Unfolding of cytochrome C upon interaction with azobenzene-modified copolymers.

Hydrophilic or amphiphilic macromolecules are common organic matrices used to encapsulate and protect fragile drugs such as proteins. Polymer cargoes are in addition designed for remote control of protein delivery, upon imparting the macromolecules with stimuli-responsive properties, such as light-triggered polarity switches. The effect of interaction between polymers and proteins on the stability of the proteins is, however, rarely investigated. Here we studied the unfolding/folding equilibrium of cytochrome c (cyt c) under its oxidized or reduced forms, in the presence of various amphiphilic copolymers (by circular dichroism and intrinsic fluorescence measurements). As models of stimuli-responsive amphiphilic chains, we considered poly(acrylic acid) derivatives, modified to contain hydrophobic, light-responsive azobenzene moieties. These copolymers are, thus, capable to develop both ionic (under their sodium forms at pH > 8) and hydrophobic associations with the basic protein cyt c (isoelectric point of 10.0). In aqueous buffer upon increasing urea concentrations, cyt c underwent unfolding, at [urea] of 9-10 M, which was analyzed under the framework of the equilibrium between two states (native-unfolded). In the presence of polymers, the native folding of cyt c was preserved at low concentrations of urea (typically <4M). However, the presence of polymers facilitated unfolding, which occurred at urea concentrations lowered by 2-4 M as compared to unfolding in the absence of polymers (polymer/cyt c ratio of 1:1 g/g). The predominant contribution of coulombic interactions was shown by both the lack of significant impact of the amount of (neutral) azobenzene moieties in the copolymers and the disappearance of destabilization at ionic strength higher than 150 mM. In addition, stability was similar to that of an isolated cyt c, in the presence of a neutral chain bearing acryloyl(oligoethyleneoxide) units instead of the ionized sodium acrylate moieties. DSC measurements showed that in the presence of polymers, cyt c is thermally unfolded in aqueous buffer at temperatures lowered by >20 °C as compared to thermal unfolding in the absence of polymers. Upon exposure to UV light, properties of the polymers chains were perturbed in situ, upon cis/trans isomerization of the azobenzene groups. In polymers displaying a photoresponsive polarity and hydrophobicity switch (conventional azobenzene), the stability of cyt c was not affected by the exposure to light. In contrast, when photoionization occurred (using an hydroxyl-azobenzene whose pK(a) can be photoshifted), unfolding was initiated upon exposure to light. Altogether, these results show that coulombic binding is a predominant driving force that facilitates unfolding in water/urea solutions. In regard to the design of light-responsive systems for protein handling and control of folding, we conclude that remote control of the coulombic interaction upon photoionization of chromophores can be more efficient than the more conventional photomodulation of polarity.

Configurational distribution of denatured phosphoglycerate kinase.

Physiochemical characterization of the denatured states of proteins is important for a complete understanding of the factors stabilizing their folded conformations. Using a combination of small angle neutron scattering (SANS), statistical mechanical modelling and molecular mechanics calculations, we examine the configurational distribution of phosphoglycerate kinase denatured in 4 M guanidine hydrochloride solution. The denaturing of the protein produces a clear change in the form of the SANS profile and a large increase of the radius of gyration. In the statistical mechanical model, the region of contrast neutron scattering density associated with the protein is pictured as a chain of freely jointed spheres. The model is fitted to the SANS data for the denatured protein. It is found that a model with a small number of spheres cannot account for the higher resolution scattering, indicating an absence of detectable structuration; a good fit is found with 100 spheres of 8.5 A radius. Single configurations of the fitted chain of spheres are used as low-resolution bounds for model-building and molecular mechanics calculations to obtain plausible atomic-detail models of the denatured chain.

Heat-induced unfolding of neocarzinostatin, a small all-beta protein investigated by small-angle X-ray scattering.

Neocarzinostatin is an all-beta protein, 113 amino acid residues long, with an immunoglobulin-like fold. Its thermal unfolding has been studied by small-angle X-ray scattering. Preliminary differential scanning calorimetry and fluorescence measurements suggest that the transition is not a simple, two-state transition. The apparent radius of gyration is determined using three different approaches, the validity of which is critically assessed using our experimental data as well as a simple, two-state model. Similarly, each step of data analysis is evaluated and the underlying assumptions plainly stated. The existence of at least one intermediate state is formally demonstrated by a singular value decomposition of the set of scattering patterns. We assume that the pattern of the solution before the onset of the transition is that of the native protein, and that of the solution at the highest temperature is that of the completely unfolded protein. Given these, actually not very restrictive, boundary constraints, a least-squares procedure yields a scattering pattern of the intermediate state. However, this solution is not unique: a whole class of possible solutions is derived by adding to the previous linear combination of the native and completely unfolded states. Varying the initial conditions of the least-squares calculation leads to very similar solutions. Whatever member of the class is considered, the conformation of this intermediate state appears to be weakly structured, probably less than the transition state should be according to some proposals. Finally, we tried and used the classical model of three thermodynamically well-defined states to account for our data. The failure of the simple thermodynamic model suggests that there is more than the single intermediate structure required by singular value decomposition analysis. Formally, there could be several discrete intermediate species at equilibrium, or an ensemble of conformations differently populated according to the temperature. In the latter case, a third state would be a weighted average of all non native and not completely unfolded states of the protein but, since the weights change with temperature, no meaningful curve is likely to be derived by a global analysis using the simple model of three thermodynamically well-defined states.

Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5.

Binding of bacteriophage T5 to Escherichia coli cells is mediated by specific interactions between the receptor-binding protein pb5 (67.8 kDa) and the outer membrane iron-transporter FhuA. A histidine-tagged form of pb5 was overproduced and purified. Isolated pb5 is monomeric and organized mostly as beta-sheets (51%). pb5 functionality was attested in vivo by its ability to impair infection of E. coli cells by phage T5 and Phi80, and to prevent growth of bacteria on iron-ferrichrome as unique iron source. pb5 was functional in vitro, since addition of an equimolar concentration of pb5 to purified FhuA prevented DNA release from phage T5. However, pb5 alone was not sufficient for the conversion of FhuA into an open channel. Direct interaction of pb5 with FhuA was demonstrated by isolating a pb5/FhuA complex using size-exclusion chromatography. The stoichiometry, 1 mol of pb5/1 mol of FhuA, was deduced from its molecular mass, established by analytical ultracentrifugation after determination of the amount of bound detergent. SDS-PAGE and differential scanning calorimetry experiments highlighted the great stability of the complex: (i) it was not dissociated by 2% SDS even when the temperature was raised to 70 degrees C; (ii) thermal denaturation of the complex occurred at 85 degrees C, while pb5 and FhuA were denatured at 45 degrees C and 74 degrees C, respectively. The stability of the complex renders it suitable for high-resolution structural studies, allowing future analysis of conformational changes into both FhuA and pb5 upon adsorption of the virus to its host.

Key interactions in the immunoglobulin-like structure of apo-neocarzinostatin: evidence from nuclear magnetic resonance relaxation data and molecular dynamics simulations.

The three-dimensional structure of apo-neocarzinostatin (apo-NCS, MW: ca.11000, antitumoral chromophore carrier protein) is based on a seven-stranded antiparallel beta-sandwich, very similar to the immunoglobulin folding domain. We investigated the backbone dynamics of apo-NCS by (13)C-NMR relaxation measurements and molecular dynamics simulation. Model-free parameters determined from the experimental data are compared with a 1.5-nsec molecular simulation of apo-NCS in aqueous solution. This comparison provides an accurate description of both local and collective movements within the protein. This analysis enabled us to correlate dynamic processes with key interactions of this beta-protein. Local motions that could be relevant for the intermolecular association with the ligand are also described.

Repulsive interparticle interactions in a denatured protein solution revealed by small angle neutron scattering.

In order to investigate the effect of concentration in biological processes such as protein folding, small angle neutron scattering measurements were used to determine the second virial coefficient of solutions of both native and strongly denatured phosphoglycerate kinase and the radius of gyration of the protein at zero concentration. The value of the second virial coefficient is a good probe of the non-ideality of a solution. The present results show that the unfolding of the protein leads to a drastic change in the repulsive intermolecular interactions. We conclude that these interactions are due mainly to the behaviour of the denatured polypeptide chain as an excluded volume polymer.

Conformational modification of muscle phosphofructokinase from Jaculus orientalis upon ligand binding.

Phosphofructokinase from Jaculus orientalis muscle is an allosteric enzyme regulated by substrates and nucleotide effectors. The conformational modifications upon ligand binding were probed by UV difference spectra and reactivities of thiol groups towards dithiobisnitrobenzoate and N-ethylmaleimide. The binding of Fru-6-P induced significant perturbations in the environment of the aromatic residues and buried the most reactive on the three accessible cysteines per protomer. The same effect on thiol reactivity was observed upon binding of the activator AMP. Various perturbations of both difference spectra and thiol reactivity were detected in the presence of either Mg-ATP, an allosteric inhibitor, or Mg-ITP which is not an effector.

The effect of phosphate on the unfolding-refolding of phosphoglycerate kinase induced by guanidine hydrochloride.

Phosphate ions were found to stabilize the native structure of phosphoglycerate kinase without modifying the folding pathway. The transition curves obtained from different signals: enzyme activity, ellipticity at 220 nm and fluorescence intensity at 336 nm (excitation at 292 nm) are shifted to smaller guanidine hydrochloride cm values in the absence of phosphate. The kinetic characteristics are qualitatively similar, unfolding rate constants being slightly smaller in the presence of phosphate. The mechanism by which the native structure of phosphoglycerate kinase is stabilized by phosphate probably occurs upon specific phosphate binding to the nucleotide beta- or gamma-phosphate binding site of nucleotides.

Cold denaturation of yeast phosphoglycerate kinase: which domain is more stable?

Under destabilising conditions both heat and cold denaturation of yeast phosphoglycerate kinase (PGK) can be observed. According to previous interpretation of experimental data and theoretical calculations, the C-terminal domain should be more stable than the N-terminal domain at all temperatures. We report on thermal unfolding experiments with PGK and its isolated domains, which give rise to a revision of this view. While the C-terminal domain is indeed the more stable one on heating, it reveals lower stability in the cold. These findings are of importance, because PGK has been frequently used as a model for protein folding and mutual domain interactions.

Role of the C-terminal extremities of the smooth muscle myosin heavy chains: implication for assembly properties.

The two light meromyosin isoforms from rabbit smooth muscle were prepared as recombinant proteins in Escherichia coli. These species which differed only by their C-terminal extremity showed the same circular dichroism spectra and endotherms in measurements of differential scanning calorimetry. Their solubility properties were different at pH 7.0 in the absence of monovalent salts. Their paracrystals formed at low pH differed by their aspect and number. These data suggest a role for the C-terminal extremity of myosin heavy chains in the assembly of myosin molecules in filaments and consequently in the contractility of smooth muscles.

Flexibility and folding of phosphoglycerate kinase.

Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.

How random is a highly denatured protein?

There has been renewed interest in determining the physicochemical properties of denatured states of proteins. In many denatured states there is evidence for the existence of nonrandom configurational distributions. Here we examine the small-angle neutron scattering profile of yeast phosphoglycerate kinase in the native state and in highly denaturing conditions. We show that the denatured protein scattering profile can be interpreted using a model developed for synthetic polymers in which the chain behaves as a random coil in a good solvent, i.e. with excluded volume interactions. The implications of this result for our appreciation of the protein folding process are discussed.

Rapid adaptive evolution of the tumor suppressor gene Pten in an insect lineage.

The Pten gene was initially identified in humans as a tumor suppressor. It has since been shown to play important roles in the control of cell size, cell motility, apoptosis, and organ size, and it has also been implicated in aging. Pten is highly conserved among organisms as diverse as nematodes, insects, and vertebrates. In contrast, a phylogenetic analysis by maximum likelihood of a 133-amino acid region showed an average nonsynonymous-to-synonymous rate ratio of 10.4 for Pten in the lineage leading to parasitoid wasps of the Nasonia genus, indicating very strong positive selection. A previous study identified Pten as a potential QTL candidate gene for differences in male wing size in Nasonia. Most of the amino acid replacements that occurred in the Nasonia lineage cluster in a small region of the protein surface, suggesting that they might be involved in an interaction between Pten and another protein. The phenotypic changes due to Pten are not yet known, although it is not associated with known differences in male wing size. Introgression of Pten from one species to another does affect longevity, but a causal relationship is not established.

Evidence for intermediates during unfolding and refolding of a two-domain protein, phage T4 lysozyme: equilibrium and kinetic studies.

Equilibrium and kinetic studies of the unfolding-refolding of phage T4 lysozyme induced by guanidine hydrochloride are reported. Tryptophan fluorescence and circular dichroism (CD) were used as observables. Several results indicated the existence of intermediates in the unfolded-folded transition, including (1) the noncoincidence of the transition observed by fluorescence and CD, (2) the asymmetry of the transition recorded by CD, and (3) triphasic kinetics, followed by both observables, accounting for the forward and reverse processes. Our data were inconsistent with an independent unfolding or refolding of each domain. They indicated the existence of residual structured regions particularly resistant to denaturant, which involved one or more of the three tryptophans located in the C-terminal domain. Kinetic analysis has made it possible to propose a minimum pathway corresponding to a sequential refolding, with dead-end species arising from an intermediate. We compared our data with the experimental results of Elwell and Schellman [Elwell, M., & Schellman, J.A. (1975) Biochim. Biophys. Acta 386, 309-313] and the theoretical analysis of B. Maigret (unpublished results) and proposed that the C-terminal domain refolds first, after which the overall structure can be formed and stabilized.

Structure and functional complementation of engineered fragments from yeast phosphoglycerate kinase.

Previous studies have shown that, although the isolated structural domains of yeast phosphoglycerate kinase recover a quasi-native structure in vitro as well as in vivo, they do not reassociate nor generate a functional enzyme. The aim of this work was first to study the folding of complementary fragments different from structural domains and second to determine the requirements for their reassociation and functional complementation. The method used for producing rigorously defined fragments consists of the introduction of a unique cysteinyl residue in the protein followed by a specific cleavage by 5'5'-dithiobis(2-nitrobenzoate)/potassium cyanide at this residue. Two pairs of complementary fragments were thus obtained, 1-96/97-415 and 1-248/249-415. The structure and stabilities of the different fragments were studied. The short fragments, i.e. 1-96 and 249-415 were found to contain some secondary structure, but to have a low stability. Each large fragment has a high structural content and a stability close to that of the corresponding domain. In contrast to that observed with the isolated domains, a weak but significant complementation was observed for the two pairs of fragments; the pair of fragments 1-248/249-415 recovered 8% of the activity of the native enzyme upon complementation. An independent refolding of the complementary fragments before reassociation decreased the yield of complementation for the pair of fragments 1-96/97-415, but did not affect the complementation for the other pair (1-248/249-415). From the present data and previous work on the isolated domains, it appears that the correct folding of the isolated fragments is not a prerequisite for their complementation.

Detection of intermediates in the unfolding transition of phosphoglycerate kinase using limited proteolysis.

The accessibility of peptide bonds to cleavage by Staphylococcus aureus V8 protease bound on a Sepharose matrix was used as a conformational probe in the study of the unfolding-folding transition of phosphoglycerate kinase induced by guanidine hydrochloride. It was shown that the protein is resistant to proteolysis below a denaturant concentration of 0.4 M. The transition curve, determined by susceptibility toward proteolysis, was similar to that obtained following the enzyme activity [Betton et al. (1984) Biochemistry 23, 6654-6661]. Proteolysis under conditions where the folding intermediates are more populated, i.e., 0.7 M Gdn.HCl, gave two major fragments of Mr 25K and 11K, respectively. The 25K polypeptide fragment was identified as the carboxy-terminal domain. Its conformation was similar to that of a folding intermediate trapped at a critical concentration of denaturant, and in this form, it was not able to bind nucleotide substrates [Mitraki et al. (1987) Eur. J. Biochem. 163, 29-34]. From the present data and those previously reported, we concluded that the intermediate detected on the folding pathway of phosphoglycerate kinase has a partially folded carboxy-terminal domain and an unfolded amino-terminal domain.

Evidence for residual structures in an unfolded form of yeast phosphoglycerate kinase.

The unfolding-refolding transition of phosphoglycerate kinase followed by steady-state fluorescence has clearly shown the existence of a hyperfluorescent form [Missiakas et al. (1990) Biochemistry 29, 8683-8689]. In order to determine the contribution of each of the two tryptophans to the fluorescence properties of the enzyme in the equilibrium transition and to characterize the hyperfluorescent form, two single tryptophan mutants in which tryptophans 308 and 333 were replaced by a tyrosine and a phenylalanine, respectively, were constructed. Neither the catalytic nor the physicochemical properties of the enzyme are significantly altered by these mutations. The unfolding-refolding transitions were studied using circular dichroism and tryptophan fluorescence emission. Both tryptophans contribute to the hyperfluorescence observed in the first transition. For guanidine hydrochloride concentrations higher than 0.9 M, it clearly appears that the second transition results from a further unfolding. It is accompanied by a decrease in fluorescence intensity and a 5 mm red shift of the maximum emission wavelength. When the unfolding is induced by urea, the end of the transition corresponds to the hyperfluorescent state. Further addition of guanidine hydrochloride induces complete unfolding. These results suggest the presence of residual microstructures around tryptophan 308 and tryptophan 333 in the hyperfluorescent state. The characterization of these clusters and their contribution as starting structures in the folding process are now under investigation.

Role of loops in the folding and stability of yeast phosphoglycerate kinase.

Yeast phosphoglycerate kinase (yPGK) is a monomeric two domain protein used as folding model representative of large proteins. We inserted short unstructured sequences (four Gly or four Thr) into the connections between secondary structure elements and studied the consequences of these insertions on the folding process and stability of yPGK. All the mutated proteins can refold efficiently. The effect per residue on stability is larger for the first inserted residue. Insertion in two long betaalpha loops (at residue positions 71 and 129) is more destabilizing than an insertion in a short alphabeta loop (at residue position 89) located on the opposite side of the N-terminal domain. The effect on stability is mainly due to a large increase of the unfolding rate rather than a decrease of the folding rate. This suggests that these connections between secondary structure elements do not play an active role in directing the folding process. Insertion into the short alphabeta loop (position 89) has limited effects on stability and results in the detection of a kinetic phase not previously seen with the wild-type protein, suggesting that insertions in this particular loop do qualitatively affect the folding process without a large effect on folding efficiency. For the two long betaalpha loops (positions 71 and 129) located in the inner surface of the N-terminal domain, the effects on stability are possibly associated with decoupling of the two domains as observed by differential scanning calorimetry during thermal unfolding.

Characterization of the denatured states distribution of neocarzinostatin by small-angle neutron scattering and differential scanning calorimetry.

The denatured states of a small globular protein, apo-neocarzinostatin (NCS), have been characterized using several techniques. Structural properties were investigated by optical spectroscopy techniques and small-angle neutron scattering (SANS), as a function of guanidinium chloride (GdmCl) concentration. SANS experiments show that in heavy water, the protein keeps its native size at GdmCl concentrations below 2.5 M. A sharp transition occurs at about 3.6 M GdmCl, and NCS behaves like an excluded volume chain above 5 M. The same behavior is observed in deuterated buffer by fluorescence and circular dichroism measurements. For the H(2)O buffer, the transition occurs with lower concentration of denaturant, the shift being about 0.6 M. 8-Anilino-1-naphthalenesulfonate (ANS) was used as a hydrophobic fluorescent probe for studying the early stages of protein unfolding. Protein denaturation modifies the fluorescence intensity of ANS, a maximum of intensity being detected close to 2 M GdmCl in hydrogenated buffer, which shows the existence of at least one intermediate state populated at the beginning of the unfolding pathway. Differential scanning calorimetry (DSC) was used to obtain thermodynamic values for NCS denaturation. The melting curves recorded between 20 and 90 degrees C in the presence of various GdmCl concentrations (0-3 M) cannot be explained by a simple two-state model. Altogether, the data presented in this paper suggest that before unfolding the protein explores a distribution of states which is centered around compact states at denaturant concentrations below 2 M in H(2)O, and then shifts to less structured states by increasing denaturant concentrations.