The low ionic strength crystal structure of horse cytochrome c at 2.1 å resolution and comparison with its high ionic strength counterpart

Sanishvili, Ruslan; Volz, Karl; Westbrook, Edwin M.; Margoliash, E.

doi:10.1016/s0969-2126(01)00205-2

Cited by 76 publications

(74 citation statements)

References 38 publications

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“…This behavior may affect the affinity between two proteins. Indeed, it has been observed that, by increasing the ionic strength of the solution, the affinity between cyt c and cyt b 5 decreases [61]. This may be related to changes in the overall electrostatic interaction energy, but the present study shows that there are indeed specific changes in the conformation of key residues involved in the interaction.…”

Section: Discussioncontrasting

confidence: 49%

Monitoring the conformational flexibility of cytochrome c at low ionic strength by ¹H‐NMR spectroscopy

Banci¹,

Bertini²,

Reddig³

et al. 1998

European Journal of Biochemistry

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Horse heart cytochrome c at pH 7 and low ionic strength is present as two conformers, as evidenced by 1 H-NMR spectroscopy. The two structures have been calculated using NOE and pseudocontact shift constraints. They have the same folding patterns and are essentially equal, within the rmsd of the families. The two average structures have rmsd values of 0.049 nm and 0.093 nm for the backbone and the heavy atoms, respectively. Such a difference has been analyzed through a detailed analysis of the NOEs. It appears that the species at low ionic strength differs from the species present at high ionic strength by the displacement of some external residues, such as Gln16, Ile81 and Glu90. Other changes are monitored by the chemical shifts but they cannot be quantified at the present level of resolution. Ionic-strengthdependent structural rearrangements may be relevant with respect to the problem of molecular recognition.Keywords : cytochrome c ; NMR; low ionic strength ; phosphate.Cytochrome c is an electron-transfer protein which contains a heme moiety, with the iron ion cycling between the oxidation states 2ϩ and 3ϩ during biological function [1,2]. The iron is low spin, six coordinated in both oxidation states, being axially coordinated by a histidine residue and a methionine residue. The protein appears to be quite flexible. When the oxidized form is dissolved in water, it gives rise to an alkaline form [3Ϫ17] in which the axial ligand methionine residue is detached from the coordination to iron(III). The amount of the alkaline form is pH dependent with a pK a of 9.5 [16]. At temperatures higher than 320 K and neutral pH, other species are detected, again with the axial methionine residue detached from coordination [18,19], which experience a somewhat lower pK a . Furthermore, ligands such as NH 3 [20] and imidazole [21,22] have been shown to substitute the methionine ligand. Guanidinium chloride unfolds oxidized cytochrome c at 3.5 M [23Ϫ25]. The reduced protein appears to be more stable as, apparently, there is no alkaline form and it is more resistant to heat and guanidinium chloride treatments [24].Recently, the NMR solution structure has been solved for horse heart cytochrome c [26Ϫ28], yeast iso-1-cytochrome c [29,30] and cytochrome c 6 from Monoraphidium braunii [31,32] in both oxidation states. A common feature is that backbone NH exchangeability is larger for the oxidized than for the reduced form. This indicates a larger solvent accessibility for the peptidic NHs. All the measurements were performed in 100 mM sodium phosphate.In the course of the various studies, we noted that, at low buffer concentration, there is splitting of some signals in horse heart cytochrome c (cyt c). In the presence of 5 mM sodium phosphate, pH 7, or of 10 mM Cl Ϫ or 20 mM Hepes, two species are present. It has already been reported in the literature [1, 33Ϫ 35] that several anions, including phosphate, bind cyt c in one or two binding sites. The effects of different buffer concentrations on the NMR spectra is relevant with resp...

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Section: Discussioncontrasting

confidence: 49%

Monitoring the conformational flexibility of cytochrome c at low ionic strength by ¹H‐NMR spectroscopy

Banci¹,

Bertini²,

Reddig³

et al. 1998

European Journal of Biochemistry

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“…These exhibit the same dominant features as the earlier measurements at low pH in strong saline solution (18). We have performed GNM calculations by using two different crystal structures (34,37) with PDB codes 1hrc and 1crc (at low ionic strength), obtained at 1.9 and 2.1 Å resolution, respectively. The heme group was not included in the calculations.…”

Section: Results and Comparison With Hx Experimentsmentioning

confidence: 56%

Correlation between Native-State Hydrogen Exchange and Cooperative Residue Fluctuations from a Simple Model

et al. 1998

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Recently, we developed a simple analytical model based on local residue packing densities and the distribution of tertiary contacts for describing the conformational fluctuations of proteins in their folded state. This so-called Gaussian network model (GNM) is applied here to the interpretation of experimental hydrogen exchange (HX) behavior of proteins in their native state or under weakly denaturing conditions. Calculations are performed for five proteins: bovine pancreatic trypsin inhibitor, cytochrome c, plastocyanin, staphylococcal nuclease, and ribonuclease H. The results are significant in two respects. First, a good agreement is reached between calculated fluctuations and experimental measurements of HX despite the simplicity of the model and within computational times 2 or 3 orders of magnitude faster than earlier, more complex simulations. Second, the success of a theory, based on the coupled conformational fluctuations of residues near the native state, to satisfactorily describe the native-state HX behavior indicates the significant contribution of local, but cooperative, fluctuations to protein conformational dynamics. The correlation between the HX data and the unfolding kinetics of individual residues further suggests that local conformational susceptibilities as revealed by the GNM approach may have implications relevant to the global dynamics of proteins.Hydrogen exchange (HX) data for proteins directly indicate the relative accessibility of various protons to solvent, typically under conditions that can be denaturing to different extents. Observed HX times can vary over extremely broad ranges, from instantaneous to months. Generally these various times have been interpreted and ascribed to several kinds of processesslocal fluctuations for the fastest, local unfolding for intermediate, and global unfolding for the slowest exchanges (1). Some disagreements have arisen between the descriptions of the underlying kinetic processes and the interpretation of HX mechanisms because of differences in the operational definitions of these processes (2). HX from native proteins was recently pointed out to monitor with a remarkable precision rapid conformational fluctuations of the order of microseconds to milliseconds, while no correlation could be observed between HX free energies of individual residues and their unfolding rates which are several orders of magnitude slower than local fluctuations (3). Global unfolding rates are expected to be the same for all residues, while the superimposed exchange rates due to residue fluctuations vary depending on local interactions (4, 1, 5). Here we will avoid ascribing rates to specific processes but instead investigate directly the extent of protection in the native state and compare this with the measured exchange rates. This analysis has become possible with the development of a new simple model of protein structure and dynamics, recently proposed for analyzing the conformational fluctuations of folded proteins (6, 7).Two limits, or two mechanisms of ex...

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“…Lys-27, which takes part in forming an unusual ␥-turn (22), all other residues are within the permitted region. The skeleton of T-Cc is similar to that of horse heart Cc (hh-Cc; Protein Data Bank ID 1HRC) with the greatest rms deviations (rmsd) at the residue regions 1-5, 21-23, 37-46, 55-58, 85-87, and 96-104.…”

Section: Resultsmentioning

confidence: 99%

Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis

Liu

Lin

et al. 2006

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

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Hydrogen peroxide (H2O2) is the major reactive oxygen species (ROS) produced in sperm. High concentrations of H 2O2 in sperm induce nuclear DNA fragmentation and lipid peroxidation and result in cell death. The respiratory chain of the mitochondrion is one of the most productive ROS generating systems in sperm, and thus the destruction of ROS in mitochondria is critical for the cell. It was recently reported that H 2O2 generated by the respiratory chain of the mitochondrion can be efficiently destroyed by the cytochrome c-mediated electron-leak pathway where the electron of ferrocytochrome c migrates directly to H 2O2 instead of to cytochrome c oxidase. In our studies, we found that mouse testisspecific cytochrome c (T-Cc) can catalyze the reduction of H 2O2 three times faster than its counterpart in somatic cells (S-Cc) and that the T-Cc heme has the greater resistance to being degraded by H 2O2. Together, these findings strongly imply that T-Cc can protect sperm from the damages caused by H 2O2. Moreover, the apoptotic activity of T-Cc is three to five times greater than that of S-Cc in a well established apoptosis measurement system using Xenopus egg extract. The dramatically stronger apoptotic activity of T-Cc might be important for the suicide of male germ cells, considered a physiological mechanism that regulates the number of sperm produced and eliminates those with damaged DNA. Thus, it is very likely that T-Cc has evolved to guarantee the biological integrity of sperm produced in mammalian testis. mouse testis ͉ antioxidation

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The low ionic strength crystal structure of horse cytochrome c at 2.1 å resolution and comparison with its high ionic strength counterpart

Cited by 76 publications

References 38 publications

Monitoring the conformational flexibility of cytochrome c at low ionic strength by ¹H‐NMR spectroscopy

Monitoring the conformational flexibility of cytochrome c at low ionic strength by ¹H‐NMR spectroscopy

Correlation between Native-State Hydrogen Exchange and Cooperative Residue Fluctuations from a Simple Model

Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis

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The low ionic strength crystal structure of horse cytochrome c at 2.1 å resolution and comparison with its high ionic strength counterpart

Cited by 76 publications

References 38 publications

Monitoring the conformational flexibility of cytochrome c at low ionic strength by 1H‐NMR spectroscopy

Monitoring the conformational flexibility of cytochrome c at low ionic strength by 1H‐NMR spectroscopy

Correlation between Native-State Hydrogen Exchange and Cooperative Residue Fluctuations from a Simple Model

Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis

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Monitoring the conformational flexibility of cytochrome c at low ionic strength by ¹H‐NMR spectroscopy

Monitoring the conformational flexibility of cytochrome c at low ionic strength by ¹H‐NMR spectroscopy