Peter Fojan

What distinguishes an esterase from a lipase: a novel structural approach.

Esterases and lipases both hydrolyse ester bonds. Whereas the lipases display high activity towards the aggregated state of its substrate, the esterases typically show highest activity towards the soluble state of its substrate. We have compared the amino acid sequence, the 3D-structure as well as the pH-dependent electrostatic signature of selected members of the two families, for which 3D-structural information is publicly available. Lipases display a statistically significant enhanced occurrence of non-polar residues close to the surface, clustering around the active-site. Lid opening appears to strengthen this pattern further. As we have proposed earlier the active site of lipases displays negative potential in the pH-range associated with their maximum activity, typically at pH values above 8. The esterases show a very similar pattern, however, at pH values around 6 correlated with their usually lower pH-activity optimum.

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light‐exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV‐induced changes in three‐dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon‐induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two‐state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH‐dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV‐induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far‐reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.

Protein Engineering the Surface of Enzymes

The protein surface is the interface through which a protein senses the external world. Its composition of charged, polar and hydrophobic residues is crucial for the stability and activity of the protein. The charge state of seven of the twenty naturally occurring amino acids is pH dependent. A total of 95% of all titratable residues are located on the surface of soluble proteins. In evolutionary related families of proteins such residues are particularly prone to substitutions, insertions and deletions. We present here an analysis of the residue composition of 4038 proteins, selected from 125 protein families with < 25% identity between core members of each family. Whereas only 16.8% of the residues were truly buried, 40.7% were > 30% exposed on the surface and the remainder were < 30% exposed. The individual residue types show distinct differences. The data presented provides an important new approach to protein engineering of protein surfaces. Guidelines for the optimization of solvent exposure for a given residue are given. The cutinase family of enzymes has been investigated. The stability of native cutinase has been studied as a function of pH, and has been compared with the cutinase activity towards tributyrin. Whereas the onset of enzymatic activity is linked with the deprotonation of the active site HIS188, destabilization of the 3D structure as determined by differential scanning calorimetry is coupled with the loss of activity at very basic pH values. A modeling investigation of the pH dependence of the electrostatic potentials reveals that the activity range is accompanied by the development of a highly significant negative potential in the active site cleft. The 3D structures of three mutants of the Fusarium solani pisi cutinase have been solved to high resolution using X-ray diffraction analysis. Preliminary X-ray data are presented.

Dynamic light scattering of cutinase in AOT reverse micelles.

The fungal lipolytic enzyme cutinase, incorporated into sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles has been investigated using dynamic light scattering. The reversed micelles form spontaneously when water is added to a solution of sodium bis-(2ethylhexyl) sulfosuccinate in isooctane. When an enzyme is previously dissolved in the water before its addition to the organic phase, the enzyme will be incorporated into the micelles. Enzyme encapsulation in reversed micelles can be advantageous namely to the conversion of water insoluble substrates and to carry out synthesis reactions. However protein unfolding occurs in several systems as for cutinase in sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles. Dynamic light scattering measurements of sodium bis-(2ethylhexyl) sulfosuccinate reversed micelles with and without cutinase were taken at different water to surfactant ratios. The results indicate that cutinase was attached to the micellar wall and that might cause cutinase unfolding. The interactions between cutinase and the bis-(2ethylhexyl) sulfosuccinate interface are probably the driving force for cutinase unfolding at room temperature. Twenty-four hours after encapsulation, when cutinase is unfolded, a bimodal distribution was clearly observed. The radii of reversed micelles with unfolded cutinase were determined and found to be considerable larger than the radii of the empty reversed micelles. The majority of the reversed micelles were empty (90-96% of mass) and the remainder (4-10%) containing unfolded cutinase were larger by 26-89 A.

Synthesis and photovoltaic properties from inverted geometry cells and roll-to-roll coated large area cells from dithienopyrrole-based donor–acceptor polymers

A series of donor–acceptor low band gap polymers composed of alternating dithienopyrrole or its derivative as donors and phthalimide or thieno[3,4-c]pyrrole-4,6-dione as acceptors (P1–P4) are synthesized by Stille coupling polymerization. All polymers show strong absorption in the visible region, for P2 and P4 possessing thieno[3,4-c]pyrrole-4,6-dione as an acceptor, their film absorption covers the region of 500–800 nm and 500–750 nm respectively, which makes them attractive as low band gap polymer solar cell (PSC) materials. With the incorporation of thiophene bridges, P3 and P4 have 0.24 and 0.21 eV higher HOMO energy levels than P1 and P2, respectively. A bandgap as low as 1.66 eV is obtained for P2. An up-scaling experiment is performed on bulk-heterojunction PSCs with an inverted device geometry fabricated on a small scale by spin coating and on a large scale using roll-to-roll (R2R) slot-die coating and screen printing. In both cases the best performing polymer is P2 with a Voc of 0.56 V, a Jsc of −12.6 mA cm−2, a FF of 40.3%, and a PCE of 2.84% for small spin coated devices, and a Voc of 0.56 V, a Jsc of −8.18 mA cm−2, a FF of 30.7%, and a PCE of 1.40% are obtained for R2R-fabricated devices with a significantly better performance than a standard P3HT/PCBM driven device.

Patterned poly(lactic acid) films support growth and spontaneous multilineage gene expression of adipose-derived stem cells

Conventional culture surfaces do not provide optimal environmental cues for expansion or differentiation of adult stem cells. Aiming to increase the efficiency of the in vitro culture conditions, biocompatible and biodegradable biomaterials such as poly(lactic acid) (PLA) have been proposed to engineer the stem cell microenvironment. In this study, we explored the feasibility of using PLA substrates to control the responses of adipose-derived stem cells (ASCs). The substrates consisted of flat and patterned PLA films fabricated by casting a chloroform-PLA solution on a glass surface. Patterning was achieved through the condensation of nano-sized water droplets during chloroform evaporation, which resulted in films displaying irregularly distributed circular indentations with a mean diameter of 248±65 nm. Both types of PLA substrates were assessed for protein adsorption using fibronectin and in vitro cell culturing. Tissue-culture polystyrene (TCPS) plates were used as control surfaces. The experiments demonstrated that the patterned PLA substrates had a significantly higher fibronectin adsorption capacity when compared with the flat counterparts. For the entire duration of the culture period, there was no significant difference in cell growth rate on the PLA surfaces with respect to TCPS despite signs of reduced adhesion. In addition, the semi-quantitative real-time RT-PCR analysis of a set of 14 lineage-specific genes revealed that the PLA-related transcriptional activity significantly surpassed that of TCPS. Remarkably, when assessing the effect of patterning, the patterned films proved superior regarding the activation of genes involved in the skeletal myogenic, cardiomyogenic, chondrogenic, and adipogenic pathways. Taken together, our data provide evidence that the surface patterning can exert such an influence on the stem cell microenvironment that the differentiation process can be effectively modulated. Consequently, the patterned PLA surfaces could potentially be used as a platform for localized delivery and engraftment of stem cells.

Cutinase-AOT interactions in reverse micelles: the effect of 1-hexanol.

Cutinase encapsulated in dioctyl sulfosuccinate reverse micelles displays very low stability, undergoing fast denaturation due to an anchoring at the micellar interface. The denaturation process and the structure of the reverse micelle were characterized using biophysical techniques. The kinetics of denaturation observed from fluorescence match the increase of the hydrodynamic radius of reverse micelles. Denaturation in reverse micelles is mainly the unfolding of the three-dimensional structure since the decrease in the circular dichroism ellipticity in the far-UV range is very small. The process is accompanied by an increase in the steady-state anisotropy, as opposed to what happens for denaturation in aqueous solution. Since 1-hexanol used as co-surfactant in dioctyl sulfosuccinate reverse micelles slows or even prevents cutinase denaturation, its effect on cutinase conformation and on the size of reverse micelles was analyzed. When 1-hexanol is present, cutinase is encapsulated in a large reverse micelle, as deduced from dynamic light scattering. The large reverse micelle filled with cutinase was built from the fusion of reverse micelles according to a pseudo-unimolecular process ranging in time from a few minutes to 2h depending on the reverse micellar concentration. This slow equilibrium driven by the encapsulated cutinase has not been reported previously. The encapsulation of cutinase in dioctyl sulfosuccinate reverse micelles establishes a completely new equilibrium characterized by a bimodal population of empty and filled reverse micelles, whose characteristics depend greatly on the interfacial characteristics, that is, on the absence or presence of 1-hexanol.

The insect defensin lucifensin from Lucilia sericata.

Today, the use of maggot debridement therapy has undergone a renaissance as it is increasingly being used when treating chronic wounds. However, it has also become a target in the search for new antibiotics as the disinfecting abilities of L. sericata maggots also apply to multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) (Kerridge et al. 2005). The wound healing properties of the larvae occurs mainly through debridement and disinfection (Dumville et al. 2009). The removal of bacteria occurs through ingestion and degradation of the bacteria, however, the larvae also produce excretions/secretions (ES) which confer reduction in the amount of especially Gram positive bacteria (Jaklic et al. 2008). Recently a 40 residue antimicrobial peptide (lucifensin) has been found in various tissues and the extracellular secretion (ES) of L. sericata, isolated and sequenced (Čeřovský et al. 2009). It was found by studying radial diffusion assays (RDA) of fractions from reverse phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography. The peptide has high sequence similarity with insect defensins from other insects. The sequences of insect defensins are highly conserved, especially the 6 cysteines which have the pairing Cys1–Cys4, Cys2–Cys5 and Cys3–Cys6 of which the first connects the loop and second b-strand while the two remaining pairs connect the a-helix and first b-strand. Sapecin from Sarcophaga peregrina (Hanzawa et al. 1990, PDB entry: 1L4V) and insect defensin A from Phormia terranova (Cornet et al. 1995, PDB entry: 1ICA) are two well studied insect defensins, for which the 3D structure has already been determined. Many of the studies have focused on elucidating the defensin-membrane interactions as these are believed to cause antimicrobial activity, which for insect defensins primarily is observed against Gram-positive bacteria. A study of the effect of insect defensin A on Micrococcus luteus revealed that the defensin permeabilized the cytoplasmic membrane leading to loss of cytoplasmic potassium, partial depolarization of the inner membrane and decrease in cytoplasmic ATP (Cociancich et al. 1993). It was proposed that the cause of these effects is a pore formation in the cytoplasmic membrane by defensin A oligomers. However, a recent study of the fungal defensin plectasin, which also has an insect defensin motif, showed that it had a bacterial killing mechanism which did not involve membrane permeabilisation (Schneider et al. 2010). Instead plectasin was found to target the cell wall precursor lipid II, which it binds to, thus inhibiting synthesis of cell walls. In the same study it was also found that lucifensin is able to bind to lipid II in a stoichiometric ratio and therefore possibly has two mechanisms of action. M. K. E. Nygaard P. Fojan Institute for Physics and Nanotechnology, Aalborg University, Skjernvej 4A, 9220 Aalborg Ø, Denmark

Overexpression of NRPS4 leads to increased surface hydrophobicity in Fusarium graminearum.

The plant pathogen Fusarium graminearum is the infamous cause of Fusarium head blight worldwide resulting in significant losses of yield and reduced grain feed quality. It also has the potential to produce a range of small bioactive peptides produced by the non ribosomal peptide synthetases (NRPSs). Most of these are unknown as F. graminearum contains 19 NRPS encoding genes, but only three have been assigned products. For the first time, we use deletion and overexpression mutants to investigate the functions and product of NRPS4 in F. graminearum. Deletion of NRPS4 homologues in Alternaria brassicicola and Cochloibolus heterostrophus has been shown to result in mutants unable to repel water. In a time study of surface hydrophobicity we observed that water droplets could penetrate 7 d old colonies of the NRPS4 deletion mutants. Loss in ability to repel water was first observed on 13 d old cultures of the wild type strain, whereas the overexpression strain remained water repellant throughout the 38 d time study. The conidia of both mutants were examined and those of the overexpression mutant showed distinct morphological differences in form of collapsed cells. These observations might suggest that the peptide product of NRPS4 could be an architectural factor in the cell walls of Fusarium or an indirect regulator of hydrophobicity.

Fouling of enhanced biological phosphorus removal-membrane bioreactors by humic-like substances

Fouling by free extracellular polymeric substances was studied in an enhanced biological phosphorus removal-membrane bioreactor. It was demonstrated that the free extracellular polymeric substances, primarily consisting of humic-like substances, were adsorbed to the membrane used in the enhanced biological phosphorus removal-membrane bioreactor plant. Infrared analyses indicated the presence of the humic-like substances on the membranes active surface after filtration of the free extracellular polymeric substances suspension. Scanning electron microscopy showed the presence of a gel layer on the membrane surface after filtration of the free extracellular polymeric substances suspension. The gel layer caused a significant decline in water flux. This layer was not entirely removed by a backwashing, and the membranes water flux could not be re-established. The membrane used in the enhanced biological phosphorus removal-membrane bioreactor plant showed infrared spectra similar to that fouled by the free extracellular polymeric substances suspension in the laboratory. Thus, the results of this study show the importance of humic-like substances in irreversible fouling of enhanced biological phosphorus removal-membrane bioreactor systems.