Protein chemistry

Scientific publications - Protein chemistry

Faber, C., Hobley, T.J., Mollerup, J., Thomas, O.R.T., Kaasgaard, S.G.

"Factors affecting the solubility of Bacillus halmapalus α-amylase"

Chemical Engineering and Processing: Process Intensification, 47 (6), pp. 1007-1017. (2008)

A detailed study of the solubility of recombinant Bacillus halmapalus α-amylase has been conducted. A semi-purified preparation from a bulk crystallisation was chos. en that contained six isoforms with pI-values of between 5.5 and 6.1. The solubility was strongly affected by pH and could be reduced approximately 200-fold at pH 6 as compared to pH 10, leaving only 0.1 mg/mL in solution. Solubility could also be dramatically manipulated using salts. The choice of anions was found to be more important than of the cations, and the lowest solubility was found using sodium sulphate. For the anions, solubility followed the order expected from the Hofmeister series, however, a more complex behaviour was seen for the cations. With the exception of lithium, their efficiency to influence the solubility was reversed to what was expected. The polydispersity of the solution was reduced by salt addition and zeta potential measurements indicated a shift in pI caused by lithium. Possible explanations for the observations are discussed, extending our present understanding of how salts affect the solubility of proteins, one that to date is primarily based on experiments with lysozyme. © 2007 Elsevier B.V. All rights reserved.

Sonesson, A.W., Callisen, T.H., Brismar, H., Elofsson, U.M.

"Adsorption and activity of Thermomyces lanuginosus lipase on hydrophobic and hydrophilic surfaces measured with dual polarization interferometry (DPI) and confocal microscopy"

Colloids and Surfaces B: Biointerfaces, 61 (2), pp. 208-215. (2008)

The adsorption and activity of Thermomyces lanuginosus lipase (TLL) was measured with dual polarization interferometry (DPI) and confocal microscopy at a hydrophilic and hydrophobic surface. In the adsorption isotherms, it was evident that TLL both had higher affinity for the hydrophobic surface and adsorbed to a higher adsorbed amount (1.90 mg/m2) compared to the hydrophilic surface (1.40-1.50 mg/m2). The thickness of the adsorbed layer was constant (∼3.5 nm) on both surfaces at an adsorbed amount >1.0 mg/m2, but decreased on the hydrophilic surface at lower surface coverage, which might be explained by partially unfolding of the TLL structure. However, a linear dependence of the refractive index of the adsorbed layer on adsorbed amount of TLL on C18 surfaces indicated that the structure of TLL was similar at low and high surface coverage. The activity of adsorbed TLL was measured towards carboxyfluorescein diacetate (CFDA) in solution, which upon lipase activity formed a fluorescent product. The surface fluorescence intensity increase was measured in a confocal microscope as a function of time after lipase adsorption. It was evident that TLL was more active on the hydrophilic surface, which suggested that a larger fraction of adsorbed TLL molecules were oriented with the active site facing the solution compared to the hydrophobic surface. Moreover, most of the activity remained when the TLL surface coverage decreased. Earlier reports on TLL surface mobility on the same surfaces have found that the lateral diffusion was highest on hydrophilic surfaces and at low surface coverage of TLL. Hence, a high lateral mobility might lead to a longer exposure time of the active site towards solution, thereby increasing the activity against a water-soluble substrate. © 2007 Elsevier B.V. All rights reserved.

Roca, M., De Maria, L., Wodak, S.J., Moliner, V., Tuñón, I., Giraldo, J.

"Coupling of the guanosine glycosidic bond conformation and the ribonucleotide cleavage reaction: Implications for barnase catalysis"

Proteins: Structure, Function and Genetics, 70 (2), pp. 415-428. (2008)

To examine the possible relationship of guanine-dependent GpA conformations with ribonucleotide cleavage, two potential of mean force (PMF) calculations were performed in aqueous solution. In the first calculation, the guanosine glycosidic (Gχ) angle was used as the reaction coordinate, and computations were performed on two GpA ionic species: protonated (neutral) or deprotonated (negatively charged) guanosine ribose O2′. Similar energetic profiles featuring two minima corresponding to the anti and syn Gχ regions were obtained for both ionic forms. For both forms the anti conformation was more stable than the syn, and barriers of ∼4 kcal/mol were obtained for the anti → syn transition. Structural analysis showed a remarkable sensitivity of the phosphate moiety to the conformation of the G χ angle, suggesting a possible connection between this conformation and the mechanism of ribonucleotide cleavage. This hypothesis was confirmed by the second PMF calculations, for which the O2′-P distance for the deprotonated GpA was used as reaction coordinate. The computations were performed from two selected starting points: the anti and syn minima determined in the first PMF study of the deprotonated guanosine ribose O2′. The simulations revealed that the O2′ attack along the syn Gχ was more favorable than that along the anti Gχ: energetically, significantly lower barriers were obtained in the syn than in the anti conformation for the O-P bond formation; structurally, a lesser O2′-P initial distance, and a better suited orientation for an in-line attack was observed in the syn relative to the anti conformation. These results are consistent with the catalytically competent conformation of barnase-ribonucleotide complex, which requires a guanine syn conformation of the substrate to enable abstraction of the ribose H2′ proton by the general base Glu73, thereby suggesting a coupling between the reactive substrate conformation and enzyme structure and mechanism. © 2007 Wiley-Liss, Inc.

Sonesson, A.W., Blom, H., Hassler, K., Elofsson, U.M., Callisen, T.H., Widengren, J., Brismar, H.

"Protein-surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS)"

Journal of Colloid and Interface Science, 317 (2), pp. 449-457. (2008)

The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 μM C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption. © 2007 Elsevier Inc. All rights reserved.

Rodriguez-Larrea, D., Ibarra-Molero, B., De Maria, L., Borchert, T.V., Sanchez-Ruiz, J.M.

"Beyond Lumry-Eyring: An unexpected pattern of operational reversibility/irreversibility in protein denaturation"

Proteins: Structure, Function and Genetics, 70 (1), pp. 19-24. (2008)

We have found that, contrary to naïve intuition, the degree of operational reversibility in the thermal denaturation of lipase from Thermomyces lanuginosa (an important industrial enzyme) in urea solutions is maximum when the protein is heated several degrees above the end of the temperature-induced denaturation transition. Upon cooling to room temperature, the protein seems to reach a state with enzymatic activity similar to that of the initial native state, but with higher denaturation temperature and radically different behavior in terms of susceptibility to irreversible denaturation. These results show that patterns of operational reversibility/irreversibility in protein denaturation may be more complex than the often-taken-for-granted, two-situation classification (reversible vs. irreversible). Furthermore, they are consistent with the possibility of existence of different native or native-like states separated by high kinetic barriers under native conditions and they suggest experimental procedures to reach and study such "alternative" native states. © 2007 Wiley-Liss, Inc.

Jauffred, L., Callisen, T.H., Oddershede, L.B.

Visco-elastic membrane tethers extracted from Escherichia coli by optical tweezers

Biophysical Journal, 93 (11), pp. 4068-4075. (2007)

Tethers were created between a living Escherichia coli bacterium and a bead by unspecifically attaching the bead to the outer membrane and pulling it away using optical tweezers. Upon release, the bead returned to the bacterium, thus showing the existence of an elastic tether between the bead and the bacterium. These tethers can be tens of microns long, several times the bacterial length. Using mutants expressing different parts of the outer membrane structure, we have shown that an intact core lipopolysaccharide is a necessary condition for tether formation, regardless of whether the beads were uncoated polystyrene or beads coated with lectin. A physical characterization of the tethers has been performed yielding visco-elastic tether force-extension relationships: for first pull tethers, a spring constant of 10-12 pN/μm describes the tether visco-elasticity, for subsequent pulls the spring constant decreases to 6-7 pN/μm, and typical relaxation timescales of hundreds of seconds are observed. Studies of tether stability in the presence of proteases, lipases, and amylases lead us to propose that the extracted tether is primarily composed of the asymmetric lipopolysaccharide containing bilayer of the outer membrane. This unspecific tethered attachment mechanism could be important in the initiation of bacterial adhesion. © 2007 by the Biophysical Society.

Bagger, H.L., Øgendal, L.H., Westh, P.

"Solute effects on the irreversible aggregation of serum albumin"

Biophysical Chemistry, 130 (1-2), pp. 17-25. (2007)

Thermal stress on bovine serum albumin (BSA) promotes protein aggregation through the formation of intermolecular β-sheets. We have used light scattering and chromatography to study effects of (< 1 M) Na2SO4, NaSCN, sucrose, sorbitol and urea on the rate of the thermal aggregation. Both salts were strong inhibitors of BSA aggregation and they reduced both the size and number (concentration) of aggregate particles compared to non-ionic solutes (or pure buffer). Hence, the salts appear to suppress both nucleation- and growth rate. The non-electrolyte additives reduced the initial aggregation rate (compared to pure buffer), but did not significantly limit the extent of aggregation in samples quenched after 27 min. heat exposure (40-50% aggregation in all samples). The non-electrolytes did, however, modify the aggregation process as they consistently brought about smaller but more concentrated aggregates than pure buffer. The results are discussed along the lines of linkage- and transition state theories. In this framework, the rate of the aggregation process is governed by the equilibrium between a thermally denatured state (D) and the transition state D≠. Thus, the effect of a solute relies on its preferential interactions with respectively D and D≠. The current results do not show any correlation between the solutes' preferential interactions with native BSA and their effect on the rate of aggregation. This suggests that non-specific, "Hofmeister-type" interactions, which scale with the solvent accessible surface area, are of minor importance. Rather, salt induced suppression of aggregation is suggested to depend on the modulation of specific electrostatic forces in the D≠ state. © 2007 Elsevier B.V. All rights reserved.

Nordkvist, M., Nielsen, P.M., Villadsen, J.

"Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase: Kinetics and operational stability"

Biotechnology and Bioengineering, 97 (4), pp. 694-707. (2007)

Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase was studied. The Km-value for lactose, obtained by a traditional enzymatic assay, was 0.066 mM at pH 6.4 and 38°C. The effect of oxygen on the enzymatic rate of reaction as well as the operational stability of the enzyme was studied by performing reactions at constant pH and temperature in a stirred tank reactor. Catalase was included in all reactions to avoid inhibition and deactivation of the oxidase by hydrogen peroxide. At pH 6.4 and 38°C, Km for oxygen was 0.97 mM, while the catalytical rate constant, kcat, was 94 s-1. Furthermore, we found that the operational stability of the oxidase was dependent on the type of base used for neutralization of the acid produced. Thus, when 2 M NaOH was used for neutralization of a reaction medium containing 50 mM phosphate buffer, significant deactivation of the oxidase was observed. Also, we found that the oxidase was protected against deactivation by base at high lactose concentrations. A simple model is proposed to explain the obtained results. © 2006 Wiley Periodicals, Inc.

Bagger, H.L., Hoffmann, S.V., Fuglsang, C.C., Westh, P.

"Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system"

Biophysical Chemistry, 129 (2-3), pp. 251-258. (2007)

The interactions of sodium dodecyl sulfate (SDS) and two glyco-variants of the enzyme phytase from Peniophora lycii were investigated. One variant (Phy) was heavily glycosylated while the other (dgPhy) was enzymatically deglycosylated. Effects at 24 °C of titrating SDS to Phy and dgPhy were studied by Isothermal Titration Calorimetry (ITC) and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. Comparisons of results for the two variants were used to elucidate glycan-surfactant interrelationships. The CD spectra suggested that both the native and the SDS-denatured states of the two variants were mutually similar, and hence that the denaturation process was structurally equivalent for the two glyco-variants. The denatured state was far from fully unfolded and probably retained a substantial content of native-like structure. Furthermore, it was found that the glycans brought about only a small increase in the resistance towards SDS induced denaturation. The SDS concentration required to denature half of the protein molecules differed less than 1 mM for the two variants. The affinity for SDS of both variants was unusually low. The amount of bound SDS (w/w) at different stages of the binding isotherm was 3-10 times lower than that reported for the most previously investigated globular proteins. Analysis of the relative affinity of the glycan and peptide moieties suggested that the carbohydrates bind much less surfactant. At saturation, glycans adsorbed about half as much SDS (in g/g) as the peptide moiety of Phy and about five times less than average proteins. © 2007 Elsevier B.V. All rights reserved.

Sonesson, A.W., Elofsson, U.M., Callisen, T.H., Brismar, H.

"Tracking single lipase molecules on a trimyristin substrate surface using quantum dots"

Langmuir, 23 (16), pp. 8352-8356. (2007)

The mobility of single lipase molecules has been analyzed using single molecule tracking on a trimyristin substrate surface. This was achieved by conjugating lipases to quantum dots and imaging on spin-coated trimyristin surfaces by means of confocal laser scanning microscopy. Image series of single lipase molecules were collected, and the diffusion coefficient was quantified by analyzing the mean square displacement of the calculated trajectories. During no-flow conditions, the lipase diffusion coefficient was (8.0 ±5.0) ×10-10cm2/s. The trajectories had a "bead on a string" appearance, with the lipase molecule restricted in certain regions of the surface and then migrating to another region where the restricted diffusion continued. This gave rise to clusters in the trajectories. When a flow was applied to the system, the total distance and average step length between the clusters increased, but the restricted diffusion in the cluster regions was unaffected. This can be explained by the lipase operating in two different modes on the surface. In the cluster regions, the lipase is likely oriented with the active site toward the surface and hydrolyzes the substrate. Between these regions, a diffusion process is proposed where the lipase is in contact with the surface but affected by the external flow. © 2007 American Chemical Society.

Nordkvist, M., Nielsen, P.M., Villadsen, J.

"Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase: Kinetics and operational stability"

Biotechnology and Bioengineering, 97 (4), pp. 694-707. (2007)

Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase was studied. The Km-value for lactose, obtained by a traditional enzymatic assay, was 0.066 mM at pH 6.4 and 38°C. The effect of oxygen on the enzymatic rate of reaction as well as the operational stability of the enzyme was studied by performing reactions at constant pH and temperature in a stirred tank reactor. Catalase was included in all reactions to avoid inhibition and deactivation of the oxidase by hydrogen peroxide. At pH 6.4 and 38°C, Km for oxygen was 0.97 mM, while the catalytical rate constant, kcat, was 94 s-1. Furthermore, we found that the operational stability of the oxidase was dependent on the type of base used for neutralization of the acid produced. Thus, when 2 M NaOH was used for neutralization of a reaction medium containing 50 mM phosphate buffer, significant deactivation of the oxidase was observed. Also, we found that the oxidase was protected against deactivation by base at high lactose concentrations. A simple model is proposed to explain the obtained results. © 2006 Wiley Periodicals, Inc.

Faber, C., Hobley, T.J., Mollerup, J., Thomas, O.R.T., Kaasgaard, S.G.

"Study of the solubility of a modified bacillus licheniformis α-amylase around the isoelectric point"

Journal of Chemical and Engineering Data, 52 (3), pp. 707-713. (2007)

The solubility of a modified recombinant Bacillus licheniformis α-amylase (mBLA) has been studied by batch crystallization. A semi-pure preparation was chosen containing five isoforms with pi values from 6 to 7.3 (weighted average of 6.6). Small amounts (<1%) of protein impurities were also present. Solubility was studied in the pH range of 6 to 8. The lowest solubility without added salts was 60 mg·mL-1 at pH 7. The addition of 0.1 mol·L-1 sodium salts of nitrate, sulfate, and thiocyanate had a small effect on solubility. However, solubility was lowered significantly by adding 0.5 mol·L-1 sodium sulfate at all pH values and increased with 0.5 mol·L-1 sodium thiocyanate at pH 7 and pH 8. The effect of anions on α-amylase solubility followed the Hofmeister series, and only weak evidence of reversal was seen below the isoelectric point. Cations had little effect on solubility. The sign and magnitude of the α-amylase ζ potential was determined in the presence and absence of 0.1 mol·L-1 salt. Qualitatively, ζ potential correctly predicted the different salts influence on mBLA solubility. © 2007 American Chemical Society.

Nielsen, A.D., Borch, K., Westh, P.

"Thermal stability of Humicola insolens cutinase in aqueous SDS"

Journal of Physical Chemistry B, 111 (11), pp. 2941-2947. (2007)

Cutinase from Humicola insolens (HiC) has previously been shown to bind anomalously low amounts of the anionic surfactant sodium dodecylsulfate (SDS). In the current work, we have applied scanning and titration calorimetry to investigate possible relationships between this weak interaction and the effect of SDS on the equilibrium and kinetic stability of HiC. The results are presented in a "state-diagram," which specifies the stable form of the protein as a function of temperature and SDS concentration. In comparison with other proteins, the equilibrium stability HiC is strongly decreased by SDS. For low SDS concentrations (SDS:HiC molar ratio, MR < 8) this trait is also found for the kinetically controlled thermal aggregation of the protein. At higher MR, however, SDS stabilizes noticeably against irreversible aggregation. We suggest that this relies on electrostatic repulsion of the increasingly negatively charged HiC-SDS complexes. The combined interpretation of calorimetric and binding data allowed the calculation of the changes in enthalpy and heat capacity for the association of HiC and SDS near the saturation point. The latter function was about -410 J mol-1 K-1 or similar to the heat capacity change for micelle formation (-470 J mol-1 K-1). This suggests that SDS is hydrated to a similar extent in the micellar and protein associated forms. The results are discussed in terms of the Wyman theory for linked equilibria. Quantitative analysis along these lines suggests that the reversible thermal unfolding of the protein couples to the binding of 2-3 additional SDS molecules. This corresponds to a 15-20% increase in the binding number. Wyman theory also rationalizes relationships between low affinity and high susceptibility observed in this study. © 2007 American Chemical Society.

Bertram, H.C., Kristensen, M., Østdal, H., Baron, C.P., Young, J.F., Andersen, H.J.

"Does oxidation affect the water functionality of myofibrillar proteins?"

Journal of Agricultural and Food Chemistry, 55 (6), pp. 2342-2348. (2007)

Water-binding properties of myofibrils extracted from porcine muscle, and added hemoglobin with and without exposure to H2O2, were characterized using low-field proton NMR T2 relaxometry. The effects of pH and ionic strength in the samples were investigated as pH was adjusted to 5.4, 6.2, and 7.0 and ionic strength was adjusted to 0.29, 0.46, and 0.71 M, respectively. The formation of dityrosine as a measure of oxidative protein cross-linking revealed a significant increase in dityrosine concentrations upon H2O2 activation. The formation of dityrosine was strongly pH-dependent and increased with decreasing pH. In addition, increased levels of thiobarbituric acid reactive substances were observed upon addition of H 2O2, implying that lipid oxidation was enhanced, however, with a different oxidation pattern as compared to the myofibrillar proteins. Low-field NMR relaxation measurements revealed reduced T2 relaxation times upon H2O2 activation, which corresponds to reduced water-holding capacity upon oxidation. However, a direct relationship between degree of oxidation and T2 relaxation time was not observed with various pH values and ionic strengths, and further studies are needed for a complete understanding of the effect of oxidation on myofibrillar functionality. © 2007 American Chemical Society.

Sonesson, A.W., Brismar, H., Callisen, T.H., Elofsson, U.M.

"Mobility of Thermomyces lanuginosus lipase on a trimyristin substrate surface"

Langmuir, 23 (5), pp. 2706-2713. (2007)

We have studied the mobility of active and inactive Thermomyces lanuginosus lipase (TLL) on a spin-coated trimyristin substrate surface using fluorescence recovery after photobleaching (FRAP) in a confocal microscopy setup. By photobleaching a circular spot of fluorescently labeled TLL adsorbed on a smooth trimyristin surface, both the diffusion coefficient D and the mobile fraction f could be quantified. FRAP was performed on surfaces with different surface density of lipase and as a function of time after adsorption. The data showed that the mobility of TLL was significantly higher on the trimyristin substrate surfaces compared to our previous studies on hydrophobic model surfaces. For both lipase variants, the diffusion decreased to similar rates at high relative surface density of lipase, suggesting that crowding effects are dominant with higher adsorbed amount of lipase. However, the diffusion coefficient at extrapolated infinite surface dilution, D0, was higher for the active TLL compared to the inactive (D0 = 17.9 × 10-11 cm2/s vs D0 = 4.1 × 10-11 cm 2/s, data for the first time interval after adsorption). Moreover, the diffusion decreased with time after adsorption, most evident for the active TLL. We explain the results by product inhibition, i.e., that the accumulation of negatively charged fatty acid products decreased the diffusion rate of active lipases with time. This was supported by sequential adsorption experiments, where the adsorbed amount under flow conditions was studied as a function of time after adsorption. A second injection of lipase led to a significantly lower increase in adsorbed amount when the trimyristin surface was pretreated with active TLL compared to pretreatment of inactive TLL. © 2007 American Chemical Society.

Xu, F., Ding, H.

"A new kinetic model for heterogeneous (or spatially confined) enzymatic catalysis: Contributions from the fractal and jamming (overcrowding) effects"


Applied Catalysis A: General, 317 (1), pp. 70-81. (2007)

Heterogeneous catalysis involves catalysts and reactants separated in different phases. In these systems, the interaction between the reactant and catalyst can be quite different from its homogeneous counterpart, because of the peculiarity of the diffusion and molecular collision processes constricted in spaces with dimension less than three. The fractal theory, developed for mathematic, physical, chemical, and biological processes with inherent irregularity and complexity, can be applied to heterogeneous catalysis. To better understand the heterogeneous enzymatic reactions, a fractal Michaelis kinetics was reformulated, after applying the general fractal formalism to the classical model of homogeneous enzymatic reactions. A kinetic "jamming" effect caused by the overcrowding of enzyme/substrate in confined space was also studied. The new kinetic model was applied to the hydrolysis of cellulose by cellobiohydrolase, a representative heterogeneous biocatalytic system highly fractal due to the strong surface adsorption of the enzyme onto the insoluble substrate as well as to the one-dimensional "processive" enzymatic mechanism. The usefulness of the model for the study and application of other enzymatic reactions was discussed. © 2006 Elsevier B.V. All rights reserved.

Balashev, K., John DiNardo, N., Callisen, T.H., Svendsen, A., Bjørnholm, T.

"Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A2 and Humicola lanuginosa lipase"

Biochimica et Biophysica Acta - Biomembranes, 1768 (1), pp. 90-99. (2007)

An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A2 (PLA2) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir-Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates. © 2006 Elsevier B.V. All rights reserved.

Seema, Kumari, R., Gupta, G., Saluja, D., Kumar, A., Goel, S., Tyagi, Y.K., Gulati, R., Vinocha, A., Muralidhar, K., Dwarakanth, B.S., Rastogi, R.C., Parmar, V.S., Patkar, S.A., Raj, H.G.

"Characterization of protein transacetylase from human placenta as a signaling molecule calreticulin using polyphenolic peracetates as the acetyl group donors"

Cell Biochemistry and Biophysics, 47 (1), pp. 53-64. (2007)

We have earlier shown that a unique membrane-bound enzyme mediates the transfer of acetyl group(s) from polyphenolic peracetates (PA) to functional proteins, which was termed acetoxy drug: protein transacetylase (TAase) because it acted upon several classes of PA. Here, we report the purification of TAase from human placental microsomes to homogeneity with molecular mass of 60 kDa, exhibiting varying degrees of specificity to several classes of PA confirming the structure-activity relationship for the microsome-bound TAase. The TAase catalyzed protein acetylation by a model acetoxy drug, 7,8-diacetoxy-4-methyl coumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with anti-acetyl lysine antibody. TAase activity was severely inhibited in calcium-aggregated microsomes as well as when Ca2+ was added to purified TAase, suggesting that TAase could be a calcium binding protein. Furthermore, the N-terminal sequence analysis of purified TAase (EPAVYFKEQFLD) using Swiss Prot Database perfectly matched with calreticulin (CRT), a major microsomal calcium binding protein of the endoplasmic reticulum (ER). The identity of TAase with CRT was substantiated by the observation that the purified TAase avidly reacted with commercially available antibody raised against the C-terminus of human CRT (13 residues peptide, DEEDATGQAKDEL). Purified TAase also showed Ca2+ binding and acted as a substrate for phosphorylation catalyzed by protein kinase C (PKC), which are hallmark characteristics of CRT. Further, purified placental CRT as well as the commercially procured pure CRT yielded significant TAase catalytic activity and were also found effective in mediating the acetylation of the target protein NADPH cytochrome P-450 reductase by DAMC as detected by Western blot using anti-acetyl lysine antibody. These observations for the first time convincingly attribute the transacetylase function to CRT. Hence, this transacetylase function of CRT is designated calreticulin transacetylase (CRTAase). We envisage that CRTAase plays an important role in protein modification by way of acetylation independent of Acetyl CoA. © Copyright 2007 by Humana Press Inc. All rights of any nature whatsoever reserved.

Rathore, N., Gellman, S.H., De Pablo, J.J.

"Thermodynamic stability of β-peptide helices and the role of cyclic residues"

Biophysical Journal, 91 (9), pp. 3425-3435. (2006)

Beta-peptides are emerging as an attractive class of peptidomimetic molecules. In contrast to naturally occurring α-peptides, short oligomers of β-amino acids (comprising just 4-6 monomers) exhibit stable secondary structures that make them amenable for quantitative, concerted experimental and theoretical studies of the effects of particular chemical interactions on structure. In this work, molecular simulations are used to study the thermodynamic stability of helical conformations formed by β-peptides containing varying proportions of acyclic (β3) and cyclic (ACH) residues. More specifically, several β-peptides differing only in their content of cyclic residues are considered in this work. Previous computational studies of β-peptides have relied mostly on energy minimization of molecular dynamics simulations. In contrast, our study relies on density-of-states based Monte Carlo simulations to calculate the free energy and examine the stability of various folded structures of these molecules along a well-defined order parameter. By resorting to an expanded-ensemble formalism, we are able to determine the free energy required to unfold specific molecules, a quantity that could be measured directly through single-molecule force spectroscopy. Simulations in both implicit and explicit solvents have permitted a systematic study of the role of cyclic residues and electrostatics on the stability of secondary structures. The molecules considered in this work are shown to exhibit stable H-14 helical conformations and, in some cases, relatively stable H-12 conformations, thereby suggesting that solvent quality may be used to manipulate the hydrogen-bonding patterns and structure of these peptides. © 2006 by the Biophysical Society.

Ransbarger, D., Xu, F.

"Activation of laccase by penicillin and derivatives"

Process Biochemistry, 41 (9), pp. 2082-2086. (2006)

Capable of oxidizing a wide range of reducing substances, laccase has a great potential for various biocatalytic applications. Extensive research effort is being made for improving laccase's activity, specificity, and stability. We observed that pre-incubating with penicillin led to a significant activation of a Rhizoctonia solani laccase, although penicillin was not a redox substrate of the enzyme. The activation impacted mainly the kcat of the laccase, and the effect was optimal at pH 5. Ampicillin and penicilloic acid were also active on the laccase (although their effect were weaker than that of penicillin), but 6-aminopenicillanic acid, phenylacetic acid, and cephalexin were not active. The apparent effect from penicillin was attributed to a non-redox interaction with the polypeptide backbone of the laccase. The effect was significantly weaker on Myceliophthora thermophila and Trametes villosa laccases. © 2006 Elsevier Ltd. All rights reserved.

Rodriguez-Larrea, D., Minning, S., Borchert, T.V., Sanchez-Ruiz, J.M.

"Role of Solvation Barriers in Protein Kinetic Stability"

Journal of Molecular Biology, 360 (3), pp. 715-724. (2006)

The stability of several protein systems of interest has been shown to have a kinetic basis. Besides the obvious biotechnological implications, the general interest of understanding protein kinetic stability is emphasized by the fact that some emerging molecular approaches to the inhibition of amyloidogenesis focus on the increase of the kinetic stability of protein native states. Lipases are among the most important industrial enzymes. Here, we have studied the thermal denaturation of the wild-type form, four single-mutant variants and two highly stable, multiple-mutant variants of lipase from Thermomyces lanuginosa. In all cases, thermal denaturation was irreversible, kinetically controlled and conformed to the two-state irreversible model. This result supports that the novel molecular-dynamics-focused, directed-evolution approach involved in the preparation of the highly stable variants is successful likely because it addresses kinetic stability and, in particular, because heated molecular dynamics simulations possibly identify regions of disrupted native interactions in the transition state for irreversible denaturation. Furthermore, we find very large mutation effects on activation enthalpy and entropy, which were not accompanied by similarly large changes in kinetic urea m-value. From this we are led to conclude that these mutation effects are associated to some structural feature of the transition state for the irreversible denaturation process that is not linked to large changes in solvent accessibility. Recent computational studies have suggested the existence of solvation/desolvation barriers in at least some protein folding/unfolding processes. We thus propose that a solvation barrier (arising from the asynchrony between breaking of internal contacts and water penetration) may contribute to the kinetic stability of lipase from T. lanuginosa (and, possibly, to the kinetic stability of other proteins as well). © 2006 Elsevier Ltd. All rights reserved.

Sonesson, A.W., Elofsson, U.M., Brismar, H., Callisen, T.H.

"Adsorption and mobility of a lipase at a hydrophobic surface in the presence of surfactants"

Langmuir, 22 (13), pp. 5810-5817. (2006)

With the aim of being able to manipulate the processes involved in interfacial catalysis, we have studied the effects of a mixture of nonionic/anionic surfactants, C12E1/LAS (1:2 mol %), on the adsorption and surface mobility of a lipase obtained from Thermomyces lanuginosus (ILL). Surface plasmon resonance (SPR) and ellipsometry were used to analyze the competitive adsorption process between surfactants and TLL onto hydrophobic model surfaces intended to mimic an oily substrate for the lipase. We obtained the surface diffusion coefficient of a fluorescently labeled TLL variant on silica silanized with octadecyltrichlorosilane (OTS) by fluorescence recovery after photobleaching (FRAP) on a confocal laser scanning microscope. By means of ellipsometry we calibrated the fluorescence intensity with the surface density of the lipase. The TLL diffusion was measured at different surface densities of the enzyme and at two time intervals after coadsorption with different concentrations of C12E6/LAS. The surfactant concentrations were chosen to represent concentrations below the critical micelle concentration (CMC), in the CMC region, and above the CMC. The apparent TLL surface diffusion was extrapolated to infinite surface dilution, D 0. We found that the presence of surfactants strongly modulated the surface mobility of TLL: with D0 = 0.8 × 10-11 cm2/s without surfactants and D0 = 13.1 × 10 -11 cm2/s with surfactants above the CMC. The increase in lipase mobility on passing the CMC was also accompanied by a 2-fold increase in the mobile fraction of TLL. SPR analysis revealed that surface bound TLL was displaced by C12E6/LAS in a concentration-dependent manner, suggesting that the observed increase in surface mobility imparts bulk-mediated diffusion and so-called rebinding of TLL to the surface. Our combined results on lipase/surfactant competitive adsorption and lipase surface mobility show how surfactants may play an important role in regulating interfacial catalysis from physiological digestion to technical applications such as detergency. © 2006 American Chemical Society.

Raj, H.G., Kumari, R., Seema, Gupta, G., Kumar, R., Saluja, D., Muralidhar, K.M., Kumar, A., Dwarkanath, B.S., Rastogi, R.C., Prasad, A.K., Patkar, S.A., Watterson, A.C., Parmar, V.S.

"Novel function of calreticulin: Characterization of calreticulin as a transacetylase-mediating protein acetylator independent of acetyl CoA using polyphenolic acetates"

Pure and Applied Chemistry, 78 (5), pp. 985-992. (2006).

Our earlier investigations culminated in the discovery of a unique membrane-bound enzyme in mammalian cells catalyzing the transfer of acetyl group from polyphenolic acetates (PAs) to certain functional proteins, resulting in the modulation of their activities. This enzyme was termed acetoxy drug:protein transacetylase (TAase) since it acted upon several classes of PAs. TAase was purified from rat liver microsomes to homogeneity and exhibited the molecular weight of 55 KDa. TAase-catalyzed protein acetylation by PAs was evidenced by the demonstration of immunoreactivity of the acetylated target protein such as nitric oxide synthase (NOS) with anti-acetyl lysine. The possible acetylation of human platelet NOS by PA as described above resulted in the enhancement of intracellular levels of nitric oxide (NO). PAs unlike the parent polyphenols were found to exhibit NO-related physiological effects. The N-terminal sequence was found to show 100% homology with N-terminal sequence of mature calreticulin (CRT). The identity of TAase with CRT, an endoplasmic reticulum (ER) protein, was evidenced by the demonstration of the properties of CRT such as immunoreactivity with anti-calreticulin, binding to Ca2+ ions and being substrate for phosphorylation by protein kinase c (PKC), which are the hallmark characteristics of CRT. These observations for the first time convincingly attribute the transacetylase function to CRT, which possibly plays an important role in protein modification by way of carrying out acetylation of various enzymes through a biochemical mechanism independent of acetyl CoA. © 2006 IUPAC.

Høiberg-Nielsen, R., Fuglsang, C.C., Arleth, L., Westh, P.

"Interrelationships of glycosylation and aggregation kinetics for Peniophora lycii phytase"

Biochemistry, 45 (15), pp. 5057-5066. (2006)

The kinetics of thermally induced aggregation of the glycoprotein Peniophora lycii phytase (Phy) and a deglycosylated form (dgPhy) was studied by dynamic (DLS) and static (SLS) light scattering. This provided a detailed insight into the time course of the formation of small aggregates (∼10-100 molecules) of the enzyme. The thermodynamic stability of the two forms was also investigated using scanning calorimetry (DSC). It was found that the glycans strongly promoted kinetic stability (i.e., reduced the rate of irreversible denaturation) while leaving the equilibrium denaturation temperature, T d, defined by DSC, largely unaltered. At pH 4.5-5.0, for example, dgPhy aggregated ∼200 times faster than Phy, even though the difference in Td was only 1-3 °C. To elucidate the mechanism by which the glycans promote kinetic stability, we measured the effect of ionic strength and temperature on the aggregation rate. Also, the second virial coefficients (B22) for the two forms were measured by SLS. These results showed that the aggregation rate of Phy scaled with the concentration of thermally denatured protein. This suggested first-order kinetics with respect to the concentration of the thermally denatured state. A similar but less pronounced correlation was found for dgPhy, and it was suggested that while the aggregation process for the deglycosylated form is dominated by denatured protein, it also involves a smaller contribution from associating molecules in the native state. The measurements of B22 revealed that dgPhy had slightly higher values than Phy. This suggests that dgPhy interacts more favorably with the buffer than Phy and hence rules out strong hydration of the glycans as the origin of their effect on the kinetic stability. On the basis of this and the effects of pH and ionic strength, we suggest that the inhibition of aggregation is more likely to depend on steric hindrance of the glycans in the aggregated form of the protein. © 2006 American Chemical Society.

Bagger, H.L., Fuglsang, C.C., Westh, P.

"Hydration of a glycoprotein: Relative water affinity of peptide and glycan moieties"

European Biophysics Journal, 35 (4), pp. 367-371. (2006)

Glycosylation, the most prevalent post-translational modification of proteins, affects a number of physical properties including the interactions with the surrounding aqueous solvent. Such glycan-water interactions have been discussed with respect to the increased solubility generally observed for glycoproteins, but experimental support of this correlation remains sparse. We have applied a two-channel calorimetric method to measure the free energy and enthalpy of hydration at 25°C for the glycoprotein phytase (Phy) and a deglycosylated form (dgPhy) of the same protein. Comparisons of results for Phy and dgPhy show that the polypeptide moiety has a higher affinity for water than the glycans. In fact, at moderate hydration levels (∼0.3 g water/g macromolecule) the water uptake appears to be entirely governed by adsorption to the peptide groups. We conclude that strengthened interaction with the solvent is unlikely to be the mechanism underlying the increased solubility and lowered propensity of aggregation often reported to result from the glycosylation of proteins. © EBSA 2005.

Hedin, E.M.K., Høyrup, P., Patkar, S.A., Vind, J., Svendsen, A., Hult, K.

"Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy"

Biochemistry, 44 (50), pp. 16658-16671. (2005)

The triglyceride lipase (EC Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 14185-14196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TLL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface. © 2005 American Chemical Society.

Sonesson, A.W., Callisen, T.H., Brismar, H., Elofsson, U.M.

"Lipase surface diffusion studied by fluorescence recovery after photobleaching"

Langmuir, 21 (25), pp. 11949-11956. (2005)

We have analyzed surface diffusion properties of a variant of Thermomyces lanuginosa lipase (TLL) on hydrophilic silica and silica methylated with dichlorodimethylsilane (DDS) or octadecyltrichlorosilane (OTS). For this study a novel method for analysis of diffusion on solid surfaces was developed. The method is based on fluorescence recovery after photobleaching using confocal microscopy. When a rectangular area of the sample was photobleached, fluorescence recovery could be analyzed as one-dimensional diffusion, resulting in simplified mathematical expressions for fitting the data. The method was initially tested by measuring bovine serum albumin diffusion on glass, which led to a diffusion coefficient in good correspondence to earlier reports. For the analysis of TLL diffusion, ellipsometry data of TLL adsorption were used to calibrate fluorescence intensity to surface density of lipase, enabling measurements of the diffusion coefficient at different surface densities. The average diffusion coefficient was calculated in two time intervals after adsorption. Mobile fraction and diffusion coefficient were lowest on the OTS surface, when extrapolated to infinite surface dilution. Moreover, the diffusion rate decreased with time on the hydrophobic surfaces. Our observations can be explained by the surface dependence on the distribution of orientations and conformations of adsorbed TLL, where the transition from the closed to the catalytically active open and more hydrophobic structure is important. © 2005 American Chemical Society.

Rønne, T.H., Pedersen, L.S., Xu, E.

"Triglycéride selectivity of immobilized thermomyces lanuginosa lipase in interesterification"

JAOCS, Journal of the American Oil Chemists' Society, 82 (10), pp. 737-743. (2005)

The triglyceride (fatty acid) selectivity of an immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) was investigated in lipase-catalyzed interesterification reactions between two mono-acid TG in n-hexane. Tristearin (tri-C18:0) was used as a reference in a series of TG with saturated FA from tri-C4:0 to tri-C20:0, except for tri-C6:0, and in a series of unsaturated FA from tri-C18:1 to tri-C18:3. The quantification was performed by HPLC, and different methods of selectivity evaluation were used. None of the methods used showed any significant differences between the performances of the lipase on the different TG, indicating that Lipozyme TL IM is nonselective toward FA or TG in the system used. A response surface design was used to investigate the influence of water activities (aw) and reaction temperatures on the reactivity of Lipozyme TL IM with a system of tripalmitin (tri-C16:0) and trilaurin (tri-C12:0) in n-hexane. An increase in temperature (40 to 60°C) was found to affect the reactivity of the lipase significantly. The reactivity of Lipozyme TL IM was unaffected by the change in aw from 0.1130 to 0.5289. An increase in aw only led to an increase in FFA formation. Copyright © 2005 by AOCS Press.

Nielsen, A.D., Arleth, L., Westh, P.

"Analysis of protein-surfactant interactions - A titration calorimetric and fluorescence spectroscopic investigation of interactions between Humicola insolens cutinase and an anionic surfactant"

Biochimica et Biophysica Acta - Proteins and Proteomics, 1752 (2), pp. 124-132. (2005)

We have studied interactions of cutinase (HiC) from Humicula insolens and sodium dodecyl sulphate (SDS) by parallel calorimetric and fluorescence investigations of systems in which the concentration of both components was changed systematically. Results from the two methods exhibit a number of synchronous characteristics, when plotted against the total SDS concentration, [SDS]tot. The molecular origin of several of these anomalies was assigned, and five intervals of [SDS]tot in which different modes of interactions dominated were identified. Going from low to high [SDS] tot, these modes were: binding of (a few) SDS to native HiC, formation of oligomeric protein aggregates, denaturation of HiC and adsorption of SDS on denatured protein. For [SDS]tot > 3-6 mM (depending on the protein concentration), the adsorption saturated, and no further protein-detergent interaction could be detected. Two particularly conspicuous anomalies in the calorimetric data were ascribed to respectively denaturation and saturation. It was found that [SDS]tot at these points depended linearly on the (total) protein concentration, [HiC]. We suggest that this reflects the balance between bound and free SDS [SDS]tot = [SDS] aq + [HiC] Nb where [SDS]aq and Nb are, respectively, the aqueous ("free") concentration of SDS and the average number of SDS bound per protein. Interpretation of the results along these lines showed that at 22°C and pH 7.0, HiC denaturesRosgaard, L., Zygadlo, A., Scheller, H.V., Mant, A., Jensen, P.E.

"Insertion of the plant photosystem I subunit G into the thylakoid membrane: In vitro and in vivo studies of wild-type and tagged versions of the protein"

FEBS Journal, 272 (15), pp. 4002-4010. (2005)

Subunit G of photosystem I is a nuclear-encoded protein, predicted to form two transmembrane α-helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma, whilst the N- and C-termini most likely face the lumen. PSI-G constructs in which a His- or Strep-tag is placed at the C-terminus or in the loop region insert with the same topology as wild-type photosystem I subunit G (PSI-G). However, the presence of the tags in the loop make the membrane-inserted protein significantly more sensitive to trypsin, apparently by disrupting the interaction between the loop and the PSI core. Knock-out plants lacking PSI-G were transformed with constructs encoding the C-terminal and loop-tagged PSI-G proteins. Experiments on thylakoids from the transgenic lines show that the C-terminally tagged versions of PSI-G adopt the same topology as wild-type PSI-G, whereas the loop-tagged versions affect the sensitivity of the loop region to trypsin, thus confirming the in vitro observations. Furthermore, purification of PSI complexes from transgenic plants revealed that all the tagged versions of PSI-G are incorporated and retained in the PSI complex, although the C-terminally tagged variants of PSI-G were preferentially retained. This suggests that the loop region of PSI-G is important for proper integration into the PSI core. Our experiments demonstrate that it is possible to produce His- and Strep-tagged PSI in plants, and provide further evidence that the topology of membrane proteins is dictated by the distribution of positive charges, which resist translocation across membranes. © 2005 FEBS.

Nielsen, A.D., Arleth, L., Westh, P.

"Interactions of Humicola insolens cutinase with an anionic surfactant studied by small-angle neutron scattering and isothermal titration calorimetry"

Langmuir, 21 (10), pp. 4299-4307. (2005)

The interaction of cutinase from Humicula insolens (HiC) and sodium dodecyl sulfate (SDS) has been investigated by small-angle neutron scattering (SANS) and isothermal titration calorimetry (ITC). The concerted interpretation of structural and thermodynamic information for identical systems proved valuable in attempts to elucidate the complex modes of protein-detergent interaction. Particularly so at the experimental temperature 22°C, where the formation of SDS micelles is athermal (ΔH = 0), and the effects of protein-detergent interactions stand out clearly in the thermograms. It was found that the effect of SDS on cutinase depended strongly on the sample composition. Thus, addition of SDS corresponding to a molar ratio, nS = nSDS/n HiC of about 10, was associated with the formation of HiC/SDS aggregates, which include more than one protein molecule. The SANS results suggested that on the average such adducts contained two HiC, and the ITC traces showed that they form and break down slowly. At slightly higher SDS concentrations (ns = 10-25) these "dimers" dissociated, and the protein denatured. The denaturation showed the characteristic positive enthalpy change, but the SDS denatured state of HiC was unusually compact with a radius of gyration close to that of the native conformation. Further titration with SDS was associated with exothermic binding to the denatured protein until the saturation point at about ns = 90. At this point, the free monomer concentration was 2.2 mM and the binding number was ∼40 SDS/HiC. Interestingly, this degree of SDS binding (∼0.5 g of SDS/g of HiC) is less than half the amount bound to typical water-soluble proteins. © 2005 American Chemical Society.

Eriksson, J., Malmsten, M., Tiberg, F., Callisen, T.H., Damhus, T., Johansen, K.S.

"Model cellulose films exposed to H. insolens glucoside hydrolase family 45 endo-cellulase - The effect of the carbohydrate-binding module"

Journal of Colloid and Interface Science, 285 (1), pp. 94-99. (2005)

The effects of enzyme structure and activity on the degradation of model cellulose substrates were investigated by ellipsometry for the cellulase Humicola insolens GH45. The inactive variant D10N was found to adsorb at the cellulose surface but also to be incorporated into the cellulose films to an extent that depended on pH. For the native protein, the initial adsorption monitored for the inactive variant D10N was followed by enzyme-mediated degradation of the cellulose films. Again, a dependence on pH was found, such that higher pH resulted in slower enzymatic degradation. Removing the carbohydrate-binding module eliminated this pH dependence but also resulted in a decreased adsorption to the cellulose surface, and in a decreased net catalytic effect. © 2004 Elsevier Inc. All rights reserved.

Ternström, T., Svendsen, A., Akke, M., Adlercreutz, P.

"Unfolding and inactivation of cutinases by AOT and guanidine hydrochloride"

Biochimica et Biophysica Acta - Proteins and Proteomics, 1748 (1), pp. 74-83. (2005)

We present a comparative analysis of the unfolding and inactivation of three cutinases in the presence of guanidine hydrochloride (GdnHCl) and bis(2-ethylhexyl) sodium sulfosuccinate (AOT). Previous investigations have focused on the cutinase from Fusarium solani pisi (FsC). In addition to FsC, the present study includes the cutinase from Humicola insolens (HiC) and a mutant variant of HiC (μHiC) with increased activity and decreased surfactant sensitivity. Equilibrium and time-resolved denaturation by AOT were studied in aqueous solution and reverse micelles, and were compared with GdnHCl denaturation. The far-UV CD and fluorescence denaturation profiles obtained in the aqueous solutions of the two denaturants coincide for all three cutinases, indicating that unfolding is a co-operative two-state process under these conditions. In reverse micelles, the cutinases unfold with mono-exponential rates, again indicating a two-state process. The free energy of denaturation in water was calculated by linear extrapolation of equilibrium data, yielding very similar values for the three cutinases with averages of -11.6 kcal mol -1 and -2.6 kcal mol-1 for GdnHCl and AOT, respectively. Hence, the AOT denatured state (DAOT) is less destabilised than the GdnHCl denatured state (DGdnHCl), relative to the native state in water. Far-UV CD spectroscopy revealed that DAOT retains some secondary structure, while DGdnHCl is essentially unstructured. Similarly, fluorescence data suggest that DAOT is more compact than DGdnHCl. Activity measurements reveal that both DAOT and DGdnHCl are practically inactive (catalytic activity <1% of that of the native enzyme). The fluorescence spectrum of DAOT in reverse micelles did not differ significantly from that observed in aqueous AOT. NMR studies of DAOT in reverse micelles indicated that the structure is characteristic of a molten globule, consistent with the CD and fluorescence data. © 2004 Elsevier B.V. All rights reserved.

Von Ossowski, I., Eaton, J.T., Czjzek, M., Perkins, S.J., Frandsen, T.P., Schülein, M., Panine, P., Henrissat, B., Receveur-Bréchot, V.

"Protein disorder: Conformational distribution of the flexible linker in a chimeric double cellulase"

Biophysical Journal, 88 (4), pp. 2823-2832. (2005)

The structural properties of the linker peptide connecting the cellulose-binding module to the catalytic module in bimodular cellulases have been investigated by small-angle x-ray scattering. Since the linker and the cellulose-binding module are relatively small and cannot be readily detected separately, the conformation of the linker was studied by means of an artificial fusion protein, Cel6BA, in which an 88-residue linker connects the large catalytic modules of the cellulases Cel6A and Cel6B from Humicola insolens. Our data showed that Cel6BA is very elongated with a maximum dimension of 178 A, but could not be described by a single conformation. Modeling of a series of Cel6BA conformers with interdomain separations ranging between 10 Å and 130 Å showed that good Guinier and P(r) profile fits were obtained by a weighted average of the scattering curves of all the models where the linker follows a nonrandom distribution, with a preference for the more compact conformers. These structural properties are likely to be essential for the function of the linker as a molecular spring between the two functional modules. Small-angle x-ray scattering therefore provides a unique tool to quantitatively analyze the conformational disorder typical of proteins described as natively unfolded. © 2005 by the Biophysical Society.

Maurus, R., Begum, A., Kuo, H.-H., Racaza, A., Numao, S., Andersen, C., Tams, J.W., Vind, J., Overall, C.M., Withers, S.G., Brayer, G.D.

"Structural and mechanistic studies of chloride induced activation of human pancreatic α-amylase"

Protein Science, 14 (3), pp. 743-755. (2005)

The mechanism of allosteric activation of α-amylase by chloride has been studied through structural and kinetic experiments focusing on the chloride-dependent N298S variant of human pancreatic α-amylase (HPA) and a chloride-independent TAKA-amylase. Kinetic analysis of the HPA variant clearly demonstrates the pronounced activating effect of chloride ion binding on reaction rates and its effect on the pH-dependence of catalysis. Structural alterations observed in the N298S variant upon chloride ion binding suggest that the chloride ion plays a variety of roles that serve to promote catalysis. One of these is having a strong influence on the positioning of the acid/base catalyst residue E233. Absence of chloride ion results in multiple conformations for this residue and unexpected enzymatic products. Chloride ion and N298 also appear to stabilize a helical region of polypeptide chain from which projects the flexible, substrate binding loop unique to chloride-dependent α-amylases. This structural feature also serves to properly orient the catalytically essential residue D300. Comparative analyses show that the chloride-independent α-amylases compensate for the absence of bound chloride by substituting a hydrophobic core, altering the manner in which substrate interactions are made and shifting the placement of N298. These evolutionary differences presumably arise in response to alternative operating environments or the advantage gained in a particular product profile. Attempts to engineer chloride-dependence into the chloride-independent TAKA-amylase point out the complexity of this system, and the fact that a multitude of factors play a role in binding chloride ion in the chloride-dependent α-amylases.

Høyrup, P., Callisen, T.H., Jensen, M.Ø., Halperin, A., Mouritsen, O.G.

"Lipid protrusions, membrane softness, and enzymatic activity"

Physical Chemistry Chemical Physics, 6 (7), pp. 1608-1615. (2004)

The activity of phospholipase A2 on lipid bilayers displays a characteristic lag-burst behavior that has previously been shown to reflect the physical properties of the substrate. It has remained unclear which underlying molecular mechanism is responsible for this phenomenon. We propose here that protrusions of single lipid molecules out of the bilayer plane could provide such a mechanism. The proposal is supported by a combination of atomic-scale molecular dynamics simulations, theory, and experiments that have been performed in order to investigate the relationship between on the one side lipid protrusion modes and mechanical softness of phospholipid bilayers and on the other side the activity of enzymes acting on lipid bilayers composed of different unsaturated lipids. Specifically, our experiments show a correlation between the bilayer bending rigidity and the apparent Arrhenius activation energy extracted from systematic lag-time versus temperature analyses.

H.L. Bagger; C.C. Fuglsang; P. Westh. (2003)

"Preferential binding of two compatible solutes to the glycan moieties of Peniophora Lycii phytase."

Biochemistry, 42, 10295-10300 (2003)

Regulation of hydration behavior, and the concomitant effects on solubility and other properties, has been suggested as a main function of protein glycosylation. In this work, we have studied the hydration of the heavily glycosylated Peniophora lycii phytase in solutions (0.15-1.1 m) of the two compatible solutes glycerol and sorbitol. Osmometric measurements showed that glycerol preferentially binds to phytase (i.e., glycerol-glycoprotein interactions are more favorable than water-glycoprotein interactions resulting in a preferential accumulation of glycerol near the protein interface), while sorbitol is preferentially excluded from the hydration sphere (water-glycoprotein interactions are the more favorable). To assess contributions from carbohydrate and peptide moieties, respectively, we compared phytase (Phy) and a modified, yet enzymatically active form (dgPhy) in which 90% of the glycans had been removed. This revealed that both polyols showed a pronounced and approximately equal degree of preferential binding to the carbohydrate moiety. This preferential binding of polyols to glycans is in contrast to the exclusion from peptide interfaces observed here (for dgPhy) and in numerous previous reports on nonglycosylated proteins. Despite the distinct differences between peptide and carbohydrate groups, glycosylation had no effect on the stabilizing action provided by glycerol and sorbitol. On the basis of this, it was concluded that the carbohydrate mantle of Phy is equally accessible in the native and thermally denatured states, respectively (most likely fully accessible in both), and thus that its interactions with compatible solutes have little or no effect on conformational equilibria of the glycoprotein. For solubility and aggregation equilibria, on the other hand, the results suggest a polyol-induced stabilization of monomeric forms.

A.D. Nielsen; C.C. Fuglsang; P. Westh.

"Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus alpha-amylase: a calorimetric investigation."

Biochem. J., 373, 337-343 (2003)

The effect of temperature and calcium ions on the denaturation of a recombinant á-amylase from Bacillus halmapalus á-amylase (BHA) has been studied using calorimetry. It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability. Thus an apparent denaturation temperature (Td) of 83 °C in the presence of excess calcium ions was observed, whereas Td decreased to 48 °C when calcium was removed. The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA. An activation energy EA of 101 kJ/mol was found for the denaturation of calcium-depleted BHA. The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures. The two denaturation reactions are coupled with the calcium-binding equilibrium between calciu m-bound and -depleted amylase. A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration. Thus it is estimated that the apparent EA can be increased to approx. 123 kJ/mol by increasing the free-calcium concentration.

A.D. Nielsen; M.L. Pusey; C.C. Fuglsang; P. Westh.

"A proposed mechanism for the thermal denaturation of a recombinant Bacillus halmapalus alpha-amylase ? the effect of calcium ions."

Biochim. Biophys. Acta., 1652, 52-63 (2003)

The thermal stability of a recombinant a-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure. Denaturation of BHA is irreversible with a T-m of approximately 89 degreesC and DSC thermograms can be described using a one-step irreversible model. A 5 degreesC increase in T-m in the presence of 10- fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol- bis[beta-aminoethyl ether] N, N, N', N'-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 degreesC lower T-m as deter mined by both CD and DSC. Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes. Combined interpretation of these observations and structural information on homologous a-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA. The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria. Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.

F. Xu; E.J. Golightly; K.R. Duke; S.F. Lassen; B. Knusen; S. Christensen; K.M. Brown; S.H. Brown; M. Schulein.

"Humicola insolens cellobiose dehydrogenase: cloning, redox chemistry, and "logic gate"-like dual functionality."

Enzyme and Microbial Technology, 28(9-10), 744-753 (2001)

Cellobiose dehydrogenase is a hemoflavoenzyme that catalyzes the sequential electron-transfer from an electron-donating substrate (e.g. cellobiose) to a flavin center, then to an electron-accepting substrate (e.g. quinone) either directly or via a heme center after an internal electron-transfer from the flavin to heme. We cloned the dehydrogenase from Humicola insolens, which encodes a protein of 761 amino acid residues containing an N-terminal heme domain and a C- terminal Ravin domain, and studied how the catalyzed electron transfers are regulated. Based on the correlation between the rate and redox potential, we demonstrated that with a reduced flavin center, the enzym e, as a reductase, could export electron from its heme center by a "outer-sphere" mechanism. With the "resting" flavin center, however, the enzyme could have a peroxidase-like function and import electron to its heme center after a peroxidative activation. The dual functionality of its heme center makes the enzyme a molecular "logic gate", in which the electron how through the heme center can be switched in direction by the redox state of the coupled flavin center.

C.C. Fuglsang; R.M. Berka; J.A. Wahleithner; S. Kauppinen; J.R. Shuster; G. Rasmussen; T. Halkier,; H. Dalbøge B. Henrissat.

"Biochemical analysis of recombinant fungal mutanases."

J. Biol. Chem. 275, 2009-2018 (2000)

Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum  1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH2-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong  -amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 °C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with KD around 0.11 and 0.13 °M at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mut anase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.