Stop-flow kinetics studies of the interaction of surfactant, sodium dodecyl sulfate, with acid-denatured cytochrome c

Interactions of sodium dodecyl sulfate (SDS) at submicellar and micellar concentration, with the globular protein, horse heart cytochrome c, at low pH have been shown to stabilize two molten globule-like intermediates. These dynamic studies were performed using far-UV, near-UV, and Soret-band circular dichroism (CD) as well as fluorescence methods. Stopped-flow CD and fluorescence studies of acid-denatured cytochrome c refolding with SDS were performed at both submicellar and micellar concentrations. Distinctive refolding mechanisms (from analysis of both CD and fluorescence) were found under these two conditions, and an obvious refolding intermediate was evident in the fluorescence traces. In addition, stopped-flow CD in the Soret region showed multistep kinetics, suggesting that the spectral changes in this region are not only solvent effect related but also connected with the change of secondary structure. A possible folding mechanism is proposed to rationalize the kinetics results.     

Optical spectroscopic differentiation of various equilibrium denatured states of horse cytochrome c

Thermal unfolding of cytochrome c (cyt c) from several states has been studied using equilibrium spectroscopic techniques. CD in the uv, vibrational circular dichroism, infrared, and uv-vis absorption spectra measured at various temperatures, pHs, salt concentrations, and GuHCl concentrations are used to show the conformational as well as heme structural differences between native and various denatured states. The difference in thermal denaturation behaviors of cyt c starting from acid denatured, molten globule (MG), and the A and native states are explored. Different final high temperature states were observed for cytochrome c unfolding from four different initial states (native, MG, A, and acid denatured state) by electronic CD, Fourier transform infrared (FTIR), and vibrational CD (VCD). Consistent with this, different thermal unfolding pathways for the MG and A states are suggested by the FTIR and VCD data for this process.     

Fast folding of cytochrome c.

Native iso-2 cytochrome c contains two residues (His 18, Met 80) coordinated to the covalently attached heme. On unfolding of iso-2, the His 18 ligand remains coordinated to the heme iron, whereas Met 80 is displaced by a non-native heme ligand, His 33 or His 39. To test whether non-native His-heme ligation slows folding, we have constructed a double mutant protein in which the non-native ligands are replaced by asparagine and lysine, respectively (H33N,H39K iso-2). The double mutant protein, which cannot form non-native histidine-heme coordinate bonds, folds significantly faster than normal iso-2 cytochrome c: gamma = 14-26 ms for H33N,H39K iso-2 versus gamma = 200-1,100 ms for iso-2. These results with iso-2 cytochrome c strongly support the hypothesis that non-native His-heme ligation results in a kinetic barrier to fast folding of cytochrome c. Assuming that the maximum rate of a conformational search is about 10(11) s-1, the results imply that the direct folding pathway of iso-2 involves passage through on the order of 10(9) or fewer partially folded conformers.     

Solvent-induced collapse of alpha-synuclein and acid-denatured cytochrome c

The effects of solution conditions on protein collapse were studied by measuring the hydrodynamic radii of two unfolded proteins, alpha-synuclein and acid-denatured ferricytochrome c, in dilute solution and in 1 M glucose. The radius of alpha-synuclein in dilute solution is less than that predicted for a highly denatured state, and adding 1 M glucose causes further collapse. Circular dichroic data show that alpha-synuclein lacks organized structure in both dilute solution and 1 M glucose. On the other hand, the radius of acid-denatured cytochrome c in dilute solution is consistent with that of a highly denatured state, and 1 M glucose induces collapse to the size and structure of native cytochrome c. Taken together, these data show that alpha-synuclein, a natively unfolded protein, is collapsed even in dilute solution, but lacks structure.     

Thermal stability of hydrophobic heme pocket variants of oxidized cytochrome c

Microcalorimetry has been used to measure the stabilities of mutational variants of yeast iso-1 cytochrome c in which F82 and L85 have been replaced by other hydrophobic amino acids. Specifically, F82 has been replaced by Y and L85 by A. The double mutant F82Y,L85A iso-1 has also been studied, and the mutational perturbations are compared to those for the two single mutants, F82Y iso-1 and L85A iso-1. Results are interpreted in terms of known crystallographic structures. The data show that (1) the destabilization of the mutant proteins is similar in magnitude to that which is theoretically predicted by the more obvious mutation-induced structural effects; (2) the free energy of destabilization of the double mutant, F82Y,L85A iso-1, is less than the sum of those of the two single mutants, almost certainly because, in the double mutant, the -OH group of Y82 is able to protrude into the cavity formed by the L85A substitution. The more favorable structural accommodation of the new -OH group in the double mutant leads to additional stability through (1) further decreases in the volumes of internal cavities and (2) formation of an extra protein-protein hydrogen bond.     

Denaturation of MM-creatine kinase by sodium dodecyl sulfate

The denaturation of dimeric cytoplasmic MM-creatine kinase by sodium dodecyl sulfate (SDS) has been investigated using activity measurements, far-ultraviolet circular dichroism, SEC-HPLC, electric birefringence, intrinsic probes (cysteine and tryptophan residues), and an extrinsic fluorescent probe (ANS). Our results show that inactivation is the first detectable event; the inactivation curve midpoint is located around 0.9 mM SDS. The second event is dissociation and it occurs in parallel to tertiary and secondary perturbations, as demonstrated by the coincidence (near 1.3 mM) of the midpoints of the transition curves monitoring dissociation and structural changes. At high total SDS concentration (concentration higher than 2.5 mM), the monomer had bound 170 mol of SDS per mol of protein. In these conditions, electric birefringence experiments suggest that the SDS-CK complex may be described as a prolate ellipsoid with an axial ratio of 1.27 (14 nm×11 nm). These results are compatible with recent models of SDS-protein complexes: the protein decorated micelle structure or the necklace structure.     

Folding barrier in horse cytochrome c: support for a classical folding pathway

Native-state structures and conformations of ferrocytochrome c, nitrosylcytochrome c, and carbonmonoxycytochrome c are very similar. They are, however, immensely different from each other in terms of thermodynamic stability. The dramatic destabilization of ferrocytochrome c to the extent of 12 kcal mol(-1) produces no effect on the folding rate, and this is so in spite of the fact that all three test-tube variants fold in an apparent two-state manner. For all three proteins the folding barrier is early in time, sizable in energy, and is of the same magnitude (approximately 6.5 kcal mol(-1)). These results raise some challenges to the "new view" of protein folding. An early transition state, the search for which consumes most of the observed folding time, is suggested.     

Equilibrium unfolding of a small low-potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris.

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.     

Protein folding: Hypotheses and experiments

The current state of the problem of protein folding is reviewed with special attention to the novel molten globule state of the protein molecule, intermediate between the native and unfolded states. Experimental evidence on the existence of this state and its role in protein folding are compared with the sequential model of protein folding proposed by the author in 1972–1973.     

Cooperative omega loops in cytochrome c: role in folding and function

Hydrogen exchange experiments under slow exchange conditions show that an omega loop in cytochrome c (residues 40-57) acts as a cooperative unfolding/refolding unit under native conditions. This unit behavior accounts for an initial step on the unfolding pathway, a final step in refolding, and a number of other structural, functional and evolutionary properties.     

IR spectra of cytochrome c denatured with deuterated guanidine hydrochloride show increase in beta sheet

Attenuated total reflectance Fourier transform IR (ATR-FTIR) spectra are obtained for horse heart ferricytochrome c in solutions of 0-7M guanidine hydrochloride and deuterated guanidine hydrochloride. Substitutions of deuterium for hydrogen in both the denaturant and protein provide resolvable amide I spectra over a wide range of denaturant concentrations. Deuteration enhances the ability to measure the true protein IR spectrum in the amide I region in which the secondary structure can be deduced, because spectra in D(2)O are less prone to spectral distortion upon background denaturant subtraction than spectra in H(2)O. Other investigators studying equilibrium unfolded cytochrome c were limited to guanidine concentrations below 3.0M because of detector saturation. Detector saturation is avoided with the use of ATR-FTIR spectroscopy, allowing one to obtain protein spectra at high denaturant concentrations. Second derivative spectra of samples show reductions in alpha helix and increases in beta sheet at high denaturant concentrations, contrary to expectations of finding primarily a random coil secondary structure. Using this new technique, the protein was estimated to consist of 51% beta sheet and only 15% random coil in the presence of 6.6M deuterated guanidine hydrochloride.     

The cytochrome c folding landscape revealed by electron-transfer kinetics

We have investigated the folding energy landscape of cytochrome c by exploiting the widely different electron-transfer (ET) reactivities of buried and exposed Zn(II)-substituted hemes. An electronically excited Zn-porphyrin in guanidine hydrochloride denatured Zn-substituted cytochrome c (Zn-cyt c) reduces ruthenium(III) hexaammine about ten times faster than when embedded in the fully folded protein. Measurements of ET kinetics during Zn-cyt c folding reveal a burst intermediate in which one-third of the ensemble has a protected Zn-porphyrin and slow ET kinetics; the remaining fraction exhibits fast ET characteristic of a solvent-exposed redox cofactor. The ET data show that, under solvent conditions favoring the folded protein, collapsed non-native structures are not substantially more stable than extended conformations, and that the two populations interchange rapidly. Most of the folding free energy, then, is released when compact structures evolve into the native fold.     

Thermodynamics of the reconstitution of tuna cytochrome c from two peptide fragments

Two peptide fragments from tuna cytochrome c (cyt c), N-fragment (residues 1-44 containing the heme) and C-fragment (residues 45-103), combine to form a 1:1 fragment complex. This was clearly proved by ion-spray mass spectrometry. It was found from CD and NMR spectra that the structure of the fragment complex formed is similar to that of an intact cyt c, although each isolated fragment itself is unstructured. Binding constants and enthalpies upon the complex formation were directly observed by isothermal titration calorimetry. Thermodynamic parameters (deltaG(o)b, deltaHb, deltaS(o)b, and deltaC(b)p)) associated with the complex formation were determined at various pHs and temperatures. DeltaHb was found to be almost independent of pH values. The change in heat capacity accompanying the complex formation (deltaC(b)p) was directly determined from the temperature dependence of deltaHb. In addition, the change in heat capacity and enthalpy upon tuna cyt c unfolding were determined by differential scanning calorimetry. Thermodynamic parameters for the unfolding/dissociation process of the fragment complex were compared with those for cyt c unfolding at pH 3.9 and 303 K. In a comparison of two unfolding processes, the heat capacity change of each was very close to the other, while both the unfolding enthalpy and entropy of the fragment complex were larger than those of tuna cyt c. These thermodynamic data suggest that the internal interactions between polar groups (hydrogen bonding) and nonpolar groups (van der Waals interactions) are preserved in the fragment complex as well as in the native state of cyt c.     

Conformational diversity of acid-denatured cytochrome c studied by a matrix analysis of far-UV CD spectra.

The singular value decomposition (SVD) analysis was applied to a large set of far-ultraviolet circular dichroism (far-UV CD) spectra (100-400 spectra) of horse heart cytochrome c (cyt c). The spectra were collected at pH 1.7-5.0 in (NH4)2SO4, sorbitol and 2,2,2-trifluoroethanol (TFE) solutions. The present purpose is to develop a rigorous matrix method applied to far-UV CD spectra to resolve in details conformational properties of proteins in the non-native (or denatured) regions. The analysis established that three basis spectral components are contained in a data set of difference spectra (referred to the spectrum of the native state) used here. By a further matrix transformation, any observed spectrum could be decomposed into fractions of the native (N), the molten-globule (MG), the highly denatured (D), and the alcohol-induced helical (H) spectral forms. This method could determine fractional transition curves of each conformer as a function of solution conditions, which gave the results consistent with denaturation curves of cyt c monitored by other spectroscopic methods. The results in sorbitol solutions, for example, suggested that the preferential hydration effect of the co-solvent stabilizes the MG conformer of cyt c. This report has found that the systematic SVD analysis of the far-UV CD spectra is a powerful tool for the conformational analysis of the non-native species of a protein when it is suitably supplemented with other experimental methods.     

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length

Reversible denaturation of Pseudomonas aeruginosa cytochrome c₅₅₁ (PAc₅₅₁) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc₅₅₁ denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc₅₅₁ denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc₅₅₁, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.     

Misfolded proteins and neurodegeneration: role of non-native cytochrome c in cell death

Intermediates play a relevant role in the protein-folding process, because the onset of diseases of genetic nature is usually coupled with protein misfolding and the formation of stable intermediate species. This article describes and briefly discusses the mechanisms considered responsible, at molecular level, for a number of neurodegenerative diseases. In particular, interest is focused on the newly discovered role of cytochrome c in programmed cell death (apoptosis), consisting of acquisition of powerful cardiolipin-specific peroxidase action. Cardiolipin oxidation induces cytochrome c detachment from the mitochondrial membrane and favors the accumulation of products releasing proapoptotic factors. Cytochrome c showing peroxidase activity has non-native structure, and shows enhanced access of the heme catalytic site to small molecules, such as H₂O₂. The strict correlation linking cytochrome c with the onset of neurodegenerative disorders is described and the therapeutic approach discussed.     

Complete observation of all structural,conformational, and fibrillation transitions of monomeric globular proteins at submicellar sodium dodecyl sulfate concentrations

Although considerable information is available regarding protein–sodium dodecyl sulfate (SDS) interactions, it is still unclear as to how much SDS is needed to denature proteins. The role of protein charge and micellar surfactant concentration on amyloid fibrillation is also unclear. This study reports on equilibrium measurements of SDS interaction with six model proteins and analyzes the results to obtain a general understanding of conformational breakdown, reorganization and restructuring of secondary structure, and entry into the amyloid fibrillar state. Significantly, all of these responses are entirely resolved at much lower than the critical micellar concentration (CMC) of SDS. Electrostatic interaction of the dodecyl sulfate anion (DS⁻) with positive surface potential on the protein can completely unfold both secondary and tertiary structures, which is followed by protein chain restructuration to α-helices. All SDS-denatured proteins contain more α-helices than the corresponding native state. SDS interaction stochastically drives proteins to the aggregated fibrillar state.     

Stability of yeast iso-1-ferricytochrome c as a function of pH and temperature.

Absorbance-detected thermal denaturation studies of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c were performed between pH 3 and 5. Thermal denaturation in this pH range is reversible, shows no concentration dependence, and is consistent with a 2-state model. Values for free energy (delta GD), enthalpy (delta HD), and entropy (delta SD) of denaturation were determined as functions of pH and temperature. The value of delta GD at 300 K, pH 4.6, is 5.1 +/- 0.3 kcal mol-1. The change in molar heat capacity upon denaturation (delta Cp), determined by the temperature dependence of delta HD as a function of pH (1.37 +/- 0.06 kcal mol-1 K-1), agrees with the value determined by differential scanning calorimetry. pH-dependent changes in the Soret region indicate that a group or groups in the heme environment of the denatured protein, probably 1 or both heme propionates, ionize with a pK near 4. The C102T variant exhibits both enthalpy and entropy convergence with a delta HD of 1.30 kcal mol-1 residue-1 at 373.6 K and a delta SD of 4.24 cal mol-1 K-1 residue-1 at 385.2 K. These values agree with those for other single-domain, globular proteins.