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The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases.

Crustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we show that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed that the molecular weight of a protein with hyalorunidase activity is 40–50 kDa. Analysis of the hepatopancreas transcriptome and results of cloning and sequencing of cDNA revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. Further analysis showed that hyaluronat enzymatic cleavage follows the $$\beta $$ β -elimination mechanism, which is well known for bacterial hyaluronidases. The results of ion-exchange chromatography showed that the final product of hyaluronate degradation is unsaturated tetrasaccharide. Thus, we identified a new hyaluronidase of higher eukaryotes, which is not integrated into the modern classification of hyaluronidases.

Crustacean hyaluronidases are 1 poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we have shown that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed the hyaluronidase activity of the protein roughly from40 to 50 kDa relative to the molecular marker used in electrophoresis. Analysis of the hepatopancreas transcriptome revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. It turned out that the reaction of cleavage of hyaluronate in the presence of a homogenate proceeds by the mechanism of b-elimination, which is well known for bacterial hyaluronidases. Thus, a new hyaluronidase of higher eukaryotes was found and described, which is not integrated into the modern classification of hyaluronidases.

Pseudomonas microorganisms are used for bioremediation of soils contaminated with petroleum hydrocarbons. The overall remediation efficiency is largely dependent on the presence of macro- and micronutrients. Widely varying concentrations of available nitrogen and iron (Fe) in soils were shown to affect residual hydrocarbons in the course of biodegradation. The regulatory mechanisms of expression of hydrocarbon catabolic genes in low nitrogen/low iron conditions remain unclear. The catabolism of naphthalene, a two-ring polycyclic aromatic hydrocarbon, has been well studied in pseudomonads in terms of the involvement of specific transcriptional activators, thus making it useful in revealing additional regulatory control of the adaptation of hydrocarbon destructors to a low level of the essential nutrients. The Pseudomonas putida strain BS3701 is a component of the “MicroBak” preparation for soil remediation. Previously, this strain was shown to contain genes encoding the key enzymes for naphthalene catabolism: naphthalene 1,2-dioxygenase, salicylate hydroxylase, catechol 2,3-dioxygenase, and catechol 1,2-dioxygenase. Our study aimed to clarify whether the naphthalene catabolic gene expression is dependent on the amount of nitrogen and iron in the growth culture medium, and if so, at exactly which stages the expression is regulated. We cultivated the strain in low nitrogen/low iron conditions with the concurrent evaluation of the activity of the key enzymes and the mRNA level of genes encoding these enzymes. We are the first to report that naphthalene catabolic genes are subject not only to transcriptional but also post-transcriptional regulation.

An original procedure for mapping hydrogeological windows is based on studying the geological structure of the massif overlaying the Podol’sko-Myachkovskii aquifer, changes in the hydrodynamic and hydrogeochemical conditions in the zone of “windows”; as well as calculating the travel time of contaminants in the vertical flow from the Meso-Cenozoic to Podol’sko-Myachkovskii Carboniferous aquifer. Both the areal and vertical distributions of low-permeability deposits are taken into account along with the variations of their thickness and permeability. The results obtained can be used in the development of groundwater protection measures and economic activity regulations in areas with hydrogeological windows.

Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5′-C-C-3′. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.

This paper presents the results of assessing the hydrogeological windows’ impact on the Podol’sko-Myachkovskii aquifer contamination in Moscow as the most important reserve source of drinking water supply. Groundwater contamination in these territories can occur due to rapid mixing of contaminated groundwater of the overlying above-Jurassic aquifer and pure groundwater of the Podolsko-Myachkovskii aquifer. The study consisted in the analysis of the water chemistry data of these aquifers; the choice of the elements—indicators of groundwater contamination; the analysis of the indicators’ distribution schemes and comparison with the boundaries of hydrogeological windows. The research results testified to the significantly elevated concentrations of contamination indicators in the Podolsko-Myachkovskii aquifer at the sites of hydrogeological windows allocated within the specific geostructural and hydrogeological conditions.

PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS). In contrast, there was no universal trend regarding the effect of PEGylation on the thermal stability of a protein based on data generated by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In addition, DSF was validated as a fast and inexpensive screening method for thermal unfolding studies of PEGylated proteins. Multivariate data analysis revealed clear trends in biophysical properties upon PEGylation for a subset of proteins, although no universal trends were found. Taken together, these findings are important in the consideration of biophysical methods and evaluation of second-generation biopharmaceutical drug candidates.

This paper presents the results of assessing the hydrogeological windows impact on the Podol’sko-Myachkovskii aquifer contamination in Moscow as the most important reserve source of drinking water supply. In the areas of hydrogeological windows, the contamination of the Podolsko-Myachkovskii aquifer can occur due to rapid mixing of contaminated groundwater of the overlying above-Jurassic aquifer and pure groundwater of the Podolsko-Myachkovskii aquifer. The study consisted of analyzing the water chemistry data of these aquifers; choice of the elements—indicators of groundwater contamination; analysis of the indicators distribution schemes in the studied aquifers and comparison with the boundaries of hydrogeological windows. The research results testified to the significantly elevated concentrations of contamination indicators in the Podolsko-Myachkovskii aquifer at the sites of hydrogeological windows allocated within the specific geostructural, hydrogeological and tectonic conditions.

Currently, many automobile tunnels are being designed and put into operation in urban areas including Moscow. This is associated with the need of the city to unload the motorways in the absence of sufficient-free areas for the construction of land routes. Experience of operation has shown the expediency of building such facilities on the city territory. Several deep tunnels were commissioned in Moscow including the northwestern tunnel, 27 m deep and 3.1 km long.

In this chapter, a probabilistic approach to forecasting of the risk caused by groundwater contamination is considered. The general conceptual model submitted for consideration includes two parts: (1) a model of the impact of contaminated groundwater resulting from urban sources; and (2) a model of the impact of contaminated groundwater on the recipients. Examples of the probabilistic approach to forecasting of the risk of groundwater contamination are considered in the territories of the leaching fields of domestic wastewater, the gas station and radioactive waste repository. Stochastic modeling was used for risk forecasting. The probability of water intake contamination, damage to the population from contamination of drinking water, and the value of the risk were calculated.

The article describes the problems of the modern development of towns in Russia, their education, development and extinction of the XX century. Highlighted the key types of settlements established during the Soviet Union. Analyzed the planning structure of settlements in Russia. The analysis of the existing situation urban settlement in the Samara region Novosemeykino, its development and reconstruction at the level of urban development. The conclusion about the feasibility of development and reconstruction of towns in Russia.

The research results of the hydrodynamic situation, as well as the chemical and isotopic compositions of target aquifers in the vicinity of radioactive waste storage are considered. The surpassing of water quality standards for Ra- 226, α-activity, Si, Fe, F, and some heavy metals was detected in target Upper Carboniferous aquifers. It was ascertained that the main reason for these changes was disturbance of hydrodynamic conditions as a result of withdrawal enhancement. Intensification of water-rock interactions caused the values of Ra-226 and α-activity to increase in target aquifers, because the main natural sources of Ra-226 are water-bearing rocks containing flints with natural radioactivity and overlying Jurassic layers containing phosphorite. Descending or ascending groundwater leakage caused contaminant incursion from the adjacent aquifers.

Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP–PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.

Enzymes catalyze biochemical reactions in highly crowded environments where the amount of macromolecules may occupy up to 40% of the volume. Here we report how cell-like conditions tune catalytic parameters for the monomeric multi-copper oxidase, Saccharomyces cerevisiae Fet3p, in vitro. At low amounts of crowding agent, we detect increases in both of K(M) (weaker substrate binding) and k(cat) (improved catalytic efficiency), whereas at higher crowding levels, both parameters were reduced. Presence of crowding agents does not affect Fet3p structural content but increases thermal resistance. The observations are compatible with ordering of a non-optimal substrate-binding site and restricted internal dynamics as a result of excluded volume effects making the protein less structurally 'strained'.

SummaryFragile X syndrome is the most common form of inherited mental retardation in humans, with an estimated prevalence of about 1 in 4000 males. Although several observations indicate that the absence of functional Fragile X Mental Retardation Protein (FMRP) is the underlying basis of Fragile X syndrome, the structure and function of FMRP are currently unknown. Here, we present an X-ray crystal structure of the tandem KH domains of human FMRP, which reveals the relative orientation of the KH1 and KH2 domains and the location of residue Ile304, whose mutation to Asn is associated with a particularly severe incidence of Fragile X syndrome. We show that the Ile304Asn mutation both perturbs the structure and destabilizes the protein.

Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation., Intrinsically disordered proteins can phase separate from the soluble intracellular space. Here the authors show that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing and orchestrates the earliest cellular responses to DNA breakage.

Thermodynamics of the Na1−xLixNbO3 system is investigated by high-temperature drop-solution calorimetry in molten 3Na2O–4MoO3 solvent at 973 K. Standard molar enthalpies of formation are derived. The estimated heats of transition between hypothetical and stable structures, lithium niobate and perovskite for NaNbO3 and vice versa for LiNbO3 are −6 kJ/mol and −10 kJ/mol, respectively. X-ray diffraction studies at room temperature showed for 0 ≤x≤ 0.14 there are three phases based on different ordering of the perovskite type lattice: orthorhombic with a quadrupled reduced perovskite cell at 0 ≤x≤ 0.02, orthorhombic with a doubled reduced perovskite cell at 0.015 ≤x≤ 0.14, and rhombohedral at 0.08 ≤x≤ 0.13. There are two two-phase (morphotropic) regions with coexistence of the two orthorhombic phases at 0.015 ≤x≤ 0.02 and with the second orthorhombic phase coexisting with the rhombohedral phase at 0.08 ≤x≤ 0.13. A reproducible anomaly in specific heat at ∼600 K, not reported previously, has been observed in pure NaNbO3. Heat-capacity measurements confirm a phase transition at 553 K for 0.07 ≤x≤ 0.09. With increasing lithium concentration, a gradual disappearance of high-temperature phase transitions associated with tilting of oxygen octahedra has been observed.

Azurin is a blue-copper protein with a beta-barrel structure of Greek Key topology. In vitro, copper can be substituted with zinc without change in protein structure. We here analyze the kinetic folding behavior of zinc-substituted Pseudomonas aeruginosa azurin. Our findings can be summarized in three key conclusions: first, zinc remains strongly bound to the polypeptide upon unfolding, suggesting that the cofactor may bind to the protein before polypeptide folding in vivo. Second, the semi-logarithmic plot of folding and unfolding rates for zinc-substituted azurin as a function of denaturant concentration exhibits curvature due to a changing transition-state structure. Third, the extrapolated folding speed in water for zinc-substituted azurin is similar to that of other proteins with the same topology, implying that there is a speed limit that can be modulated by stability-driven transition-state movement for formation of beta-barrel structures with Greek Key topology.

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.

BACKGROUND: Characterisation of the specific antibody response, including the epitope binding pattern, is an essential task for understanding the molecular mechanisms of food allergy. Examination of antibody formation in a controlled environment requires animal models. The purpose of this study was to examine the amount and types of antibodies raised against three cow's milk allergens; β-lactoglobulin (BLG), α-lactalbumin (ALA) and β-casein upon oral or intraperitoneal (i.p.) administration. A special focus was given to the relative amount of antibodies raised against linear versus conformational epitopes.METHODS: Specific antibodies were raised in Brown Norway (BN) rats. BN rats were dosed either (1) i.p. with the purified native cow's milk allergens or (2) orally with skimmed milk powder (SMP) alone or together with gluten, without the use of adjuvants. The allergens were denatured by reduction and alkylation, resulting in unfolding of the primary structure and a consequential loss of conformational epitopes. The specific IgG1 and IgE responses were analysed against both the native and denatured form of the three cow's milk allergens, thus allowing examination of the relative amount of linear versus conformational epitopes.RESULTS: The inherent capacity to induce specific IgG1 and IgE antibodies were rather similar upon i.p. administration for the three cow's milk allergens, with BLG = ALA > β-casein. Larger differences were found between the allergens upon oral administration, with BLG > ALA > β-casein. Co-administration of SMP and gluten had a great impact on the specific antibody response, resulting in a significant reduced amount of antibodies. Together results indicated that most antibodies were raised against conformational epitopes irrespectively of the administration route, though the relative proportions between linear and conformational epitopes differed remarkably between the allergens.CONCLUSIONS: This study showed that the three-dimensional (3D) structure has a significant impact on the antibodies raised for both systemic and orally administered allergens. A remarkable difference in the antibody binding patterns against linear and conformational epitope was seen between the allergens, indicating that the structural characteristics of proteins may heavily affect the induced antibody response.

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.

Ovarian cancer is a leading cause of deaths, yet many aspects of the biology of the disease and a routine means of its detection are lacking. We have used protein microarrays and autoantibodies from cancer patients to identify proteins that are aberrantly expressed in ovarian tissue. Sera from 30 cancer patients and 30 healthy individuals were used to probe microarrays containing 5,005 human proteins. Ninety-four antigens were identified that exhibited enhanced reactivity from sera in cancer patients relative to control sera. The differential reactivity of four antigens was tested by using immunoblot analysis and tissue microarrays. Lamin A/C, SSRP1, and RALBP1 were found to exhibit increased expression in the cancer tissue relative to controls. The combined signals from multiple antigens proved to be a robust test to identify cancerous ovarian tissue. These antigens were also reactive with tissue from other types of cancer and thus are not specific to ovarian cancer. Overall our studies identified candidate tissue marker proteins for ovarian cancer and demonstrate that protein microarrays provide a powerful approach to identify proteins aberrantly expressed in disease states.

Lack of functional Fragile X mental retardation protein (FMRP) is the primary cause of the Fragile-mental retardation syndrome in humans. In most cases, the disease results from transcriptional silencing of fragile mental retardation gene 1, fmr1, which encodes FMRP. However, a single missense mutation (I304N) in the second KH domain of FMRP gives rise to a particularly severe case of Fragile X syndrome. A Drosophila homolog of FMRP has been identified, Drosophila Fragile X related protein (dFXRP). The corresponding missense mutation in dFXRP, the I307N, has pronounced effects on the in vivo activity of the protein. The effect of the point mutation on the structure and function of FMRP is unclear, and published data are contradictory. No in vitro structural or stability studies have been performed on dFXRP. Here we show that a construct that contains only the tandem KH1-KH2 domains is a stable, well-folded unit suitable for detailed structural and functional characterization. Using this KH1-KH2 construct we explicitly test a hypothesis that has been proposed to explain the effect of the Ile-->Asn mutation: that it causes complete unfolding of the protein. Here we show that the I307N point mutation does not completely unfold the KH domain. The KH1-KH2 construct bearing I307N substitution is stable in isolation and adopts a native-like fold. Thus our data favor alternative explanations for the in vivo observed loss of dFXRP activity associated with I307N mutation: (a) the point mutation might affect intra and/or inter-molecular interactions of dFXRP; or (b) it might impair dFXRP's interactions with its RNA target(s).

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.

Pseudomonas aeruginosa azurin is a blue-copper protein with a Greek-key fold. Removal of copper produces an apoprotein with the same structure as holoazurin. To address the effects on thermodynamic stability and folding dynamics caused by small cavities in a β-barrel, we have studied the behavior of the apo-forms of wild-type and two mutant (His-46-Gly and His-117-Gly) azurins. The equilibrium- and kinetic-folding and unfolding reactions appear as two-state processes for all three proteins. The thermodynamic stability of the two mutants is significantly decreased as compared with the stability of wild-type azurin, in accord with cavities in or near the hydrophobic interior having an overall destabilizing effect. Large differences are also found in the unfolding rates: the mutants unfold much faster than wild-type azurin. In contrast, the folding-rate constants are almost identical for the three proteins and closely match the rate-constant predicted from the native-state topology of azurin. We conclude that the topology is more important than equilibrium stability in determining the folding speed of azurin.

Azurin is a single-domain beta-barrel protein with a redox-active copper cofactor. Upon Pseudomonas aeruginosa azurin unfolding, the cofactor remains bound to the polypeptide, coordinating three ligands: cysteine-112, one histidine imidazole, and a third, unknown ligand. In order to identify which histidine (histidine-117 and histidine-46 both coordinate copper in native azurin) is involved in copper coordination in denatured azurin, two single-site (histidine to glycine) mutants, His117Gly and His46Gly azurin, are investigated here. Equilibrium denaturation experiments of His46Gly azurin loaded with copper demonstrate that copper remains bound to this mutant in high urea concentrations where the protein's secondary structure is lost. In contrast, for copper-loaded His117Gly azurin, copper does not stay coordinated upon polypeptide unfolding. The copper absorption at 370 nm in denatured His46Gly azurin agrees with that for copper in complex with a peptide corresponding to residues 111-123 in azurin, suggesting similar metal coordination. We conclude that histidine-117 (and not histidine-46) is the histidine copper ligand in denatured azurin. This is also in accord with the proximity of histidine-117 to cysteine-112 in the primary sequence.

Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.