptophan, various acidic residues, the N and C termini, and the two propionic acid substituents of the heme. Among the charged groups of the protein, the lysine side-chain groups on the surface of the protein which appear to be very mobile (3) are proposed to play an essential role in electron transfer (4). Local motion of other groups and segmental motion of the N-terminal a-helix are thought to be important characteristics of the protein's structure (5).Recent studies on cytochrome structure include one-and two-dimensional proton NMR studies of the mitochondrial cytochrome (6-9). These studies, which are approaching complete assignment of the 1H NMR spectrum of the protein, follow an abundant literature.Although the structures of cytochromes are well studied by x-ray crystallography (10-12) and are known to be conserved (13,14), their dynamic structures in solution and pH dependence are not well understood. In previous work (15), we have assigned the important 15N resonances and observed conformational changes of the reduced state of cytochrome c2 induced by changes in pH using 15N NMR. In the present paper, we report the observation of pH-induced denaturation and refolding of cytochrome c2. MATERIALS AND METHODSGrowth of Rhodospirlum rubrum. '5NH4Cl was obtained from MSD isotopes. The G-9 (blue-green) mutant of R. rubrum was kindly provided by P. F. Weaver (SERI). Growth media and conditions were as described (15). The 15N-enriched cytochrome c2 was purified from 15NH4CI-grown R. rubrum by standard methods (16,...

show abstract

“…As a result of these many roles as well as its favorable relaxation characteristics, [methyl- 13 C]methionine has proven popular as an NMR label (Jones et al, 1975, 1976; Blakley et al 1978; Deber et al, 1978; Jaeck & Benz, 1979; Stollery et al, 1980; Wooten et al 1981; Hardy & Dill, 1982; Rosevear 1988; Seigneuret et al 1991; Lin et al, 1994; Krudy et al 1994; Duewel et al 1995; Howarth et al 1995; Siivari et al 1995; Beatty et al 1996; Kleerekoper et al 1998; Cox et al 1999; He et al 1999; Kleerekoper and Putkey 1999; Yuan et al 1999; Skrynnikov et al, 2001; Duewel et al 2001; Bose-Basu et al 2004; Yuan et al 2004; Kirby et al 2005; DellaVecchia et al 2007; Gelis et al, 2007; Kloiber et al, 2007; Zheng et al 2009; Zheng et al 2010; Religa et al, 2010). Nevertheless, there have been few theoretical analyses of the conformational dependence of its NMR parameters.…”

Section: Introductionmentioning

confidence: 99%

Conformational dependence of 13C shielding and coupling constants for methionine methyl groups

et al. 2010

View full text Add to dashboard Cite

Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of 3JCSCC, 3JCSCH, and the isotropic shielding, σiso. Results have been compared with experimental data reported in the literature, as well as data obtained on [methyl-13C]methionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, 3JCSCC/3JCOCC ~ .7. It is further demonstrated that the 3JCSCH coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of δ13Cε. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large ~ 100° χ3 values. The utility of the δ13Cε as a basis for estimating the gauche/trans ratio for χ3 is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are discussed.

show abstract

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

Cited by 29 publications

References 46 publications

NMR study of the alkaline isomerization of ferricytochrome c

NMR study of the alkaline isomerization of ferricytochrome c

pH-induced changes in Rhodospirillum rubrum cytochrome c2 and subsequent renaturation: an 15N NMR study.

Conformational dependence of 13C shielding and coupling constants for methionine methyl groups

Contact Info

Product

Resources

About