Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c

Titration to high pH converts yeast iso-2 cytochrome c to an inactive but more stable alkaline form lacking a 695-nm absorbance band [Osterhout, J. J., Jr., Muthukrishnan, K., & Nall, B. T. (1985) Biochemistry 24, 6680-6684]. The kinetics of absorbance-detected refolding of the alkaline form have been measured by dilution of guanidine hydrochloride in a stopped-flow instrument. Fast-folding species (tau 2) are detected, as in refolding to the native state at neutral pH. An additional kinetic phase (tau a) is observed with an amplitude opposite in sign to the fast phase. The amplitude of this phase increases and the rate increases with increasing pH. Comparison to pH-jump measurements of the fully folded protein shows that phase tau a has the same sign, rate, and pH dependence as the alkaline isomerization reaction, suggesting that this new phase involves isomerization of native or nativelike species following fast folding. Absorbance difference spectra are taken at 5-s intervals during refolding at high pH. The spectra verify that nativelike species--with a 695-nm absorbance band--are formed transiently, before conversion of the protein to the alkaline form. Refolding in the presence of ascorbate shows that the transient, nativelike species are reducible, unlike alkaline iso-2. Thus, (1) refolding to the alkaline form of iso-2 cytochrome c proceeds through transient native or nativelike species, and (2) a folding pathway leading to native or nativelike forms is maintained at high pH, where native species are no longer the thermodynamically favored product.(ABSTRACT TRUNCATED AT 250 WORDS)     

A transient spin-state change during alkaline isomerization of ferricytochrome c

Kinetic difference spectra during the alkaline isomerization of ferricytochrome c were obtained by the pH-jump method in the range of 540 to 655 nm. The spectrum of the transient intermediate, which appears during the course of the isomerization, was reproduced from the spectra. The intermediate showed an intense absorption band at 600 nm, indicating that it is a high spin or mixed spin species. This is in contrast to the stable neutral and alkaline forms which are low spin species. The transient spin-state change during the isomerization was also observed upon rapid oxidation of ferrocytochrome c at alkaline pH.     

Kinetic studies on isomerization of ferricytochrome c in alkaline and acid pH ranges by the circular dichroism stopped-flow method

The isomerization of horse-heart ferricytochrome c caused by varying pH was kinetically studied by using circular dichroism (CD) and optical absorption stopped-flow techniques. In the pH range of 7--13, the existence of the three different forms of ferricytochrome c (pH less than 10, pH 10--12, and pH greater than 12) was indicated from the statistical difference CD spectra. On the basis of analyses of the stopped-flow traces in the near-ultraviolet and Soret wavelength regions, the isomerization of ferricytochrome c from neutral form to the above three alkaline forms was interpreted as follows (1) below pH 10, the replacement of the intrinsic ligand of methionine residue by lysine residue occurs; (2) between pH 10 and 12, the uncoupling of the polypeptide chain from close proximity of the heme group occurs first, followed by the interconversion of the intrinsic ligands; and (3) above pH 12, hydroxide form of ferricytochrome c is formed, though the replacement of the intrinsic ligand by extrinsic ligands may occur via different routes from those below pH 12. The CD changes at 288 nm and in the Soret region caused by the pH-jump (down) from pH 6.0 to 1.6 were compared with the appearance of the 620-nm absorption band ascribed to the formation of the high-spin form of ferricytochrome c. Both CD and absorption changes indicated that the isomerization at pH 1.6 consisted of two processes: one proceeded within the dead-time (about 2 ms) of the stopped-flow apparatus and the other proceeded at a determinable rate with the apparatus. On the basis of these results, the isomerization of ferricytochrome c at pH 1.6 was explained as follows: (1) the transition from the low-spin form to the high-spin forms occurs within about 2 ms, the dead-time of the stopped-flow apparatus; and (2) the polypeptide chain is unfolded after the formation of the high-spin form.     

Low temperature phototransformations of protochlorophyll(ide) in etiolated leaves

By methods of difference and derivative spectroscopy it was shown that in etiolated leaves at 77 K three photoreactions of P650 protochlorophyllide take place which differ in their rates and positions of spectral maxima of the intermediates formed in the process: P650R668, P650R688, and P650R697. With an increase of temperature up to 233 K, in the dark, R688 and R697 are transformed into the known chlorophyllide forms C695/684 and C684/676, while R668 disappears with formation of a shorter wavelength form of protochlorophyllide with an absorption maximum at 643–644 nm.Along with these reactions, at 77 K phototransformations of the long-wave protochlorophyllide forms with absorption maxima at 658–711 nm into the main short-wave forms of protochlorophyllide are observed. At 233 K in the dark this reaction is partially reversible. This process may be interpreted as a reversible photodisaggregation of the pigment in vivo.The mechanism of P650 reactions and their role in the process of chlorophyll photobiosynthesis are discussed.Abbreviations P650 protochlorophyll(ide) with absorption maximum at 650 nm - C697/684 chlorophyllide with fluorescence maximum at 695 nm and absorption maximum at 684 nm - R697 intermediate with absorption maximum at 697 nm     

Photoconversion of long-wavelength protochlorophyll native form Pchl 682/672 into chlorophyll Chl 715/696 in Chlorella vulgaris B-15

By spectral methods, the final stages of chlorophyll formation from protochlorophyll (ide) were studied in heterotrophic cells of Chlorella vulgaris B-15 mutant, where chlorophyll dark biosynthesis is inhibited. It was shown that during the dark cultivation, in the mutant cells, in addition to the well-known protochlorophyll (ide) forms Pchlide 655/650, Pchl(ide) 640/635, Pchl(ide) 633/627, a long-wavelength protochlorophyll form is accumulated with fluorescence maximum at 682 nm and absorption maximum at 672 nm (Pchl 682/672). According to the spectra measured in vivo and in vitro, illumination of dark grown cells leads to the photoconversion of Pchl 682/672 into the stable long wavelength chlorophyll native form Chl 715/696. This reaction was accompanied by well-known photoreactions of shorter-wavelength Pchl (ide) forms: Pchlide 655/650Chlide 695/684 and Pchl (ide) 640/635Chl (ide) 680/670. These three photoreactions were observed at room temperature as well as at low temperature (203–233 K).Abbreviations Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - Pchl protochlorophyll - PS I RC Photosystem I reaction centres. Abbreviations for native pigment forms: the first number after the pigment symbol corresponds to maximum position of low-temperature (77 K) fluorescence band (nm), second number to maximum position of long-wavelength absorption band     

One-electron reactions in biochemical systems as studied by pulse radiolysis. VI. Stages in the reduction of ferricytochrome c

When ferricytochrome c at pH about 9 is reduced by hydrated electrons and/or CO₂^?, it gives rise to an unstable form of ferrocytochrome c whose absorption spectrum, particularly in the Soret region, differs from that of normal ferrocytochrome c. This form changes intramolecularly (life-time about 0.1 s at ambient temperature) to yield normal ferrocytochrome c, and by 0.5 s the change in absorption spectrum in the range 225–600 nm produced by e^?_aq and/or CO₂^? is identical to the final change produced by reduction with an equivalent amount of sodium dithionite. This shows that both e^?_aq and CO^?₂ reduce cytochrome c with practically 100% efficiency. In the range 600–800 nm the spectrum of the unstable form is the same as that of normal ferrocytochrome c, both having small absorptions at 695 nm as compared with ferricytochrome c. As the unstable form disappears however a further loss of absorption at 695 nm occurs. This is taken to imply that the unstable form decays to a second unstable form which then rapidly donates an electron to the unchanged neutral form of ferricytochrome c, so reducing absorption in the 695 nm band. Subsequent to this process the absorption in the 695 nm band increases over a period of minutes owing to re-equilibration between the neutral and alkaline formes of ferricytochrome c. Between pH 7 and 10 the effect of pH on the absorption changes is consistent with the hypothesis of a second unstable form of ferrocytochrome c. Additional phenomena arise in more alkaline solutions. The rates of the various unimolecular processes are thought to be determined by the rates of change of conformation of the protein parts of the molecule following the change in oxidation state.     

Test of the extended two-state model for the kinetic intermediates observed in the folding transition of ribonuclease A

A test has been made of the proposal that: (a) the extended two-state model describes the kinetic intermediates seen in the folding transition of RNAase A, i.e. that the only species present in folding experiments are the native protein and multiple forms of the completely unfolded protein; and (b) that the interconversion between the two known unfolded forms of RNAase A (the U₁

U₂ reaction) is described solely by the cis-trans isomerization of the proline residues. The test is to measure the rate of the U₁

U₂ reaction in a wide range of refolding conditions and to compare these data with the kinetic properties of proline isomerization.The main results are as follows. (1) The activation enthalpy of the U₁

U₂ reaction in refolding conditions (pH 6, 20 ° to 40 °C) is less than 5 kcal/mol. This is much too small to be explained as proline isomerization. (2) Both the rate and the activation enthalpy change sharply at guanidine hydrochloride concentrations below 2 m. There appear to be two pathways for the U₁

U₂ reaction in refolding conditions, and the slower pathway is favored by adding guanidine hydrochloride. (3) The rate and activation enthalpy for proline isomerization in l-alanyl-l-proline are unaffected by 2 m-guanidine hydrochloride.The results show that the proline isomerization hypothesis and the extended two-state model cannot both be correct for RNAase A. They suggest that partial folding occurs rapidly in refolding conditions and that the extended two-state model is invalid. They leave open the question of whether or not proline isomerization is the rate-limiting step in the U₁

U₂ reaction.Another possible source of slow configurational reactions in the unfolded state is mentioned. The three major, overlapping, disulfide-bonded loops of RNAase A can exist in two isomeric configurations. Interconversion of these isomers requires pulling one loop, or one end of the polypeptide chain, through a second loop and this is likely to be a slow process.In some conditions, heat-unfolded but not guanidine-unfolded RNAase A shows a second slow-refolding process. It may result from aggregates of the heatunfolded protein which are formed and broken up slowly. Conditions are given for eliminating this reaction.     

In vivo chlorophyll forms in higher plants and algae sensitive to treatment with lutein

Lamella preparations of spinach, Chlorella, Phaeodactylum, Anabatnaand Porphyra were treated with a hydrophobic reagent, lutein,and the absorption and fluorescence spectra in the red regionbefore and after treatment were compared for changes causedby the treatment. Absorption spectra of all these preparationsunderwent the same spectral change, transformation of a bandat 684 nm into a band at 666 nm. The longer the maximum wavelengthof the red peak, the greater was the fractional absorbance decreaseat 684 nm. The content of C684 (the chlorophyll form responsiblefor the 684 nm band) in the lamellae was estimated from thefractional decreases as being progressively higher in the orderof Phaeodactylum, Porphyra, Anabatna, Chlorella and spinach.The fluorescence spectra at liquid nitrogen temperature beforetreatment showed two bands. The longer wavelength band was transformedby the treatment into a shorter wavelength band(s), as describedbelow, according to the maximum wavelengths: spinach, F735F695(or F686); Chlorella, F715F700 (or F686); Phaeodactylum, anunidentified componentF690; Anabaena, F732F685 (or F695); Porphyra,F726F683. These chlorophyll forms with fluorescence maxima between715 and 735 nm were, therefore, designated C684 based on absorptionspectrophotometry, and are considered to play a role in photosystemII. (Received August 15, 1972; )     

Histidine-mediated pH-sensitive regulation of M-ficolin:GlcNAc binding activity in innate immunity examined by molecular dynamics simulations

Background

M-ficolin, a pathogen recognition molecule in the innate immune system, binds sugar residues including N-acetyl-D-glucosamine (GlcNAc), which is displayed on invading microbes and on apoptotic cells. The cis and trans Asp282-Cys283 peptide bond in the M-ficolin, which was found to occur at neutral and acidic pH in crystal structures, has been suggested to represent binding and non-binding activity, respectively. A detailed understanding of the pH-dependent conformational changes in M-ficolin and pH-mediated discrimination mechanism of GlcNAc-binding activity are crucial to both immune-surveillance and clearance of apoptotic cells.

Methodology/Principal Findings

By immunodetection analysis, we found that the pH-sensitive binding of GlcNAc is regulated by a conformational equilibrium between the active and inactive states of M-ficolin. We performed constant pH molecular dynamics (MD) simulation at a series of pH values to explore the pH effect on the cis-trans isomerization of the Asp282-Cys283 peptide bond in the M-ficolin fibrinogen-like domain (FBG). Analysis of the hydrogen bond occupancy of wild type FBG compared with three His mutants (H251A, H284A and H297A) corroborates that His284 is indispensible for pH-dependent binding. H251A formed new but weaker hydrogen bonds with GlcNAc. His297, unlike the other two His mutants, is more dependent on the solution pH and also contributes to cis-trans isomerization of the Asp282-Cys283 peptide bond in weak basic solution.

Conclusions/Significance

Constant pH MD simulation indicated that the cis active isomer of Asp282-Cys283 peptide bond was predominant around neutral pH while the trans bond gradually prevailed towards acidic environment. The protonation of His284 was found to be associated with the trans-to-cis isomerization of Asp282-Cys283 peptide bond which dominantly regulates the GlcNAc binding. Our MD simulation approach provides an insight into the pH-sensitive proteins and hence, ligand binding activity.     

Photo-sensitized Isomerization of Retinal by Dyes

THE initial reaction following absorption of light in the retina is the isomerization of the 11-cis retinal chromophore of the visual pigment¹. Isolated 11cis retinal will undergo the same isomerization to the all-trans form when excited by light of wavelength shorter than about 450 nm and this reaction can be sensitized to light of longer wavelengths by the addition of trace amounts of iodine to the solution².     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715–740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50-4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the lightharvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (F_v) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm.From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emittting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

Alkalitolerant micromycetes in acidic and neutral soils of the temperate zone

A wide diversity of micromycetes from various taxonomic groups in acidic and neutral soils is known from the literature data. In the present work, the fungi isolated from these soils and capable of growth at high pH are analyzed. The fungi were isolated from acidic sod-podzol and neutral cultivated soils by plating on alkaline agar (pH 10.0–10.5). Their identification was carried out using morphological, cultural, and molecular genetic criteria. Phylogenetic analysis was performed and the rates of linear growth within a broad pH range (4.0–10.4) were determined. The isolates represented a polyphyletic group of ascomycetes (Sordariomycetes), which included members of Plectosphaerellaceae (5 species) and various families of Hypocreales (4 species). The most common species were Gibellulopsis nigrescens, Acrostalagmus luteoalbus, Chordomyces antarcticum, and Plectosphaerella spp. Investigation of fungal growth at different pH values revealed all isolates to be alkalitolerant, with no alkaliphilic fungi isolated from acidic sod-podzol and neutral cultivated soils. Although the group of isolates was polyphyletic and its members originated from different ecological and trophic niches, most alkalitolerant isolates exhibited common morphological traits with acremonium- and verticillium-like conidial spore formation, abundant slime formation, and a tendency for aggregation of their mycelium in bundles. Our research confirmed the presence of fungi with alkalitolerant adaptation to external pH in the sod-podzolic and cultivated soils of the Moscow region.     

Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentum l-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.     

Chlorophyll fluorescence characteristics of system I chlorophyll {alpha}-protein complex and system II particles at room and liquid nitrogen temperatures

Emission spectra of a system I chlorophyll (Chl) -protein complex(SI Chl-P)³ and system II particles, prepared by the methodof Dietrich and Thornber (25), and by the method of Huzisigeet al. (24), respectively, were measured at room and liquidnitrogen temperatures to characterize the emission bands originatingfrom system I and system II. Room temperature and 77°K spectra clearly show that theF695 (690–697 nm) fluorescence band originates from bothphotosystems. In SI Chl-P the F695 band was observed both atroom and at liquid nitrogen temperatures. At 77°K, the Chl fluorescence at 685 nm is nearly as intenseas that at 720 nm (long-wavelength band) in dilute samples ofSI Chl-P. Reabsorption of 685 nm fluorescence has distortedconsiderably the shape of emission spectra of system I publishedthus far. In dilute samples of system II, the F695 is as (ormore) intense as F685, and the F735 is drastically decreased. Additionally, it is reported here that in Cyanidium caldarium,studied to compare the in vivo system with isolated SI Chl-Pand system II preparations, the 695 nm band is present uponexcitation in both system I and system II; the ratio of thelong-wave length fluorescence (F735) to the short-wavelengthfluorescence (F685) is much higher than those in the purifiedpreparations. Conceivably, the high values, obtained in thedilute samples of algae, are due to the reabsorption of thefluorescence from the short-wavelength form of Chl in the chloroplastin vivo. Furthermore, in this alga the phycocyanin fluorescenceband is split with maxima at 655 (phycocyanin) and 665 nm (allophycocyanin)at 77°K. At room temperature, however, the allophycocyaninfluorescence predominates having a peak at about 670 nm. Therelative increase in phycocyanin fluorescence at 77°K maybe due to a decrease in the energy transfer from it to allophycocyaninin agreement with slow Förster type transfer. ² Department of Botanical Sciences, University of California,Los Angeles, California 90024, U. S. A. (Received September 7, 1971; )     

Proline isomerization in the C-terminal region of HSP27

In mammals, small heat-shock proteins (sHSPs) typically assemble into interconverting, polydisperse oligomers. The dynamic exchange of sHSP oligomers is regulated, at least in part, by molecular interactions between the α-crystallin domain and the C-terminal region (CTR). Here we report solution-state nuclear magnetic resonance (NMR) spectroscopy investigations of the conformation and dynamics of the disordered and flexible CTR of human HSP27, a systemically expressed sHSP. We observed multiple NMR signals for residues in the vicinity of proline 194, and we determined that, while all observed forms are highly disordered, the extra resonances arise from cis-trans peptidyl-prolyl isomerization about the G193-P194 peptide bond. The cis-P194 state is populated to near 15% at physiological temperatures, and, although both cis- and trans-P194 forms of the CTR are flexible and dynamic, both states show a residual but differing tendency to adopt β-strand conformations. In NMR spectra of an isolated CTR peptide, we observed similar evidence for isomerization involving proline 182, found within the IPI/V motif. Collectively, these data indicate a potential role for cis-trans proline isomerization in regulating the oligomerization of sHSPs.