Andrea M. Santos

Activity, conformation and dynamics of cutinase adsorbed on poly(methyl methacrylate) latex particles

The adsorption of a recombinant cutinase from Fusarium solani pisi onto the surface of 100 nm diameter poly(methyl methacrylate) (PMMA) latex particles was evaluated. Adsorption of cutinase is a fast process since more than 70% of protein molecules are adsorbed onto PMMA at time zero of experiment, irrespective of the tested conditions. A Langmuir-type model fitted both protein and enzyme activity isotherms at 25 degrees C. Gamma(max) increased from 1.1 to 1.7 mg m(-2) and U(max) increased from 365 to 982 U m(-2) as the pH was raised from 4.5 to 9.2, respectively. A decrease (up to 50%) in specific activity retention was observed at acidic pH values (pH 4.5 and 5.2) while almost no inactivation (eta(act) congruent with 87-94%) was detected upon adsorption at pH 7.0 and 9.2. Concomitantly, far-UV circular dichroism (CD) spectra evidenced a reduction in the alpha-helical content of adsorbed protein at acidic pH values while at neutral and alkaline pH the secondary structure of adsorbed cutinase was similar to that of native protein. Fluorescence anisotropy decays showed the release of some constraints to the local motion of the Trp69 upon protein adsorption at pH 8.0, probably due to the disruption of the tryptophan-alanine hydrogen bond when the tryptophan interacts with the PMMA surface. Structural data associated with activity measurements at pH 7.0 and 9.2 showed that cutinase adsorbs onto PMMA particles in an end-on orientation with active site exposed to solvent and full integrity of cutinase secondary structure. Hydrophobic interactions are likely the major contribution to the adsorption mechanism at neutral and alkaline pH values, and a higher amount of protein is adsorbed to PMMA particles with increasing temperature at pH 9.2. The maximum adsorption increased from 88 to 140 mg cutinase per g PMMA with temperature raising from 25 to 50 degrees C, at pH 9.2.

Fluorescence of the Single Tryptophan of Cutinase: Temperature and pH Effect on Protein Conformation and Dynamics {

The cutinase from Fusarium solani pisi is an enzyme with a single l‐tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala–Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge–transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10‐fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling‐in‐cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein‐melting temperature and to protein‐backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.

Molecular characterization of the interaction of crotamine-derived nucleolar targeting peptides with lipid membranes

A novel class of cell-penetrating, nucleolar-targeting peptides (NrTPs), was recently developed from the rattlesnake venom toxin crotamine. Based on the intrinsic fluorescence of tyrosine or tryptophan residues, the partition of NrTPs and crotamine to membranes with variable lipid compositions was studied. Partition coefficient values (in the 10(2)-10(5) range) followed essentially the compositional trend POPC:POPG≤POPG<POPC≤POPC:cholesterol. Leakage assays showed that NrTPs induce minimal lipid vesicle disruption. Fluorescence quenching of NrTPs, either by acrylamide or lipophilic probes, revealed that NrTPs are buried in the lipid bilayer only for negatively-charged membranes. Adoption of partial secondary structure by the NrTPs upon interaction with POPC and POPG vesicles was demonstrated by circular dichroism. Translocation studies were conducted using a novel methodology, based on the confocal microscopy imaging of giant multilamellar vesicles or giant multivesicular liposomes. With this new procedure, which can now be used to evaluate the membrane translocation ability of other molecules, it was demonstrated that NrTPs are able to cross lipid membranes even in the absence of a receptor or transmembrane gradient. Altogether, these results indicate that NrTPs interact with lipid bilayers and can penetrate cells via different entry mechanisms, reinforcing the applicability of this class of peptide as therapeutic tools for the delivery of molecular cargoes.

Orientation of cutinase adsorbed onto PMMA nanoparticles probed by tryptophan fluorescence.

The fluorescence of the single tryptophan (Trp69) of cutinase from Fusarium solani pisi, free in aqueous solution and adsorbed onto the surface of poly(methyl methacrylate) (PMMA) latex particles, was studied at pHs of 4.5 and 8.0. The monodisperse PMMA particles (d=106.0+/-0.1 nm) were coated with a quite compact monolayer of cutinase at both pH values. The Trp decay curve of the folded free cutinase in solution can only be fitted with a sum of four exponentials with lifetimes of 0.05, 0.3-0.4, 2-3, and 6-7 ns, irrespective of pH. The 50 ps lifetime is attributed to the population of Trp residues hydrogen bonded with the Ala32 and strongly quenched by a close disulfide bridge, while the other lifetimes are due to the non-hydrogen-bonded Trp rotamers. The 50 ps Trp lifetime component disappears by temperature melting and upon protein adsorption, owing to the disruption of the Trp-Ala hydrogen bond and the release of the Trp residue from the vicinity of the disulfide bridge. This shows that cutinase adsorption occurs by the region of the protein where the Trp is located, which agrees with the retention of cutinase enzymatic activity by adsorption at basic pH.

Rubredoxin mutant A51C unfolding dynamics: a Förster Resonance Energy Transfer study.

The unfolding dynamics of the rubredoxin mutant A51C (RdA51C) from Desulfovibrio vulgaris (DvRd) was studied on the temperature range from 25 degrees C to 90 degrees C and by incubation at 90 degrees C. By Förster Resonance Energy Transfer (FRET) the donor (D; Trp37) to acceptor (A; 1,5-IAEDANS) distance distribution was probed at several temperatures between 25 degrees C and 90 degrees C, and incubation times at 90 degrees C. From 25 degrees C to 50 degrees C the half-width distributions values (hw) are small and the presence of a discrete D-A distance was considered. At temperatures higher than 60 degrees C broader hw values were observed reflecting the existence of a distance distribution. The protein denaturation was only achieved by heating the solution for 2h at 90 degrees C, as probed by the increase of the D-A mean distance. From Trp fluorescence it was shown that its vicinity was maintained until approximately 70 degrees C, being the protein hydrodynamic radius invariant until 50 degrees C. However, at approximately 70 degrees C a change in the partial unfolding kinetics indicates the disruption of specific H-bonds occurring in the hydrophobic core. The red shift of 13nm, observed on the Trp37 emission, confirms the exposition of Trp to solvent after protein incubation at 90 degrees C for 2.5h.