Refolding of trypsin-subtilisin inhibitor from marine turtle eggwhite

Trypsin-subtilisin inhibitor from marine turtle eggwhite refolded quantitatively from its fully reduced state atpH 8.5 in the presence of reduced and oxidized glutathione. The refolding process was studied by following the accompanying changes in inhibitory activity, fluorescence, sulfhydryl group titer, and hydrodynamic volume. The refolding process followed second-order kinetics with rate constants of 4.80×10² M^–1 sec^–1 for trypsin-inhibiting domain and 0.77× 10² M^–1 sec^–1 for subtilisin-inhibiting domain of the inhibitor at 30°C and their respective activation energies of the refolding process were 15.9 and 21.6 kcal/mol. Fluorescence intensity of the reduced inhibitor decreased with time of refolding until it corresponded to the intensity of the native inhibitor. The inhibitor contained 1–2%-helix, 40–42%-sheet, and 57–58% random coil structure. Refolded inhibitor gave a circular dichroic spectrum identical to that of the native inhibitor. A number of principal intermediates were detected as a function of the refolding time. Size-exclusion chromatography separated the intermediates differing in hydrodynamic volume (Stokes radius). The Stokes radius ranged from 23 Å (fully reduced inhibitor) to 18.8 Å (native inhibitor). Results indicated the independent refolding of two domains of the inhibitor and multiple pathways of folding were followed rather than an ordered sequential pathway.     

Sensitivity of Superfolder GFP to Ionic Agents

Superfolder variant of the green fluorescent protein (sfGFP) became a favorite probe for examination of the unfolding–refolding processes of fluorescent proteins with beta-barrel structure owing to its reversible unfolding in comparison with other fluorescent proteins. Its benefit is the proper folding even in fusion constructions with poorly folded polypeptides. We noticed that guanidine thiocyanate affects not only the structure of protein but its chromophore directly. Therefore we studied the influence of ionic denaturants and salts including guanidine thiocyanate, guanidine hydrochloride, sodium chloride and sodium thiocyanate on spectral features of sfGFP. It was shown that moderate amounts of the studied agents do not disrupt sfGFP structure but provoke pronounced alteration of its spectral characteristics. Changes in absorption and CD spectra in visible spectral range indicate the specific binding of SCN⁻ and Cl⁻ anions in the sfGFP chromophore vicinity. The anion binding results in the redistribution of sfGFP molecules with neutral and anionic chromophores. This also hinders the proton transfer in the chromophore excited state, considerably decreasing the fluorescence intensity of sfGFP. Our results indicate that when ionic denaturants are used in the studies of fluorescent protein folding their effect on fluorophore charge state should be taken into account.     

Heteronuclear three-dimensional NMR spectroscopy of a partially denatured protein: The A-state of human ubiquitin

Summary Human ubiquitin is a 76-residue protein that serves as a protein degradation signal when conjugated to another protein. Ubiquitin has been shown to exist in at least three states: native (N-state), unfolded (U-state), and, when dissolved in 60% methanol:40% water at pH 2.0, partially folded (A-state). If the A-state represents an intermediate in the folding pathway of ubiquitin, comparison of the known structure of the N-state with that of the A-state may lead to an understanding of the folding pathway. Insights into the structural basis for ubiquitin's role in protein degradation may also be obtained. To this end we determined the secondary structure of the A-state using heteronuclear three-dimensional NMR spectroscopy of uniformly ¹⁵N-enriched ubiquitin. Sequence-specific ¹H and ¹⁵N resonance assignments were made for more than 90% of the residues in the A-state. The assignments were made by concerted analysis of three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets. Because of ¹H chemical shift degeneracies, the increased resolution provided by the ¹⁵N dimension was critical. Analysis of short- and long-range NOEs indicated that only the first two strands of -sheet, comprising residues 2–17, remain in the A-state, compared to five strands in the N-state. NOEs indicative of an -helix, comprising residues 25–33, were also identified. These residues were also helical in the N-state. In the N-state, residues in this helix were in contact with residues from the first two strands of -sheet. It is likely, therefore, that residues 1–33 comprise a folded domain in the A-state of ubiquitin. On the basis of ¹H chemical shifts and weak short-range NOEs, residues 34–76 do not adopt a rigid secondary structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The secondary structure presented here differs from and is more thorough than that determined previously by two-dimensional ¹H methods [Harding et al. (1991) Biochemistry, 30, 3120–3128].     

Effect of temperature and surface potential on the electrogenic proton uptake in the QB site of the Rhodobacter sphaeroides photosynthetic reaction center: QA −. B −. → QAQBH2 transition

Direct electrometry was used to study the light-induced voltage changes in the Rhodobacter sphaeroides chromatophores adsorbed to a phospholipid-impregnated nitrocellulose film. After the second laser flash, a fast increase in the voltage associated with charge separation was followed by a slower increase attributed to the proton uptake in the Q_B site of the photosynthetic reaction centers. Kinetics and relative amplitudes of these voltage changes attributed to the Q_A ^–. _B ^–. Q_AQ_BH₂ transition, were measured as a function of pH and temperature between +4 and +40 °C. The kinetics can be approximated by a single exponent above +23 °C (100 µs at +25 °C, pH 7.2), whereas below this temperature, it was a good fit of two exponential approximation (65 µs and 360 µs with similar contributions at +10 °C, pH 7.2). The faster component diminished with an apparent pK 8.5, whereas the slower one was maintained at a constant level until pH 9.5 and then decreased. The calculated activation energy from the temperature dependence of the slower component (55 – 65 kJ/mol) was much higher than that of the faster component (< 10 kJ/mol). The two voltage components can be attributed to the transfer of the first (faster component) and the second (slower component) proton from the reaction center surface to Q_B. We suggested that higher activation energy of the slower component was due to a conformational change in the reaction center kinetically coupled to the second proton transfer to Q_BH^–.The faster component diminished in the presence of 1 M KCl, with an apparent pK 7.5. To explain this observation, we assume that: (i) the midpoint potential of the Q_A/Q_A ^–. redox pair was higher in 1 M KCl because of the reduced surface potential of chromatophores; (ii) the midpoint potential of the Q_B ^–./Q_BH^–. redox pair was insensitive to the surface potential change; (iii) the equilibrium constant of the reaction Q_A ^–.Q_B ^–. Q_AQ_BH^– decreased at high ionic strength.     

GroEL and protein disulfide isomerase each binds with folding intermediates of D-glyceraldehyde-3-phosphate dehydrogenase released from complexes formed with the other

Simultaneous presence of two chaperones, GroEL and protein disulfide isomerase (PDI), assists the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in an additive way. Delayed addition of chaperones to the refolding solution after dilution of denatured GAPDH indicates an interaction with intermediates formed mainly in the first 5 min for PDI and formed within a longer time period for GroEL-ATP. The above indicate that the two chaperones interact with different folding intermediates of GAPDH. After delayed addition of one chaperone to the refolding mixture containing the other at 4°C, GroEL binds with all GAPDH intermediates dissociated from PDI, and PDI interacts with the intermediates released from GroEL during the first 10–20 min. It is suggested that the GAPDH folding intermediates released from the chaperone-bound complex are still partially folded so as to be rebound by the other chaperone. The above results clearly support the network model of GroEL and PDI.     

Amide 1H n.m.r. study of the folding of ribonuclease C-peptide

The folding of ribonuclease A 1–13 (C-peptide) in H₂O near 0°C has been monitored by means of the amide and side chain NH proton resonances. The C-peptide carboxylate at low temperature forms, in a significant amount, a folded structure similar to the one that the 1–19 S-peptide adopts in the same conditions (3–13 α-helix). A quantitative comparison between helix stabilities of the lactone and carboxylate forms of C-peptide and S-peptide is reported. It is concluded that the proposed His 12⁺ … Hse 13 (COO^? salt bridge, which competes with the one-turn stabilizing salt bridge His 12⁺ … Glu 9^? in the C-peptide carboxylate, does not suppress helix formation as previously suggested but it merely reduces its stability. The behaviour of the N⁵-H resonance of the Arg 10⁺ side chain provides evidence for its implication in a further stabilizing interaction, most probably with Glu 2^?.     

Pulsed nuclear magnetic resonance study of39K within Halobacteria

Summary The³⁹K contents of isolated pellets and supernatant solutions from suspensions ofHalobacterium halobium were studied at 21–22°C by pulsed NMR spectroscopy. The rates of transverse relaxation were measured directly from the free induction decay (FID). The rate of longitudinal relaxation was measured by studying the FID after pairs of pulses of approximately 90°. Care was exercised to minimize the effect of magnetic field inhomogeneity; its contribution to the FID was approximately 25–30 sec^–1. The transverse relaxation process was found to consist of at least two components, whose rates were 321–449 sec^–1 and 1,122–2,067 sec^–1. In one preparation where the longitudinal relaxation process was studied, the data could be well fit to a single exponential relaxing at 253±33 (mean ±95% confidence limits) sec^–1. Comparison of the relative intensities of the NMR signals with the results of atomic absorption photometric analyses indicated that the great bulk of the intracellular³⁹K was detected by the NMR techniques used. The data obtained from the current NMR ofH. halobium are consistent with: (1) fractional binding of <3% of the total intracellular K⁺, (2) a small ordering factor characterizing all of the intracellular K⁺, or (3) some combination of the two.     

Kinetics of Histidine Dissociation From the Heme Fe(III) in N-fragment (residues 1–56) of Cytochrome <Emphasis Type="Italic">c</Emphasis>

We have here investigated the dissociation kinetics of the His side chains axially ligated to the heme-iron in the ferric (1–56 residues) N-fragment of horse cyt c. The ligand deligation induced by acidic pH-jump occurs as a biexponential process with different pre-exponential factors, consistent with a structural heterogeneity in solution and the presence of two differently coordinated species. In analogy with GuHCl-denatured cyt c, our data indicate the presence in solution of two ferric forms of the N-fragment characterized by bis-His coordination, as summarized in the following scheme: His18–Fe(III)–His26 \rightleftharpoons His18–Fe(III)–His33. We have found that the pre-exponential factors depend on the extent of the pH-jump. This may be correlated with the different pK_a values shown by His26 and His33; due to steric factors, His26 binds to the heme–Fe(III) less strongly than His33, as recently shown by studies on denatured cyt c. Interestingly, the two lifetimes are affected by temperature but not by the extent of the pH-jump. The lower pK_a for the deligation reaction required the use of an improved laser pH-jump setup, capable of inducing changes in H⁺ concentration as large as 1 mM after the end of the laser pulse. For the ferric N-fragment, close activation entropy values have been determined for the two histidines coordinated to the iron; this result significantly differs from that for GuHCl-denatured cyt c, where largely different values of activation entropy were calculated. This underlines the role played by the missing segment (residues 57–104) peptide chain in discriminating deligation of the nonnative His from the sixth coordination position of the metal.     

Simulation and experiment at high temperatures: ultrafast folding of a thermophilic protein by nucleation-condensation

We used Phi-value analysis to characterise the transition state for folding of a thermophilic protein at the relatively high temperature of 325 K. PhiF values for the folding of the three-helix bundle, peripheral subunit binding domain from Bacillus stearothermophilus (E3BD) were determined by temperature-jump experiments in the absence of chemical denaturants. E3BD folded in microseconds through a highly diffuse transition state. Excellent agreement was observed between experiment and the results from eight (independent) molecular dynamics simulations of unfolding at 373 K. We used a combination of heteronuclear NMR experiments and molecular dynamics simulations to characterise the denatured ensemble, and found that it contained very little persistent, residual structure. However, those regions that adopt helical structure in the native state were found by simulation to be poised for helix formation in the denatured state. These regions also had significant structure in the transition state for folding. The overall folding pathway appears to be nucleation-condensation.     

Local interactions favor the native 8-residue disulfide loop in the oxidation of a fragment corresponding to the sequence Ser-50-Met-79 derived from bovine pancreatic ribonuclease A

A 30-residue peptide was obtained from ribonuclease A by chemical cleavage with cyanogen bromide, subsequent sulfitolysis with concomitant S-sulfonation, and finally enzymatic cleavage withStaphylococcus aureus protease. The peptide was converted to the free thiol form by reductive cleavage of the S-sulfo-protecting groups withd,l-dithiothreitol. This peptide consisted of residues 50–79 of the native sequence of ribonuclease A, with the exception that methionine-79 had been converted to homoserine. Included in this sequence are residues cysteine-65 and cysteine-72, which form a disulfide bond in the native enzyme, as well as cysteine-58. This molecule may form one of three possible intramolecular disulfide bonds upon thiol oxidation, viz. one loop of 15 and 2 of 8 residues each. These isomeric peptides were prepared by oxidation with cystamine, 2-aminoethanethiolation of residual thiols, and fractionation by reverse-phase high-performance liquid chromatography. Disulfide pairings were established by mapping the tryptic fragments and confirming their composition by amino acid analysis. After protracted incubation under oxidizing conditions at 25.0°C andp H 8.0, the 26-member ring incorporating the native disulfide bond between residues 65 and 72 is the dominant product. Assuming that equilibrium is established, we infer that local interactions in the sequence of ribonuclease A significantly stabilize the native 8-residue disulfide loop with respect to the non-native 8-residue loop (G°=–1.1±0.1 kcal mole^–1). The implications of this observation for the oxidative folding of the intact protein are discussed.     

Solution structure of dynorphin A (1–17): a NMR study in a cryoprotective solvent mixture at 278 K1

Dynorphin A, the endogenous agonist for the κ opioid receptor, has been studied by NMR spectroscopy in methanol, acetonitrile, DMSO and in mixtures of hexafluoroacetone/water and DMSO/water. NMR data in the DMSO/water cryomixture at 278 K are consistent with a conformer in which the N‐terminal part, like the corresponding message domain of enkephalins, is poorly ordered, whereas the C‐terminal part is folded in a loop centred around Pro¹⁰. The folded structure of the C‐terminal part (address moiety) may shed light on the role of the essential residues Arg⁷, Lys¹¹ and Lys¹³. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Deciphering the Hidden Informational Content of Protein Sequences: FOLDABILITY OF PROINSULIN HINGES ON A FLEXIBLE ARM THAT IS DISPENSABLE IN THE MATURE HORMONE*

Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe^B1 in placental mammals) is variably positioned within crystal structures and exhibits ¹H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1–B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus.     

Pathway of Oxidative Folding of a 3-Disulfide α-Lactalbumin May Resemble Either BPTI Model or Hirudin Model

Pathways of oxidative folding of disulfide proteins display a high degree of diversity and vary among two extreme models. The BPTI model is defined by limited species of folding intermediates adopting mainly native disulfide bonds. The hirudin model is characterized by highly heterogeneous folding intermediates containing mostly non-native disulfide bonds. αLA-IIIA is a 3-disulfide variant of α-lactalbumin (αLA) with a 3-D conformation essentially identical to that of intact αLA. αLA-IIIA contains 3 native disulfide bonds of αLA, two of them are located at the calcium binding β-subdomain (Cys⁶¹–Cys⁷⁷ and Cys⁷³–Cys⁹¹) and the third bridge is located within the α-helical domain of the molecule (Cys²⁸–Cys¹¹¹). We investigate here the pathway of oxidative folding of fully reduced αLA-IIIA with and without stabilization of its β-subdomain by calcium binding. In the absence of calcium, the folding pathway of αLA-IIIA was shown to resemble that of hirudin model. Upon stabilization of β-sheet domain by calcium binding, the folding pathway of αLA-IIIA exhibits a striking similarity to that of BPTI model. Three predominant folding intermediates of αLA-IIIA containing exclusively native disulfide bonds were isolated and structurally characterized. Our results further demonstrate that stabilization of subdomains in a protein may dictate its folding pathway and represent a major cause for the existing diversity in the folding pathways of the disulfide-containing proteins.     

Raman spectroscopic studies of hen egg-white lysozyme at high temperatures and pressures

In situ high-temperature, high-pressure Raman experiments on 3 mM (pH 5) aqueous solutions of hen egg-white (HEW) lysozyme show a decrease in the relative height of the 505 cm^–1 band associated with S-S stretching vibrations at 72°C (1 bar). The peak height changes are accompanied by significant band broadening, and the integrated band intensity does not change within experimental error. The effect of increased pressure at 72°C was to hinder broadening of the 505 cm^–1 band. HEW lysozyme (2.4 mM,pH 5) was also heated at 76°C, 80°C, and 95°C for different periods of time, and aliquots were quenched to room temperature for Raman and enzymatic activity measurements. After 9 hr at 76°C, the protein exhibits enzyme activity less than 50% of the initial value, and approximately 50% reduction in activity is achieved after 3 hr at 80°C or 1 hr at 95°C. The Raman results suggest that different irreversibly denatured conformations are attained during prolonged exposures at these different temperatures. It is apparent from these studies that the S-S stretch intensity is decreased irreversibly.     

GroEL assisted folding of large polypeptide substrates in Escherichia coli: Present scenario and assignments for the future

Escherichia coli chaperonins GroEL and GroES are indispensable for survival and growth of the cell since they provide essential assistance to the folding of many newly translated proteins in the cell. Recent studies indicate that a substantial portion of the proteins involved in the host pathways are completely dependent on GroEL–GroES for their folding and hence providing some explanation for why GroEL is essential for cell growth. Many proteins either small-single domain or large multidomains require assistance from GroEL–ES during their lifetime. Proteins of size up to 70 kDa can fold via the cis mechanism during GroEL–ES assisted pathway, but other proteins (>70 kDa) that cannot be pushed inside the cavity of GroEL–ATP complex upon binding of GroES fold by an evolved mechanism called trans. In recent years, much work has been done on revealing facts about the cis mechanism involving the GroEL assisted folding of small proteins whereas the trans mechanism with larger polypeptide substrates still remains under cover. In order to disentangle the role of chaperonin GroEL–GroES in the folding of large E. coli proteins, this review discusses a number of issues like the range of large polypeptide substrates acted on by GroEL. Do all these substrates need the complete chaperonin system along with ATP for their folding? Does GroEL act as foldase or holdase during the process? We conclude with a discussion of the various queries that need to be resolved in the future for an extensive understanding of the mechanism of GroEL mediated folding of large substrate proteins in E. coli cytosol.     

NMR Study of Daunomycin Complexation with Hexadeoxynucleotide 5"-d(TpApCpGpTpA) in Aqueous Solution

Self-association of hexadeoxynucleotide 5"-d(TpApCpGpTpA) and its complexation with antitumor antibiotic daunomycin were studied by one- and two-dimensional homonuclear ¹H NMR spectroscopy and heteronuclear ¹H–³¹P NMR spectroscopy in water–salt solution. The concentration and temperature dependences of proton chemical shifts of the hexadeoxynucleotide and the ligand were measured, and equilibrium constants and thermodynamic parameters of corresponding reactions were calculated on this basis using models for the formation of hexadeoxynucleotide duplex and its complex with the antibiotic. The spatial structure of daunomycin–d(TACGTA)₂complex in solution was calculated using X-PLOR software on the basis of 2D NOE spectral data and the limit values of proton chemical shifts of the ligand. Comparative analysis of different intermolecular interactions in sequence-specific binding of the antibiotic to the DNA fragment was carried out.