Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c

Effect of a hydrophobic peptide on folding of oxidized cytochrome c (cyt c) is studied with trityrosine. Folding of cyt c was initiated by pH jump from 2.3 (acid-unfolded) to 4.2 (folded). The Soret band of the 2-ms transient absorption spectrum during folding decreased its intensity and red-shifted from 397 to 400 nm by interaction with trityrosine, whereas tyrosinol caused no significant effect. The change in the transient absorption spectrum by interaction with trityrosine was similar to that obtained with 100 mM imidazole, which showed that the population of the intermediate His/His coordinated species increased during folding of cyt c by interaction with trityrosine. The absorption change was biphasic, the fast phase (82+/-9s(-1)) corresponding to the transition from the His/H(2)O to the His/Met coordinated species, whereas the slow phase (24+/-3s(-1)) from His/His to His/Met. By addition of trityrosine, the relative ratio of the slow phase increased, due to increase of the His/His species at the initial stage of folding. According to the resonance Raman spectra of cyt c, the high-spin 6-coordinate and low-spin 6-coordinate species were dominated at pH 2.3 and 4.2, respectively, and these species were not affected by addition of trityrosine. These results demonstrated that the His/His species increased by interaction with trityrosine at the initial stage of cyt c folding, whereas the heme coordination structure was not affected by trityrosine when the protein was completely unfolded or folded. Hydrophobic peptides thus may be useful to study the effects of hydrophobic interactions on protein folding.     

Association of methanol and ethanol with heme proteins.

The behavior of ferrihemoglobin and ferrimyoglobin in widely varying concentrations of the lowest four alcohols has been studied by optical and electron paramagnetic resonance absorption spectroscopy. Methanol and ethanol, at concentrations too low to cause general conformational destabilization of the protein, produce both optical and electron paramagnetic resonance absorption spectral changes in ferrihemoglobin. These changes arise from equilibrium associations, characterized by dissociation constants at 25 degrees C of about 40 and 200 mM, respectively, for the methanol-ferrihemoglobin and ethanol-ferrihemoglobin complexes so formed. Other optical spectral changes appear when the methanol concentration exceeds 3.5 M and the ethanol, 1.0 M. At concentrations lower than 0.5 M, 1- and 2-propanol produce spectral changes of this second kind. At room temperature no optical evidence has been found that the propanols associate with ferrihemoglobin in the manner of methanol and ethanol. Methanol and ethanol at low concentration have specific effects, characterized by electron paramagnetic resonance spectral differences, upon ferric alphaSH chains. All four alcohols, over a wide range of concentrations, reduce the symmetry of electron paramagnetic resonance spectra from frozen solutions of ferrihemoglobin; even at the high end of this concentration range, none of the alcohols reduces the symmetry of electron paramagnetic resonance spectra from frozen ferrimyoglobin. Ferrimyoglobin and catalase association with methanol is measurable optically; the binding is about five and sixty times weaker, respectively, for these two proteins as compared with ferrihemoglobin.     

M?ssbauer spectroscopic studies of the nature of centre X of photosystem I reaction centres from the cyanobacterium Chlorogloea fritschii

Reduced photosystem I samples, which give the electron paramagnetic resonance (EPR) signals associated with A, A and B, and A, B and X centres, have been studied using M?ssbauer spectroscopy. The M?ssbauer spectra obtained from each type of sample is different, which indicates that iron is associated with all three centres. The spectra are similar to those obtained from ferredoxins with 4Fe-4S centres and were fitted with oxidized and reduced components, the relative proportions depending on the degree of reduction of the sample as monitored by EPR. The sample which gave only the A EPR signal showed about 26% of the reduced component, the sample which gave A and B EPR signals showed about 48% of the reduced component, while the sample which gave A, B and X EPR signals showed about 65% of the reduced component. The measurements are consistent with X being a 4Fe-S4 centre.     

Comparison of the transient folding intermediates in lysozyme and alpha-lactalbumin

Refolding kinetics of two homologous proteins, lysozyme and alpha-lactalbumin, were studied by following the time-dependent changes in the circular dichroism spectra in the aromatic and the peptide regions. The refolding was initiated by 20-fold dilution of the protein solutions originally unfolded at 6 M guanidine hydrochloride, at pH 1.5 for lysozyme and pH 7.0 for alpha-lactalbumin at 4.5 degrees C. In the aromatic region, almost full changes in ellipticity that were expected from the equilibrium differences in the spectra between the native and unfolded proteins were observed kinetically. The major fast phase of lysozyme folding has a decay time of 15 s. The decay time of alpha-lactalbumin depends on the presence or absence of bound Ca2+: 10 s for the holoprotein and 100 s for the apoprotein. In the peptide region, however, most of the ellipticity changes of the two proteins occur within the dead time (less than 3 s) of the present measurements. This demonstrates existence of an early folding intermediate which is still unfolded when measured by the aromatic bands but has folded secondary structure as measured by the peptide bands. Extrapolation of the ellipticity changes to zero time at various wavelengths gives a spectrum of the folding intermediate. Curve fitting of the peptide spectra to estimate the secondary structure fractions has shown that the two proteins assume a similar structure at an early stage of folding and that the intermediate has a structure similar to that of partially unfolded species produced by heat and, for alpha-lactalbumin, also by acid and a moderate concentration of guanidine hydrochloride.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni

Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis.     

Multiquantum EPR spectroscopy of spin-labeled arrestin K267C at 35 GHz

Three- and five-quantum absorption and dispersion multiquantum electron paramagnetic resonance spectra of a spin-labeled protein have been obtained for the first time at Q-band (35 GHz). Spectra of arrestin spin-labeled at site 267 were recorded at room temperature as a function of microwave power. The separation of irradiating microwave frequencies, Deltaf, was 10 kHz, and a newly-designed multiquantum Q-band electron paramagnetic resonance bridge was utilized, operating in a superheterodyne detection mode. The sample volume was 30 nL using a 3-loop-2-gap resonator. Most spectra were obtained at a 300 microM concentration in single, 2-min scans, but spectra were also successfully obtained at 30 microM, corresponding to one picomole of protein. Enhanced sensitivity to T(1) and T(2) was evident in the spectra, and linewidths varied considerably across the spectra. The pure absorption displays are beneficial relative to field modulation methods for spectral characterization. The presence of two states of the nitroxide spin-label with different relaxation times is evident, particularly in the dispersion spectra, which are expected to exhibit enhanced sensitivity to lineshape variation relative to absorption. Feasibility has been established for the use of this technique for site-directed spin-labeling studies of biologically relevant samples, particularly the study of protein structure and dynamics.     

Electron paramagnetic resonance studies on spin-labelling of pepsin: effects of temperature, pH and urea on its conformation.

Pepsin was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidyl) bromoacetamide, possibly at the active site, at a beta-catboxyl group of a reactive aspartic acid. The spectrum of the spin-labelled pepsin showed that the spin probe was strongly immobilized (correlation time is greater than or equal to 10(-8) sec). Spin-labelled pepsin was thermally denatured at various temperatures and electron paramagnetic resonance (e.p.r.) spectra were taken at various times. Rates of denaturation estimated from the e.p.r. spectra at various temperatures showed that the enthalpy and entropy of thermal denaturation of spin-labelled pepsin at pH 3.5 were 48.0+/-4.9 kcal/mole and 214.7+/-14.5 e.u. respectively. Addition of conc. NaOH or 1 M acetate buffer at pH 6.0 sharpened e.p.r. spectra of the spin-labelled pepsin, indicating that the spin probe became mobilized by alkaline denaturation. Addition of urea caused unfolding of the protein which increased with the urea concentration, although only slight transition of conformational changes was observed in the e.p.r. spectra.     

The 5-55 single-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor.

An analogue of the BPT1 folding intermediate that contains only the disulphide bond between Cys-5 and Cys-55 has been prepared by mutation of the other four Cys residues to Ser. On the basis of its circular dichroism and 1H-nuclear magnetic resonance spectra and its electrophoretic mobility, this intermediate is shown to be at least partially folded at low temperatures. This probably accounts for several of the unique properties of this intermediate observed during folding.     

Trehalose favors a cutinase compact intermediate off-folding pathway

The folding of cutinase, an enzyme displaying lipolytic activity, has been studied in the presence of trehalose. Equilibrium unfolding data show that trehalose increases the free energy change between folded and unfolded states. Unfolding kinetics reveal the presence of an intermediate which is ca. 60% folded in terms of solvent exposure. Trehalose stabilizes this intermediate relative to the folded state. In contrast, the intermediate revealed by folding kinetics is more compact than the transition state, as shown by the positive slope observed at low denaturant concentration in the chevron plot, as well as the decrease in the observable rate constant for folding with the increase in trehalose concentration. This intermediate displays more than 50% of area buried from the solvent (relative to the native state) compared to around 40% for the transition state for folding and therefore appears to be off the folding pathway. Trehalose stabilizes and guanidine hydrochloride destabilizes this compact intermediate. Both unfolding and folding kinetics show that compact conformational states are stabilized by trehalose, in agreement with current models on the effect of compatible solutes. This effect occurs even for compact states that decelerate the folding as in the case of the intermediate revealed by folding kinetics.     

The folding of alpha-1-proteinase inhibitor: kinetic vs equilibrium control

Previous folding studies of alpha-1-proteinase inhibitor (alpha1-PI), which regulates the activity of the serine protease human neutrophil elastase, show an intermediate state at approximately 1.5 M guanidine-HCl (Gu). For the normal form of alpha1-PI, we demonstrate the reversible formation of the same stable distribution of monomeric and polymeric intermediates after approximately 1 h in 1.5 M Gu at approximately 23 degrees C from fully folded or fully unfolded alpha1-PI at similar final total concentrations and show that the stable distribution of monomeric and polymeric intermediates conforms with the law of mass action. We attribute these observations to an apparent equilibrium among intermediates. Our CD data are compatible with the intermediates having slightly relaxed structures relative to that of fully folded alpha1-PI and, thus, with the polymeric intermediates having a loop-sheet structure. Furthermore, we observe that the rates of folding (fast and slow terms) from the intermediate state are the same as those from the fully unfolded state, thereby supporting the contention that this intermediate state is on the folding pathway. We attribute the tendency of the Z mutant protein to polymerize/aggregate to an increased rate of the monomeric intermediate to form the apparent equilibrium distribution of intermediate species relative to its rate of folding to give intact alpha1-PI.     

The kinetics of bovine growth hormone folding are consistent with a framework model

The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e. the formation of secondary structure precedes the tertiary structure) (Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51, 459-489). To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance). The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption. The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model. The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied. The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration. The protein concentration dependence on refolding is due to the transient formation of an associated intermediate. The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect. At dilute protein concentrations the refolding can be studied independent of the association phenomena. The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries. The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M. F., and Ellis, S. (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus. No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed. It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics.     

Reduction of salt-requirement of halophilic nucleoside diphosphate kinase by engineering S-S bond

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10 °C in 0.2 M NaCl over the wild type HsNDK. It refolded from heat-denaturation even in 0.1 M NaCl, while the wild type required 2 M NaCl to achieve the same level of activity recovery. This enhanced refolding is due to the three S-S bonds between two basic dimeric units in the hexameric HsNDK structure, indicating that assembly of the dimeric unit may be the rate-limiting step in low salt solution. Circular dichroism and native-PAGE analysis showed that heat-denatured HsNDK formed partially folded dimeric structure, upon refolding, in the absence of salt and the native-like secondary structure in the presence of salt above 0.1 M NaCl. However, it remained dimeric upon prolonged incubation at this salt concentration. In contrary, heat-denatured D148C mutant refolded into tetrameric folding intermediate in the absence of salt and native-like structure above 0.1 M salt. This native-like structure was then converted to the native hexamer with time.     

Spin-label detection of hemoglobin-membrane interaction at physiological pH

The interaction between hemoglobin and the cytoplasmic surface of human erythrocyte membranes at physiological pH was studied by monitoring the electron paramagnetic resonance (EPR) signal of spin-labeled membrane ghosts in hemoglobin solutions of various concentrations. The EPR spectra indicate the existence of a significant hemoglobin-membrane interaction which exhibits a substantial hemoglobin concentration dependence over the concentration range 0-12 mg/mL. An equilibrium binding model yields a hemoglobin-membrane dissociation constant, Kd, on the order of 10(-4) M, at and above physiological pH; the interaction is classified as very low-affinity binding. The interaction increases significantly when the pH is decreased. Half-saturation of the binding sites occurs at a ratio of about 10(8) hemoglobins per cell.     

Folding of a three-stranded coiled coil

Coiled coils consist of two or more amphipathic a-helices wrapped around each other to form a superhelical structure stabilized at the interhelical interface by hydrophobic residues spaced in a repeating 3-4 sequence pattern. Dimeric coiled coils have been shown to often form in a single step reaction in which association and folding of peptide chains are tightly coupled. Here, we ask whether such a simple folding mechanism may also apply to the formation of a three-stranded coiled coil. The designed 29-residue peptide LZ16A was shown previously to be in a concentration-dependent equilibrium between unfolded monomer (M), folded dimer (D), and folded trimer (T). We show by time-resolved fluorescence change experiments that folding of LZ16A to D and T can be described by 2M (k1)<==>(k(-1)) D and M + D (k2)<==>(k(-2)) T. The following rate constants were determined (25 degrees C, pH 7): k1 = 7.8 x 10(4) M(-1) s(-1), k(-1) = 0.015 s(-1), k2 = 6.5 x 10(5) M(-1) s(-1), and k(-2) = 1.1 s(-1). In a separate experiment, equilibrium binding constants were determined from the change with concentration of the far-ultraviolet circular dichroism spectrum of LZ16A and were in good agreement with the kinetic rate constants according to K(D) = k1/2k(-1) and K(T) = k2/k(-2). Furthermore, pulsed hydrogen-exchange experiments indicated that only unfolded M and folded D and T were significantly populated during folding. The results are compatible with a two-step reaction in which a subpopulation of association competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.     

Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor

An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser. Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein. The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable. Approximately half of the protein molecules are unfolded at 3 degrees C, and this proportion increases at higher temperatures. The folded and unfolded conformations are in slow exchange. The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.     

The component analysis of tryptophan fluorescence spectra of melittin during its oligomerization

The oligomerization of melittin with increasing ionic strength and protein concentration was investigated using the methods of decomposition of its tryptophan fluorescence spectra into "elementary" log-normal components. At high ionic strength (up to 2 M KCl), the emission spectra of tetrameric melittin are well described as the sum of two log-normal components, suggesting the presence of tryptophan residues in two sorts of environment with greatly differing polarity. Measurements of fluorescence spectra by iodide showed that these two spectral components possess different Stern-Volmer constants, that is, the tryptophans emitting them have different solvent accessibility, which does not correlate with the crystallographic structure of tetrameric melittin. Moreover, in the oligomerization transition induced by ionic strength, the tetrameric intermediate is formed, which has log-normal spectral components with relative contributions differing from those in 2 M KCl.     

The de novo design of a rubredoxin-like Fe site.

A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [Fe(S-Cys)4](-1),(-2). The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(II) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(II)-bound forms of the variant are folded correctly and are stable. The Fe(III) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(III) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.     

Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.     

ENDOR studies of VO2+: probing protein-metal ion interactions in nephrocalcin.

Nephrocalcin inhibits the growth of calcium oxalate monohydrate crystals in the mammalian kidney. Isoforms A and B contain three equivalents of gamma-carboxyglutamic acid (Gla) residues implicated in Ca2+-binding and exhibit strong inhibitor properties and high Ca2+-binding affinity (Kd approximately 10(-8) M). Isoforms C and D lack these properties and exhibit low Ca2+-binding affinity (Kd approximately 10(-6) M). With VO2+ as a structural probe, electron paramagnetic resonance (EPR) studies of the Ca2+-binding sites of isoforms B and D showed that VO2+ binds competitively with a metal ion:protein stoichiometry of 4:1. EPR spectral parameters of the VO2+ ion were consistent with only equatorial oxygen-donor ligands. EPR and angle-selected electron nuclear double resonance (ENDOR) spectra showed two equatorially positioned, metal coordinating waters in isoform D while in isoform B no ligands undergoing hydrogen exchange were found. Since isoform D showed no evidence for axially coordinated water, similarly to isoform B, it is likely that the protein residues occupying the axial sites are identical in both proteins. ENDOR spectra of VO2+-complexes of isoforms B and D were compared to spectra of the VO2+-complex with alpha-ethylmalonic acid (EMA), a molecular mimic of Gla. Spectra of the VO2+-complex of EMA showed axial water located trans to the V=O bond and outer shell water hydrogen-bonded to the vanadyl oxygen, consistent with the X-ray structure of Ca(EMA)2. We, therefore, conclude that the spatial disposition of carboxylate groups of Gla residues coordinating Ca2+ in isoforms A and B must differ from that observed in the crystal structure of Ca(EMA)2.     

Cooperativity of a protein folding reaction probed at multiple chain positions by real-time 2D NMR spectroscopy

The refolding reaction of S54G/P55N ribonuclease T1 is a two-step process, where fast formation of a partly folded intermediate is followed by the slow reaction to the native state, limited by a trans --> cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate was determined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by a series of 128 very fast 2D (15)N-HMQC spectra, to observe the kinetics of 66 individual backbone amide probes. We find that the intermediate is as compact as the native protein with many native chemical shifts. All 66 analyzed amide probes follow the rate-limiting prolyl isomerization, which indicates that this cooperative refolding reaction is fully synchronized. The stability of the folding intermediate was determined from the protection factors of 45 amide protons derived from a competition between refolding and H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding with many protected amides in not-yet-native regions.