Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements

The conformational features of native and mutant forms of sperm-whale apomyoglobin (apoMb) at neutral pH were probed by limited proteolysis experiments utilizing up to eight proteases of different substrate specificities. It was shown that all proteases selectively cleave apoMb at the level of chain segment 82-94 (HEAELKPLAQSHA), encompassing helix F in the X-ray structure of the holo form of the native protein; for example, thermolysin cleaves the Pro 88-Leu 89 peptide bond. These results indicate that helix F is highly flexible or largely disrupted in apoMb. Because helix F contains the helix-breaking Pro 88 residue, we propose that helix F is kept in place in the native holo protein by a variety of helix-heme stabilizing interactions. To modulate the stability of helix F, the Pro88Ala and Pro88Gly mutants were prepared by site-directed mutagenesis, and their conformational properties investigated by both far-UV circular dichroism spectroscopy and limited proteolysis. The helix content of the Pro88Ala mutant was somewhat enhanced with respect to that of both native and Pro88Gly mutant, as expected from the fact that Ala is the strongest helix inducer among the 20 amino acid residues. The rate of limited proteolysis of the three apoMb variants by thermolysin and proteinase K was in the order native > Pro88Gly > Pro88Ala, in agreement with the scale of helix propensity of Ala, Gly, and Pro. The possible role of the flexible/unfolded chain segment 82-94 for the function and fate of apoMb at the cellular level is discussed.     

High-pressure denaturation of apomyoglobin

The pressure denaturation of wild type and mutant apomyoglobin (apoMb) was investigated using a high-pressure, high-resolution nuclear magnetic resonance and high-pressure fluorescence techniques. Wild type apoMb is resistant to pressures up to 80 MPa, and denatures to a high-pressure intermediate, I(p), between 80 and 200 MPa. A further increase of pressure to 500 MPa results in denaturation of the intermediate. The two tryptophans, both in the A helix, remain sequestered from solvent in the high-pressure intermediate, which retains some native NOESY cross peaks in the AGH core as well as between F33 and F43. High-pressure fluorescence shows that the tryptophans remain inaccessible to solvent in the I(p) state. Thus the high-pressure intermediate has some structural properties in common with the apoMb I(2) acid intermediate. The resistance of the AGH core to pressures up to 200 MPa provides further evidence that the intrinsic stability of these alpha-helices is responsible for their presence in a number of equilibrium intermediates as well as in the earliest kinetic folding intermediate. Mutations in the AGH core designed to disrupt packing by burying a charge or increasing the size of a hydrophobic residue significantly perturbed the unfolding of native apoMb to the high-pressure intermediate. The F123W and S108L mutants both unfolded at lower pressures, while retaining some resistance to pressures below 50 MPa. The charge burial mutants, A130K and S108K, are not stable at very low pressures and both denature to the intermediate by 100 MPa, half of the pressure required for wild type apoMb. Thus a similar intermediate state is created independent of the method of perturbation, and mutations have similar effects on native state destabilization for both methods of denaturation. These data suggest that equilibrium intermediates that can be formed through different means are likely to resemble a kinetic intermediate.     

Equilibrium and pressure-jump relaxation studies of the conformational transitions of P13MTCP1

The conformational transitions of a small oncogene product, p13(MTCP1), have been studied by high-pressure fluorescence of the intrinsic tryptophan emission and high-pressure 1D and 2D 1H-15N NMR. While the unfolding transition monitored by fluorescence is cooperative, two kinds of NMR spectral changes were observed, depending on the pressure range. Below approximately 200 MPa, pressure caused continuous, non-linear shifts of many of the 15N and 1H signals, suggesting the presence of an alternate folded conformer(s) in rapid equilibrium (tau相似文献   

Native state energetics of the Src SH2 domain: evidence for a partially structured state in the denatured ensemble

We have defined the free-energy profile of the Src SH2 domain using a variety of biophysical techniques. Equilibrium and kinetic experiments monitored by tryptophan fluorescence show that Src SH2 is quite stable and folds rapidly by a two-state mechanism, without populating any intermediates. Native state hydrogen-deuterium exchange confirms this two-state behavior; we detect no cooperative partially unfolded forms in equilibrium with the native conformation under any conditions. Interestingly, the apparent stability of the protein from hydrogen exchange is 2 kcal/mol greater than the stability determined by both equilibrium and kinetic studies followed by fluorescence. Native-state proteolysis demonstrates that this "super protection" does not result from a deviation from the linear extrapolation model used to fit the fluorescence data. Instead, it likely arises from a notable compaction in the unfolded state under native conditions, resulting in an ensemble of conformations with substantial solvent exposure of side chains and flexible regions sensitive to proteolysis, but backbone amides that exchange with solvent approximately 30-fold slower than would be expected for a random coil. The apparently simple behavior of Src SH2 in traditional unfolding studies masks the significant complexity present in the denatured-state ensemble.     

Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.     

Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis

The structure of a protein molecule consists of both rigid and flexible sections to satisfy the demands for stability and catalysis. Because the flexibility of a protein segment is indispensable for a proteolytic attack, limited proteolysis is a superb tool to analyse both confined local fluctuations and global unfolding events in proteins. While the identification of the primary cleavage products allows the assignment of the flexible regions to the primary structure, the kinetics of proteolytic degradation enables differentiation between local fluctuations in the native protein molecule and the global unfolding process during denaturation. Modifications of the amino acid sequence in the concerned regions can tune proteolytic susceptibility and alter protein stability. In the present paper, we summarise our results on native-state and unfolded-state proteolysis of ribonuclease A (RNase A) and the effect of mutations in the detected flexible regions on the stability and unfolding of the RNase A molecule.     

Local fluctuations vs. global unfolding of proteins investigated by limited proteolysis

The structure of a protein molecule consists of both rigid and flexible sections to satisfy the demands for stability and catalysis. Because the flexibility of a protein segment is indispensable for a proteolytic attack, limited proteolysis is a superb tool to analyse both confined local fluctuations and global unfolding events in proteins. While the identification of the primary cleavage products allows the assignment of the flexible regions to the primary structure, the kinetics of proteolytic degradation enables differentiation between local fluctuations in the native protein molecule and the global unfolding process during denaturation. Modifications of the amino acid sequence in the concerned regions can tune proteolytic susceptibility and alter protein stability. In the present paper, we summarise our results on native-state and unfolded-state proteolysis of ribonuclease A (RNase A) and the effect of mutations in the detected flexible regions on the stability and unfolding of the RNase A molecule.     

Denatured states of human carbonic anhydrase II: an NMR study of hydrogen/deuterium exchange at tryptophan-indole-H(N) sites

Hydrogen/deuterium (H/D) exchange measurements in low and moderate concentrations of GuHCI were conducted on the side chain H(N) atoms of the seven tryptophans of pseudo wild-type human carbonic anhydrase II. Tryptophans 5, 16 and 245, situated in or close to the N-terminal domain were found to have little protection against exchange. The H/D exchange results for Trp-123, Trp-192 and Trp-209 showed that a previously identified molten globule and the native state gave a similar protection against exchange. Global unfolding of the protein is necessary for the efficient exchange at Trp-97, which is located in the central part of the beta-sheet.     

Fluctuations of protein structure as expressed in the distribution of hydrogen exchange rate constants

Hydrogen exchange kinetics of proteins provide information about the dynamics of their structure. The interpretation of experiments has been limited by difficulty in identifying individual rate constants. In order to invert the kinetic data, and to gain information about the individual reacting sites, we introduce a Laplace inversion technique and obtain the distribution function of rate constants by which the intricate reaction proceeds. A series of carefully overlapped experiments were performed on lysozyme at 25°C, an exchange profile obtained, and a distribution function extracted. This function was composed of a product of two terms, indicating two parallel pathways. The first, a power-law term, was attributed to exchange from the native state. This part of the distribution function thus describes the scope of the conformational fluctuations in proteins at constant temperature and pressure. The second, an exponential, was seen to be associated with the pathway involving thermal unfolding and subsequent free exchange with the solvent. The influence of trichlorethanol and glycerol on the total distribution function was measured. Trichloroethanol selectively increased the contribution from the thermal unfolding pathway, whereas glycerol, besides decreasing this type of contribution, increased the width of the distribution function attributed to structural fluctuations.     

Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme.

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.     

Changes in the apomyoglobin folding pathway caused by mutation of the distal histidine residue

Factors governing the folding pathways and the stability of apomyoglobin have been examined by replacing the distal histidine at position 64 with phenylalanine (H64F). Acid and urea-induced unfolding experiments using CD and fluorescence techniques reveal that the mutant H64F apoprotein is significantly more stable than wild-type apoMb. Kinetic refolding studies of this variant also show a significant difference from wild-type apoMb. The amplitude of the burst phase ellipticity in stopped-flow CD measurements is increased over that of wild-type, an indication that the secondary structure content of the earliest kinetic intermediate is greater in the mutant than in the wild-type protein. In addition, the overall rate of folding is markedly increased. Hydrogen exchange pulse labeling was used to establish the structure of the initial intermediate formed during the burst phase of the H64F mutant. NMR analysis of the samples obtained at different refolding times indicates that the burst phase intermediate contains a stabilized E helix as well as the A, G, and H helices previously found in the wild-type kinetic intermediate. Replacement of the polar distal histidine residue with a nonpolar residue of similar size and shape appears to stabilize the E helix in the early stages of folding due to improved hydrophobic packing. The presence of a hydrophilic histidine at position 64 thus exacts a price in the stability and folding efficiency of the apoprotein, but this residue is nevertheless highly conserved among myoglobins due to its importance in function.     

Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the importance of interactions between marginally stable elements of secondary structure in promoting the tertiary contacts of a native protein. Considering that apoMb has been extensively used as a paradigm in protein folding studies for the past few decades, the novel fragment complementing system of apoMb here described appears to be very useful for investigating the initial as well as late events in protein folding.     

Structural characterization of the molten globule state of apomyoglobin by limited proteolysis and HPLC-mass spectrometry

A method to characterize the structural conformation of an acidic molten globule apomyoglobin (apoMb) at pH 4.2 was developed using limited proteolysis and HPLC-mass spectrometry (HPLC-MS). Endoproteinase Glu-C, which has a double maximum activity at pH 4.0 and pH 7.8 toward glutamic acid (Glu), was used as a proteolytic enzyme. Using this method enabled us to compare the proteolytic cleavages of native apoMb (at pH 8.0) and molten globule (at pH 4.2) directly. Only the first cleavage event in each molecule was considered as reflecting original structural information since the original structure of the protein can be altered after the fist cleavage. Structural changes of apoMb in various pH conditions were studied here to elucidate the local helicity of molten globule apoMb. Among 13 Glu sites, only Glu83 and Glu85 in the F-helix were cleaved at pH 8.0, which confirms that only helix F is frayed upon removal of heme group. At acidic molten globule state, rapid cleavages at Glu38, Glu52, Glu54, Glu85, and Glu148 were detected, while the remaining eight sites were protected. Glu6 and Glu18 in the A-helix, and Glu105 in the G-helix were protected due to the helicity of the secondary structures. The cleavage at Glu38 and the protection at Glu41 in the C-helix indicate that the first half of the C-helix is frayed and the second half of the C-helix is structured. Cleavage at both Glu52 and Glu54 in the D-helix proves that the D-helix is disordered. The N-terminal end of the E-helix at Glu59 was protected, and the beginning of the F-helix was protected by aid of the pH-induced C-cap of the E-helix. The cleavage at Glu148 in H suggests that the C-terminal end of the H-helix is disordered. The A-helix and the first half of the B-helix were highly stable.     

The Conformational State of Apomyoglobin in the Presence of Phospholipid Vesicles at Neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near and far UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Conformational status of apomyoglobin in the presence of phospholipid vesicles at neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1

Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the molecule. The loss of enzyme activity was much earlier than the structural loss of the molecule during the course of unfolding, with the midpoint concentrations being 4.5 and 7.0 M for activity and spectroscopic measurements, respectively. The equilibrium unfolding transition could be adequately fitted to a three-state, two-step model (N left arrow over right arrow I left arrow over right arrow U) and the intermediate fraction was maximally populated at 6.3 M urea. The free energy change (DeltaG(H(2)O)) for the unfolding of native (N) to intermediate (I) was 14.2 +/- 0.28 kcal/mol and for the intermediate to the unfolded state (U) was 11.9 +/- 0.12 kcal/mol. The ANS binding measurements as a function of urea concentration indicated that the maximum binding of ANS was in 6.3 M urea due to the exposure of hydrophobic groups; this intermediate showed significant amount of tertiary structure and retained nearly 60% of secondary structure. The limited proteolysis measurements showed that the initiation of unfolding was from the C-terminal domain. Thus, the stable intermediate observed could be the C-terminal domain unfolded with exposed hydrophobic domain-domain interface. Limited proteolysis experiments during refolding process suggested that the intermediate refolded prior to completely unfolded LOX1. These results confirmed the role of cysteine residues and domain-domain interactions in the reversible unfolding of LOX1. This is the first report of the reversible unfolding of a very large monomeric, multi-domain protein, which also has a prosthetic group.     

The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure.

The heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75 degrees C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety. In contrast, the mature form, which lacks the propeptide composed of 91 amino acid residues, showed more complex behaviour: differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition. These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarboxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding. The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding.     

Structural stability of human butyrylcholinesterase under high hydrostatic pressure

Human butyrylcholinesterase is a nonspecific enzyme of clinical, pharmacological and toxicological significance. Although the enzyme is relatively stable, its activity is affected by numerous factors, including pressure. In this work, hydrostatic pressure dependence of the intrinsic tryptophan fluorescence in native and salted human butyrylcholinesterase was studied up to the maximum pressure at ambient temperature of about 1200 MPa. A correlated large shift toward long wavelengths and broadening observed at pressures between 200 and 700 MPa was interpreted as due to high pressure-induced denaturation of the protein, leading to an enhanced exposure of tryptophan residues into polar solvent environment. This transient process in native butyrylcholinesterase presumably involves conformational changes of the enzyme at both tertiary and secondary structure levels. Pressure-induced mixing of emitting local indole electronic transitions with quenching charge transfer states likely describes the accompanying fluorescence quenching that reveals different course from spectral changes. All the pressure-induced changes turned irreversible after passing a mid-point pressure of about 400 ± 50 MPa. Addition of either 0.1 M ammonium sulphate (a kosmotropic salt) or 0.1 M lithium thiocyanate (a chaotropic salt) to native enzyme similarly destabilized its structure.     

Increased stability upon heptamerization of the pore-forming toxin aerolysin

Aerolysin is a bacterial pore-forming toxin that is secreted as an inactive precursor, which is then processed at its COOH terminus and finally forms a circular heptameric ring which inserts into membranes to form a pore. We have analyzed the stability of the precursor proaerolysin and the heptameric complex. Equilibrium unfolding induced by urea and guanidinium hydrochloride was monitored by measuring the intrinsic tryptophan fluorescence of the protein. Proaerolysin was found to unfold in two steps corresponding to the unfolding of the large COOH-terminal lobe followed by the unfolding of the small NH(2)-terminal domain. We show that proaerolysin contains two disulfide bridges which strongly contribute to the stability of the toxin and protect it from proteolytic attack. The stability of aerolysin was greatly enhanced by polymerization into a heptamer. Two regions of the protein, corresponding to amino acids 180-307 and 401-427, were identified, by limited proteolysis, NH(2)-terminal sequencing and matrix-assisted laser desorption ionization-time of flight, as being responsible for stability and maintenance of the heptamer. These regions are presumably involved in monomer/monomer interactions in the heptameric protein and are exclusively composed of beta structure. The stability of the aerolysin heptamer is reminiscent of that of pathogenic, fimbrial protein aggregates found in a variety of neurodegenerative diseases.     

Thermal effects in stretching of Go-like models of titin and secondary structures

The effect of temperature on mechanical unfolding of proteins is studied using a Go-like model with a realistic contact map and Lennard-Jones contact interactions. The behavior of the I27 domain of titin and its serial repeats is contrasted to that of simple secondary structures. In all cases, thermal fluctuations accelerate the unraveling process, decreasing the unfolding force nearly linearly at low temperatures. However, differences in bonding geometry lead to different sensitivity to temperature and different changes in the unfolding pattern. Due to its special native-state geometry, titin is much more thermally and elastically stable than the secondary structures. At low temperatures, serial repeats of titin show a parallel unfolding of all domains to an intermediate state, followed by serial unfolding of the domains. At high temperatures, all domains unfold simultaneously, and the unfolding distance decreases monotonically with the contact order, that is, the sequence distance between the amino acids that form the native contact.