Simultaneous quantification of total and conjugated ubiquitin levels in a single immunoblot

Ubiquitin (Ub) is a posttranslational modifier, and total Ub (Ub_T) is always in dynamic equilibrium among free Ub (Ub_F), activated Ub (Ub_A), and conjugated Ub (Ub_C) in the forms of mono-Ub, thioester-bond-linked Ub, and peptide-bond-linked Ub, respectively. In this study, we developed a simple method to simultaneously determine the levels of Ub_T, Ub_F + Ub_A, and Ub_C in a single immunoblot and demonstrated its reliability and reproducibility by determining [Ub_T], [Ub_F + Ub_A], and [Ub_C] in various mouse tissues and cultured cells.     

Relative Domain Folding and Stability of a Membrane Transport Protein

There is a limited understanding of the folding of multidomain membrane proteins. Lactose permease (LacY) of Escherichia coli is an archetypal member of the major facilitator superfamily of membrane transport proteins, which contain two domains of six transmembrane helices each. We exploit chemical denaturation to determine the unfolding free energy of LacY and employ Trp residues as site-specific thermodynamic probes. Single Trp LacY mutants are created with the individual Trps situated at mirror image positions on the two LacY domains. The changes in Trp fluorescence induced by urea denaturation are used to construct denaturation curves from which unfolding free energies can be determined. The majority of the single Trp tracers report the same stability and an unfolding free energy of approximately + 2 kcal mol^− 1. There is one exception; the fluorescence of W33 at the cytoplasmic end of helix I on the N domain is unaffected by urea. In contrast, the equivalent position on the first helix, VII, of the C-terminal domain exhibits wild-type stability, with the single Trp tracer at position 243 on helix VII reporting an unfolding free energy of + 2 kcal mol^− 1. This indicates that the region of the N domain of LacY at position 33 on helix I has enhanced stability to urea, when compared the corresponding location at the start of the C domain. We also find evidence for a potential network of stabilising interactions across the domain interface, which reduces accessibility to the hydrophilic substrate binding pocket between the two domains.     

Sequential barriers and an obligatory metastable intermediate define the apparent two-state folding pathway of the ubiquitin-like PB1 domain of NBR1

The 90-residue N-terminal Phox and Bem1p (PB1) domain of NBR1 forms an α/β ubiquitin-like fold. Kinetic analysis using stopped-flow fluorescence reveals two-state kinetics; however, nonlinear effects in the denaturant dependence of the unfolding data demonstrate changes in the position of the rate-limiting barrier along the folding coordinate as the folding conditions change. The kinetics of wt-PB1 and several mutants show that this curvature is consistent with a single-pathway mechanism involving sequential transition states (TS1 and TS2) separated by a transiently populated high-energy intermediate, rather than movement of the transition state on a broad energy plateau. We show that the two transition states within the sequential model represent structurally and thermodynamically distinct species. TS1 is a collapsed state (α_TS1 = 0.71) with a large enthalpic barrier to formation that is rate-limiting under conditions that strongly favour folding. TS2 is highly native-like (α_TS2 = 0.93) and represents a late entropic barrier to formation of the native state. In support of the sequential transition state mechanism, we show that the G62A helix 2 substitution stabilises TS1 and the intermediate to such an extent that the latter becomes significantly populated, leading to the observation of a fast kinetic phase representing the initial U → I transition, with TS2 (α_TS2 = 0.87) becoming rate-limiting. The folding rate is not retarded by populating an intermediate, which would be expected for a misfold state, but is accelerated, suggesting that the I state is productive and on-pathway. The results show that the apparent two-state folding of the wt-PB1 domain occurs along a well-defined pathway involving structurally and thermodynamically distinct sequential transition states and an obligatory metastable intermediate that represents a productive local minimum in the energy landscape that increases the efficiency of barrier crossing through favourable effects on the entropy of activation.     

Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate

HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca^2 + binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca^2 + is essential for stability as well as correct folding of the whole protein. In the absence of Ca^2 +, pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca^2 + addition, indicating that these are not dead-end species and that Ca^2 + drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6–7 M urea, relates to Ca^2 + dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca^2 + induced folding is not restricted to the RTX domain. Taken together, Ca^2 + binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation.     

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~ 27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency > 90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.     

Evaluation of cytochrome c affinity to anionic phospholipids by means of surface plasmon resonance

We attempted to evaluate the affinity of the anionic phospholipids to cytochrome c by means of surface plasmon resonance (SPR) technique and to correlate it with the cytochrome c active site alterations and peroxidase activity. Our experiments showed a strong interdependence between the phospholipid fatty acid saturation degree, the active site structure alterations and peroxidase activity of the cytochrome c phospholipid complex. Cytochrome c peroxidase activity and Trp59 fluorescence increase in the sequence of phosphatidyl choline (PC) → phosphatidylserine (PS) → cardiolipin (CL) → phosphatidic acid (PA). The association constant (K_a) increased in the sequence PC → PA → PS → CL. The SPR spectroscopy data shows that K_a is independent of lipid saturation degree, but correlates with phospholipid negative charge value.     

An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.     

Conserved folding pathways of alpha-lactalbumin and lysozyme revealed by kinetic CD, fluorescence, NMR, and interrupted refolding experiments

In this report, it is shown by a combination of stopped-flow CD, fluorescence, and time-resolved NMR studies that the Ca^2 +-induced refolding of bovine α-lactalbumin (BLA) at constant denaturant concentration (4 M urea) exhibits triple-exponential kinetics. In order to distinguish between parallel folding pathways and a strictly sequential formation of the native state, interrupted refolding experiments were conducted. We show here that the Ca^2 +-induced refolding of BLA involves parallel pathways and the transient formation of a folding intermediate on the millisecond timescale. Our data furthermore suggest that the two structurally homologous proteins BLA and hen egg white lysozyme share a common folding mechanism. We provide evidence that the guiding role of long-range interactions in the unfolded state of lysozyme in mediating intersubdomain interactions during folding is replaced in the case of BLA by the Ca^2 +-binding site. Time-resolved NMR spectroscopy, in combination with fast ion release from caged compounds, enables the measurement of complex protein folding kinetics at protein concentrations as low as 100 μM and the concomitant detection of conformational transitions with rate constants of up to 8 s^− 1.     

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.     

The controlling roles of Trp60 and Trp95 in beta2-microglobulin function, folding and amyloid aggregation properties

Amyloidosis associated to hemodialysis is caused by persistently high β₂-microglobulin (β₂m) serum levels. β₂m is an intrinsically amyloidogenic protein whose capacity to assemble into amyloid fibrils in vitro and in vivo is concentration dependent; no β₂m genetic variant is known in the human population. We investigated the roles of two evolutionary conserved Trp residues in relation to β₂m structure, function and folding/misfolding by means of a combined biophysical and functional approach. We show that Trp60 plays a functional role in promoting the association of β₂m in class I major histocompatibility complex; it is exposed to the solvent at the apex of a protein loop in order to accomplish such function. The Trp60 → Gly mutation has a threefold effect: it stabilizes β₂m, inhibits β₂m amyloidogenic propensity and weakens the interaction with the class I major histocompatibility complex heavy chain. On the contrary, Trp95 is buried in the β₂m core; the Trp95 → Gly mutation destabilizes the protein, which is unfolded in solution, yielding nonfibrillar β₂m aggregates. Trp60 and Trp95 therefore play differential and complementary roles in β₂m, being relevant for function (Trp60) and for maintenance of a properly folded structure (Trp95) while affecting in distinct ways the intrinsic propensity of wild-type β₂m towards self-aggregation into amyloid fibrils.     

Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ < 30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg²⁺, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s^− 1 at 37 °C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the I_C intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.     

Kinetic and thermodynamic properties of the folding and assembly of formate dehydrogenase

The folding mechanism and stability of dimeric formate dehydrogenase from Candida methylica was analysed by exposure to denaturing agents and to heat. Equilibrium denaturation data yielded a dissociation constant of about 10⁻¹³ M for assembly of the protein from unfolded chains and the kinetics of refolding and unfolding revealed that the overall process comprises two steps. In the first step a marginally stable folded monomeric state is formed at a rate (k₁) of about 2 × 10⁻³ s⁻¹ (by deduction k₋₁ is about10⁻⁴ s⁻¹) and assembles into the active dimeric state with a bimolecular rate constant (k₂) of about 2 × 10⁴ M⁻¹ s⁻¹. The rate of dissociation of the dimeric state in physiological conditions is extremely slow (k₋₂ ∼ 3 × 10⁻⁷ s⁻¹).     

Ubiquitin is a novel substrate for human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (k_cat = 2 s^− 1) followed by a slow cleavage between residues 72 and 73 (k_cat = 0.07 s^−  1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG <  0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.     

Desolvation and Development of Specific Hydrophobic Core Packing during Im7 Folding

Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val→Thr or Phe→Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Φ-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (> 2.5 kJ mol^− 1) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.     

Optical studies of single-tryptophan B. licheniformis β-lactamase variants

β-lactamases (penicillinases) are important complicating factors in bacterial infections and excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A β-lactamase with three tryptophan residues, one located in each of the two protein domains and one located in the interface between domains. To determine the tryptophan contribution to the ESP UV-absorption, circular dichroism, and steady-state and time-resolved fluorescence, four Trp → Phe mutants were prepared and characterized. The residue substitutions had little impact on the native conformation. UV-absorption and CD features were identified and ascribed to specific aromatic residues. Time-resolved fluorescence showed that most of the fluorescence decay of ESP tryptophans is due to a discrete exponential component with a lifetime of 5-6 ns. Fluorescence polarization measurements indicated that fluorescence of Trp 210 is nearly independent of the fluorescence of Trp 229 and Trp 251, whereas a substantial energy homotransfer between the latter pair takes place. The spectroscopic information was rationalized on the basis of structural considerations and should help in the interpretation and monitoring of the changes at the sub domain level during the conformational transitions and fluctuations of ESP and other Class A β-lactamases.     

Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation

Hydrophobic interactions are known to play an important role for cold-adaptation of proteins; however, the role of amino acid residue, Trp, has not been systematically investigated. The extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W²⁰⁸ was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W²⁰⁸ to Tyr (W²⁰⁸Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40 °C compared to wild-type EstK. The structure model of W²⁰⁸Y suggested that Y²⁰⁸ could form a hydrogen bond with D³⁰⁸, which is located next to catalytic residue H³⁰⁷, stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures.     

Assessment of olive-mill wastewater as a growth medium for lipase production by Candida cylindracea in bench-top reactor

Olive-mill wastewater (OMW) was investigated for its suitability to serve as a medium for lipase production by Candida cylindracea NRRL Y-17506. The OMW that best supported enzyme production was characterized by low COD and low total sugars content. In shake flask batch cultures, OMW supplementation with 2.4 g l⁻¹ NH₄Cl and 3 g l⁻¹ olive oil led to an enzyme activity of about 10 U ml⁻¹. The addition of glucose or malt extract and supplements containing organic N (e.g., peptone, yeast extract) either depressed or did not affect the enzyme production. Further experiments were then performed in a 3-l stirred tank reactor to assess the impact of medium pH and stirring speed on the yeast enzyme activity. The lipase activity was low (1.8 U ml⁻¹) when the pH was held constant at 6.5, significantly increased (18.7 U ml⁻¹) with uncontrolled pH and was maximum (20.4 U ml⁻¹) when the pH was let free to vary below 6.5. A stirring regime, that varied depending on the dissolved oxygen concentration in the medium, both prevented the occurrence of anoxic conditions during the exponential growth phase and enabled good lipase production (i.e., 21.6 U ml⁻¹) and mean volumetric productivity (i.e., 123.5 U l⁻¹ h⁻¹).     

Membrane association and activity of 15/16-membered peptide antibiotics: Zervamicin IIB, ampullosporin A and antiamoebin I

Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K⁺ transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER > AMP >> ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC_16:1PC, diC_18:1PC, C_16:0/C_18:1PC, diC_20:1PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic-hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.     

Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state

Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16_meso) and hyperthermophilic (S16_thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16_thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔC_p⁰) of 6.4 and 3.3 kJ mol^− 1 K^− 1, respectively. A strong linear correlation between ΔC_p⁰ and melting temperature (T_m) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16_meso folds through an apparent two-state model, whereas S16_thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16_thermo is more compact relative to S16_meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16_thermo that appears to account for the large difference in quantified ΔC_p⁰ values and, in turn, parts of the observed extreme thermal stability of S16_thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.     

Time-resolved single-turnover of ba3 oxidase from Thermus thermophilus

The kinetics of the oxidation of fully-reduced ba₃ cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa₃-type oxidases. However the F intermediate in ba₃ oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P → F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F → O transition in ba₃ oxidase is close to that observed during the F → O transition in the aa₃ oxidases. However, the P_R → F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.