A pH dependence study on the unfolding and refolding of apoazurin

Azurin, a small blue copper protein from the bacterial species Pseudomonas aeruginosa, is mostly a β-sheet protein arranged into a single domain. Previous folding studies have shown that the equilibrium denaturation of the holoprotein follows a two-state process; however, upon removal of the copper, the denaturation had been reported to follow a three-state process. The two unfolding transitions measured for apoazurin had been thought to arise from two different folding domains. However, in the present work, we found that the denaturation of apoazurin occurs over a single transition and we determined the folding free energy to be −27.8±2.4 kJ mol⁻¹. From this measurement along with measurements previously reported for the unfolding of the holoazurin, we were able to determine that Cu(II) and Cu(I) stabilize the native structure by 25.1±6.9 kJ/mol and 12.9±8.1 kJ/mol, respectively. It is our contention that the second transition displayed in the denaturation curves previously reported for apoazurin arise from protein heterogeneity—in particular, from the presence of Zn(II) azurin. We extended our investigation into the denaturation of Zn(II) azurin at pH 6.0 and 7.5. The equilibrium denaturation studies show that the zinc ion significantly stabilizes the native-state structure at pH 7.5 and very little at the lower pH. We attribute the decrease in the stabilizing effect of the zinc ion with decreasing pH to the protonation of two histidinyl side chains. When protonated the ligands, His 46 and His 117, are incapable of binding a metal ion. Further, comparing the denaturation curves of Zn(II) azurin measured by circular dichroism with those measured by fluorescence indicates that the denaturation of Zn(II) azurin is far less simple than the denaturation of apoazurin.     

Temperature and pressure effects on C112S azurin: Volume,expansivity, and flexibility changes

The pressure‐induced unfolding of the mutant C112S azurin from Pseudomonas aeruginosa was monitored both under steady state and dynamic conditions. The unfolding profiles were obtained by recording the spectral shift of the fluorescence emission as well as by phosphorescence intensity measurements. We evaluated the difference in free energy, ΔG, as a function of pressure and temperature. The dependence of ΔG on temperature showed concave profile at all pressures studied. A positive heat capacity change of about 4.3 kJ mol^?1 deg^?1 fitted all the curves. The volume change of the reaction showed a moderate dependence on temperature when compared with other proteins previously studied. The kinetic activation parameters (ΔV*, ΔH*, ΔS*) were obtained from upward and downward pressure‐jump experiments and used to characterize the volumetric and energetic properties of the transition state between native and unfolded protein. Our findings suggest that the folding and unfolding reaction paths passed through different transition states. The change in the phosphorescence lifetime with pressure pointed out that pressure‐induced unfolding occurred within two steps: the first leading to an increased protein flexibility, presumably caused by water penetration into the protein. Major structural changes of the tryptophan environment occurred in a second step at higher pressures. Proteins 2014; 82:1787–1798. © 2014 Wiley Periodicals, Inc.     

Reversible Unfolding of a Thermophilic Membrane Protein in Phospholipid/Detergent Mixed Micelles

Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue α-helical membrane protein that is involved in transporting Cu⁺ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with ΔG_w° = 12.9 kJ mol^− 1, m = 4.1 kJ mol^− 1 M^− 1, C_m = 3 M, and ΔCp_w° = 0.93 kJ mol^− 1 K^− 1. These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.     

Refolding of the hyperthermophilic protein Ssh10b involves a kinetic dimeric intermediate

The α/β-mixed dimeric protein Ssh10b from the hyperthermophile Sulfolobus shibatae is a member of the Sac10b family that is thought to be involved in chromosomal organization or DNA repair/recombination. The equilibrium unfolding/refolding of Ssh10b induced by denaturants and heat was fully reversible, suggesting that Ssh10b could serve as a good model for folding/unfolding studies of protein dimers. Here, we investigate the folding/unfolding kinetics of Ssh10b in detail by stopped-flow circular dichroism (SF-CD) and using GdnHCl as denaturant. In unfolding reactions, the native Ssh10b turned rapidly into fully unfolded monomers within the stopped-flow dead time with no detectable kinetic intermediate, agreeing well with the results of equilibrium unfolding experiments. In refolding reactions, two unfolded monomers associate in the burst phase to form a dimeric intermediate that undergoes a further, slower, first-order folding process to form the native dimer. Our results demonstrate that the dimerization is essential for maintaining the native tertiary interactions of the protein Ssh10b. In addition, folding mechanisms of Ssh10b and several other α/β-mixed or pure β-sheet proteins are compared.     

Spectroscopic characterization and intramolecular electron transfer processes of native and type 2 Cu-depleted nitrite reductases

 Native nitrite reductases (NIRs) containing both type 1 and 2 Cu ions and type 2 Cu-depleted (T2D) NIRs from three denitrifying bacteria (Achromobacter cycloclastes IAM 1013, Alcaligenes xylosoxidans NCIB 11015, and Alcaligenes xylosoxidans GIFU 1051) have been characterized by electronic absorption, circular dichroism, and electron paramagnetic resonance spectra. The characteristic visible absorption spectra of these NIRs are due to the type 1 Cu centers, while the type 2 Cu centers hardly contribute in the same region. The intramolecular electron transfer (ET) process from the type 1 Cu to the type 2 Cu in native NIRs has been observed as the reoxidation of the type 1 Cu(I) center by pulse radiolysis, whereas no type 1 Cu in T2D NIRs exhibits the same reoxidation. The ET process obeys first-order kinetics, and observed rate constants are 1400–1900 s^–1 (t_1/2 = ca. 0.5 ms) at pH 7.0. In the presence of nitrite, the ET process also obeys first-order kinetics, with rate constants decreased by factors of 1/12–1/2 at the same pH. The redox potential of the type 2 Cu site is estimated to be +0.24 - +0.28 V, close to that of the type 1 Cu site. Nitrate and azide ions bound to the type 2 Cu site change the redox potential. Nitrite also would shift the redox potential of the type 2 Cu by coordination, and hence the intramolecular ET rate constant is decreased. Pulse radiolysis experiments on T2D NIRs in the presence of nitrite demonstrate that the type 1 Cu(I) site is slowly oxidized with a first-order rate constant of 0.03 s^–1 at pH 7.0, suggesting that nitrite bound to the protein accepts an electron from the type 1 Cu. This result is in accord with the finding that T2D NIRs show enzymatic activities, although they are lower than those of the native enzymes. Received: 9 July 1996 / Accepted: 30 January 1997     

Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement

Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this α–β parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native α-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin.     

Spatially resolved free-energy contributions of native fold and molten-globule-like Crambin

The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger but almost compensating enthalpic and entropic contributions. The balance can therefore easily be shifted by an external disturbance, such as a mutation of a single amino acid or a change of temperature, in which case the protein unfolds. Effects such as cold denaturation, in which a protein unfolds because of cooling, provide evidence that proteins are strongly stabilized by the solvent entropy contribution to the free-energy balance. However, the molecular mechanisms behind this solvent-driven stability, their quantitative contribution in relation to other free-energy contributions, and how the involved solvent thermodynamics is affected by individual amino acids are largely unclear. Therefore, we addressed these questions using atomistic molecular dynamics simulations of the small protein Crambin in its native fold and a molten-globule-like conformation, which here served as a model for the unfolded state. The free-energy difference between these conformations was decomposed into enthalpic and entropic contributions from the protein and spatially resolved solvent contributions using the nonparametric method Per|Mut. From the spatial resolution, we quantified the local effects on the solvent free-energy difference at each amino acid and identified dependencies of the local enthalpy and entropy on the protein curvature. We identified a strong stabilization of the native fold by almost 500 kJ mol⁻¹ due to the solvent entropy, revealing it as an essential contribution to the total free-energy difference of (53 ± 84) kJ mol⁻¹. Remarkably, more than half of the solvent entropy contribution arose from induced water correlations.     

Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate

To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying β-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of β-glucosidase at 90°C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90°C with an overall ΔG° of ∼ 20 kcal mol⁻¹. The high temperatures needed to chemically denature P. furiosus β-glucosidase and the large ΔG° of unfolding at high temperatures shows this to be one of the most stable proteins yet characterized. Unfolding proceeds via a three-state pathway that includes a stable intermediate species. Stability of the native and intermediate forms is concentration dependent, and we have identified a dimeric assembly intermediate using high temperature native gel electrophoresis. Based on this data, we have developed a model for the denaturation of β-glucosidase in which the tetramer dissociates to partially folded dimers, followed by the coupled dissociation and denaturation of the dimers to unfolded monomers. The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding.     

Reversible two-step unfolding of heme–human serum albumin: a 1H-NMR relaxometric and circular dichroism study

Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme–HSA has been investigated by ¹H-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate (thus fully saturating all available fatty acid binding sites in serum heme–albumin), 1.0 M guanidinium chloride induces some unfolding of heme–HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K _d = (1.2 ± 0.1) × 10⁻⁶ M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K _d = (3.4 ± 0.1) × 10⁻⁵ M]. Further addition of guanidinium chloride (up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme–HSA in the presence of myristate.     

Spatially resolved free-energy contributions of native fold and molten-globule-like Crambin

The folding stability of a protein is governed by the free-energy difference between its folded and unfolded states, which results from a delicate balance of much larger but almost compensating enthalpic and entropic contributions. The balance can therefore easily be shifted by an external disturbance, such as a mutation of a single amino acid or a change of temperature, in which case the protein unfolds. Effects such as cold denaturation, in which a protein unfolds because of cooling, provide evidence that proteins are strongly stabilized by the solvent entropy contribution to the free-energy balance. However, the molecular mechanisms behind this solvent-driven stability, their quantitative contribution in relation to other free-energy contributions, and how the involved solvent thermodynamics is affected by individual amino acids are largely unclear. Therefore, we addressed these questions using atomistic molecular dynamics simulations of the small protein Crambin in its native fold and a molten-globule-like conformation, which here served as a model for the unfolded state. The free-energy difference between these conformations was decomposed into enthalpic and entropic contributions from the protein and spatially resolved solvent contributions using the nonparametric method Per|Mut. From the spatial resolution, we quantified the local effects on the solvent free-energy difference at each amino acid and identified dependencies of the local enthalpy and entropy on the protein curvature. We identified a strong stabilization of the native fold by almost 500 kJ mol⁻¹ due to the solvent entropy, revealing it as an essential contribution to the total free-energy difference of (53 ± 84) kJ mol⁻¹. Remarkably, more than half of the solvent entropy contribution arose from induced water correlations.     

Methionine-121 coordination determines metal specificity in unfolded<Emphasis Type="Italic"> Pseudomonas aeruginosa</Emphasis> azurin

Pseudomonas aeruginosa azurin binds copper so tightly that it remains bound even upon polypeptide unfolding. Copper can be substituted with zinc without change in protein structure, and also in this complex the metal remains bound upon protein unfolding. Previous work has shown that native-state copper ligands Cys112 and His117 are two of at least three metal ligands in the unfolded state. In this study we use isothermal titration calorimetry and spectroscopic methods to test if the native-state ligand Met121 remains a metal ligand upon unfolding. From studies on a point-mutated version of azurin (Met121Ala) and a set of model peptides spanning the copper-binding C-terminal part (including Cys112, His117 and Met121), we conclude that Met121 is a metal ligand in unfolded copper-azurin but not in the case of unfolded zinc-azurin. Combination of unfolding and metal-titration data allow for determination of copper (Cu(II) and Cu(I)) and zinc affinities for folded and unfolded azurin polypeptides, respectively.     

Kinetic Analysis of Oligo(C) Formation from the 5′-Monophosphorimidazolide of Cytidine with Pb(II) Ion Catalyst at 10–75°C

The temperature dependence of the rate constants for the formation of oligocytidylate (oligo(C)) from the 5′-monophosphorimidazolide of cytidine (ImpC) in the presence of Pb(II) ion catalyst has been investigated at 10–75°C. The rate constants for the formation of oligo(C) increased in the order of the formation of 2-mer < 3-mer ≤ 4-mer; this trend resembles the trend in the cases of the template-directed and the clay-catalyzed formations of oligonucleotides. While the rate constants of the formation of oligo(C) increased with increasing temperature, the yield of oligo(C) decreased with increasing temperature. This is due to the fact that the relative magnitude of the rate constants of the formation of 2-mer, 3-mer, and 4-mer to that of the hydrolysis of ImpC decreased with increasing temperature. This is probably due to the fact that association between ImpC with the elongating oligo(C) decreases with increasing temperature. The apparent activation energy was 61.9 ± 8.5 kJ mol⁻¹ for the formation of 2-mer, 49.3 ± 2.9 kJ mol⁻¹ for 3-mer, 51.8 kJ mol⁻¹ for 4-mer, and 66.8 ± 4.5 kJ mol⁻¹ for the hydrolysis of ImpC. The significance of the temperature dependence of the formation rate constants of the model prebiotic formation of RNA is discussed.     

Hydrophobic effect on the stability and folding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a kinetically robust monomeric protein. The conformational stability and folding kinetics of Tk-RNase HII were measured for nine mutant proteins in which a buried larger hydrophobic side chain is replaced by a smaller one (Leu/Ile to Ala). The mutant proteins were destabilized by 8.9 to 22.0 kJ mol^− 1 as compared with the wild-type protein. The removal of each -CH₂- group burial decreased the stability by 5.1 kJ mol^− 1 on average in the mutant proteins of Tk-RNase HII examined. This is comparable with the value of 5.3 kJ mol^− 1 obtained from experiments for proteins from organisms growing at moderate temperature. We conclude that the hydrophobic residues buried inside protein molecules contribute to the stabilization of hyperthermophilic proteins to a similar extent as proteins at normal temperature. In the folding experiments, the mutant proteins of Tk-RNase HII examined exhibited faster unfolding compared with the wild-type protein. These results indicate that the buried hydrophobic residues strongly contribute to the kinetic robustness of Tk-RNase HII. This is the first report that provides a practical cause of slow unfolding of hyperthermostable proteins.     

Unfolding of CBP21, a lytic polysaccharide monooxygenase,without dissociation of its copper ion cofactor

Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu²⁺ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, t_p, for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of t_p in the presence of excess Cu²⁺ was around 75°C, indicating that Cu²⁺ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu²⁺ ⇌ U∙Cu²⁺, where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol⁻¹ in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu²⁺ complex itself.     

Equilibrium unfolding of a small low-potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris.

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.     

A comparative investigation of the thermal unfolding of pseudoazurin in the Cu(II)-holo and apo form

The contribution of the copper ion to the stability and to the unfolding pathway of pseudoazurin was investigated by a comparative analysis of the thermal unfolding of the Cu(II)-holo and apo form of the protein. The unfolding has been followed by calorimetry, fluorescence, optical density, and electron paramagnetic resonance (EPR) spectroscopy. The thermal transition of Cu(II)-holo pseudoazurin is irreversible and occurs between 60.0 and 67.3 degrees C, depending on the scan rate and technique used. The denaturation pathway of Cu(II)-holo pseudoazurin can be described by the Lumry-Eyring model: N --> U --> [corrected] F; the protein reversibly goes from the native (N) to the unfolded (U) state, and then irreversibly to the final (F) state. The simulation of the experimental calorimetric profiles, according to this model, allowed us to determine the thermodynamic and kinetic parameters of the two steps. The DeltaG value calculated for the Cu(II)-holo pseudoazurin is 39.2 kJ.mol(-1) at 25 degrees C. The sequence of events in the denaturation process of Cu(II)-holo pseudoazurin emergence starts with the disruption of the copper site and the hydrophobic core destabilization followed by the global protein unfolding. According to the EPR findings, the native type-1 copper ion shows type-2 copper features after the denaturation. The removal of the copper ion (apo form) significantly reduces the stability of the protein as evidenced by a DeltaG value of 16.5 kJ.mol(-1) at 25 degrees C. Moreover, the apo Paz unfolding occurs at 41.8 degrees C and is compatible with a two-state reversible process N --> [corrected] U.     

The effect of copper/zinc replacement on the folding free energy of wild type and Cys3Ala/Cys26Ala azurin

The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry–Eyring model, NUF, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 °C, while the unfolding free energy, ΔG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the observed ΔG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, E_a, as derived from the kinetic stability analysis.     

Increased folding stability of TEM-1 beta-lactamase by in vitro selection

In vitro selections of stabilized proteins lead to more robust enzymes and, at the same time, yield novel insights into the principles of protein stability. We employed Proside, a method of in vitro selection, to find stabilized variants of TEM-1 β-lactamase from Escherichia coli. Proside links the increased protease resistance of stabilized proteins to the infectivity of a filamentous phage. Several libraries of TEM-1 β-lactamase variants were generated by error-prone PCR, and variants with increased protease resistance were obtained by raising temperature or guanidinium chloride concentration during proteolytic selections. Despite the small size of phage libraries, several strongly stabilizing mutations could be obtained, and a manual combination of the best shifted the profiles for thermal unfolding and temperature-dependent inactivation of β-lactamase by almost 20 °C to a higher temperature. The wild-type protein unfolds in two stages: from the native state via an intermediate of the molten-globule type to the unfolded form. In the course of the selections, the native protein was stabilized by 27 kJ mol^− 1 relative to the intermediate and the cooperativity of unfolding was strongly increased. Three of our stabilizing replacements (M182T, A224V, and R275L) had been identified independently in naturally occurring β-lactamase variants with extended substrate spectrum. In these variants, they acted as global suppressors of destabilizations caused by the mutations in the active site. The comparison between the crystal structure of our best variant and the crystal structure of the wild-type protein indicates that most of the selected mutations optimize helices and their packing. The stabilization by the E147G substitution is remarkable. It removes steric strain that originates from an overly tight packing of two helices in the wild-type protein. Such unfavorable van der Waals repulsions are not easily identified in crystal structures or by computational approaches, but they strongly reduce the conformational stability of a protein.     

Copper binding before polypeptide folding speeds up formation of active (holo) Pseudomonas aeruginosa azurin.

Cofactors often stabilize the native state of the proteins; however, their effects on folding dynamics remain poorly understood. To uncover the role of one cofactor, we have examined the folding kinetics of Pseudomonas aeruginosa azurin, a small blue-copper protein with a copper cofactor uniquely coordinated to five protein residues. Copper removal produces apo-azurin which adopts a folded structure identical to that of the holo-form. The folding and unfolding kinetics for apo-azurin follow two-state behavior. The extrapolated folding time in water, tau approximately 7 ms, is in good agreement with the topology-based prediction. Copper uptake by folded apo-azurin, to govern active (holo) protein, is slow (tau approximately 14 min, 50:1 copper-to-protein ratio). In contrast, the formation of active (holo) azurin is much faster when copper is allowed to interact with the unfolded polypeptide. Refolding in the presence of 10:1, 50:1, and 100:1 copper:protein ratios yields identical time-trajectories: active azurin forms in two kinetic phases with folding times, extrapolated to water, of tau = 10 +/- 2 ms (major phase) and tau = 190 +/- 30 ms (minor phase), respectively. Correlating copper-binding studies, with a small peptide derived from the metal-binding region of azurin, support that initial cofactor binding is fast (tau approximately 3.7 ms) and thus not rate-limiting. Taken together, introducing copper prior to protein folding does not speed up the polypeptide-folding rate; nevertheless, it results in much faster (> 4000-fold) formation of active (i.e., holo) azurin. Living systems depend on efficient formation of functional biomolecules; attachment of cofactors prior to polypeptide folding appears to be one method to achieve this.     

Tryptophan-tryptophan energy migration as a tool to follow apoflavodoxin folding

Submolecular details of Azotobacter vinelandii apoflavodoxin (apoFD) (un)folding are revealed by time-resolved fluorescence anisotropy using wild-type protein and variants lacking one or two of apoFD's three tryptophans. ApoFD equilibrium (un)folding by guanidine hydrochloride follows a three-state model: native ↔ unfolded ↔ intermediate. In native protein, W128 is a sink for Förster resonance energy transfer (FRET). Consequently, unidirectional FRET with a 50-ps transfer correlation time occurs from W167 to W128. FRET from W74 to W167 is much slower (6.9 ns). In the intermediate, W128 and W167 have native-like geometry because the 50-ps transfer time is observed. However, non-native structure exists between W74 and W167 because instead of 6.9 ns the transfer correlation time is 2.0 ns. In unfolded apoFD this 2.0-ns transfer correlation time is also detected. This decrease in transfer correlation time is a result of W74 and W167 becoming solvent accessible and randomly oriented toward one another. Apparently W74 and W167 are near-natively separated in the folding intermediate and in unfolded apoFD. Both tryptophans may actually be slightly closer in space than in the native state, even though apoFD's radius increases substantially upon unfolding. In unfolded apoFD the 50-ps transfer time observed for native and intermediate folding states becomes 200 ps as W128 and W167 are marginally further separated than in the native state. Apparently, apoFD's unfolded state is not a featureless statistical coil but contains well-defined substructures. The approach presented is a powerful tool to study protein folding.