Patricia T. Campana

Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles.

The catalytic behaviour of alpha-CT (alpha-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme-micelle interaction leads to an increase in both the V(max) and the affinity for the substrate p -nitrophenyl acetate, indicating higher catalytic efficiency for bound alpha-CT. The bell-shaped profile of alpha-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in alpha-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the alpha-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in alpha-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of alpha-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased alpha-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm(-1), corresponding to the alpha-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the beta-sheet content in micelle-bound alpha-CT. Our data suggest that the higher catalytic efficiency of micelle-bound alpha-CT results from significant conformational changes.

A Flagellum-specific Calcium Sensor

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd ∼9 μm) and EF-4 the low affinity site (Kd ∼120 μm). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.

Screening for stability and compatibility conditions of recombinant human epidermal growth factor for parenteral formulation: Effect of pH, buffers, and excipients

A successful parenteral formulation can be developed by studying stability and compatibility of biopharmaceuticals as a function of solution composition. Here, we evaluate the influence of pH, buffers, ionic strength, protein concentration and presence of excipients on recombinant human epidermal growth factor (rhEGF) stability. The stability was accessed by reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC-HPLC), enzyme-linked immunosorbent assay (ELISA), Far-UV circular dichroism (CD) and light scattering. The overall maximal stability was obtained in pH near to 7.0 in phosphate, Tris and histidine buffers as the results of the different methods revealed. The CD results revealed that this protein is stable in an extensive pH range. Aggregation of rhEGF was minimized at pH values ranged from 6.0 to 8.0 as indicated the SEC-HPLC and light scattering results. Nor the ionic strength neither the rhEGF concentration had significant effect on the reaction rate constants. Most rhEGF-excipient instability occurs among this protein and reducing sugars. Polymers like poly(ethylene glycol) (PEG) and polysorbates increased methionine oxidation. The rhEGF oxidation and deamidation were the most common degradation pathways. This research identified critical solution factors to be considered for the development of a successful rhEGF parenteral formulation.

Biological and conformational evaluation of angiotensin II lactam bridge containing analogues

Angiotensin II (AII) is the active octapeptide product of the renin enzymatic cascade, which is responsible for sustaining blood pressure. In an attempt to establish the AII-receptor-bound conformation of this octapeptide, we designed conformationally constrained analogues by scanning the entire AII sequence with an i-(i+2) and i-(i+3) lactam bridge consisting of an Asp-(Xaa)(n)-Lys scaffold. Most analogues presented low agonistic activity when compared to AII in the different bioassays tested. The exceptions are cyclo(0-1a) [Asp(0), endo-(Lys(1a))]-AII (1) and [Asp(0), endo-(Lys(1a))]-AII (2), both of which showed activity similar to AII. Based on peptide 1 and the analogue cyclo(3-5)[Sar(1), Asp(3), Lys(5)]-AII characterized by Matsoukas et al., we analyzed the agonistic and antagonistic activities, respectively, through a new monocyclic peptide series synthesized by using the following combinations of residues as bridgehead elements for the lactam bond formation: D- or L-Asp combined with D- or L-Lys or L-Glu combined with L-Orn. Six analogues showed an approximately 20% increase in biological activity when compared with peptide (1) and were equipotent to AII. In contrast, six analogues presented antagonistic activity. These results suggest that the position of the lactam bridge is more important than the bridge length or chirality for recognition of and binding to the angiotensin II AT1-receptor.

Chemical reduction of carboxyl groups in heparin abolishes its vasodilatory activity

Previous studies have shown that heparin induces vascular relaxation via integrin‐dependent nitric oxide (NO)‐mediated activation of the muscarinic receptor. The aim of this study was to identify the structural features of heparin that are necessary for the induction of vasodilatation. To address this issue, we tested heparin from various sources for their vasodilatation activities in the rat aorta ring. Structural and chemical characteristics of heparin, such as its molecular weight and substitution pattern, did not show a direct correlation with the vasodilation activity. Principal component analysis (PCA) of circular dichroism (CD), 1H‐nuclear magnetic resonance (NMR) and vasodilation activity measurements confirmed that there is no direct relationship between the physico‐chemical nature and vasodilation activity of the tested heparin samples. To further understand these observations, unfractionated heparin (UFH) from bovine intestinal mucosa, which showed the highest relaxation effect, was chemically modified. Interestingly, non‐specific O‐ and N‐desulfation of heparin reduced its anticoagulant, antithrombotic, and antihemostatic activities, but had no effect on its ability to induce vasodilation. On the other hand, chemical reduction of the carboxyl groups abolished heparin‐induced vasodilation and reduced the affinity of heparin toward the extracellular matrix (ECM). In addition, dextran and dextran sulfate (linear non‐sulfated and highly sulfated polysaccharides, respectively) did not induce significant relaxation, showing that the vasodilation activity of polysaccharides is neither charge‐dependent nor backbone unspecific. Our results suggest that desulfated heparin molecules may be used as vasoactive agents due to their low side effects. J. Cell. Biochem. 113: 1359–1367, 2012.

Heterologous expression, characterization and structural studies of a hydrophobic peptide from the HIV-1 p24 protein

Proteins from the inner core of HIV-1, such as the capsid protein (p24), are involved in crucial processes during the virus life cycle. The p24 protein plays an active structural role in the Gag protein and in its mature form. This work describes the production of a peptide derived from the p24 C-terminal, TLRAEQASQEVKNWMTETLLVQNA, using recombinant technology. This region (p24-3) is involved in interfaces during the p24 dimerization, which occurs during capsid assembly. The p24-3 sequence was obtained by a synthetic gene strategy and inserted into the pET 32a expression vector to produce soluble fusion protein in Escherichia coli BL21(DE3). This strategy leads to an incorporation of three amino acid residues (AMA) in the N-terminal of the native sequence to form the recombinant p24-3 (rp24-3). The rp24-3 was purified by reverse phase chromatography to homogeneity, as inferred by mass spectrometry and protein sequence analysis. Structural studies using circular dichroism and steady-state fluorescence showed that the rp24-3 is structured by helical and beta elements. As a function of its hydrophobic character it can self-associate forming oligomers. We present in this paper the first development of a suitable expression system for rp24-3, which provides high amounts of the peptide. This strategy will allow the development of new antiviral (HIV) agents.

Development of glucose oxidase-based bioanodes for enzyme fuel cell applications

We fabricated an enzyme fuel cell (EFC) device based on glucose as fuel and glucose oxidase (GOx) as biocatalyst. As a strategy to improve GOx stability, preserving at the same time the enzyme catalytic activity, we propose an immobilization procedure to entrap GOx in a polymer matrix based on Nafion and multiwalled carbon nanotubes. Circular dichroism (CD) spectra were recorded to study changes in the 3D structure of GOx that might be generated by the immobilization procedure. The comparison between the CD features of GOx immobilized and free in solution indicates that the shape of the spectra and position of peaks do not significantly change. The bioelectrocatalytic activity toward glucose oxidation of immobilized GOx was studied by cyclic voltammetry and chronoamperometry experiments. Such electrochemical experiments allow monitoring the rate of GOx-catalyzed glucose oxidation and extrapolating GOx kinetic parameters. Results demonstrate that immobilized GOx has high catalytic efficiency, due the maintaining of regular and well-ordered structure of the immobilized enzyme, as indicated by spectroscopic findings. Once investigated the electrode structure–property relationship, an EFC device was assembled using the GOx-based bioanode, and sulfonated poly ether ether ketone as electrolyte membrane. Polarization and power density curves of the complete EFC device were acquired, demonstrating the suitability of the immobilization strategy and materials to be used in EFCs.

Conformational stability of peanut agglutinin using small angle X-ray scattering

In this work, quaternary conformational studies of peanut agglutinin (PNA) have been carried out using small-angle X-ray scattering (SAXS). PNA was submitted to three different conditions: pH variation (2.5, 4.0, 7.4 and 9.0), guanidine hydrochloride presence (0.5-2M) at each pH value, and temperature ranging from 25 to 60°C. All experiments were performed in the absence and presence of T-antigen to evaluate its influence on the lectin stability. At room temperature and pH 4.0, 7.4 and 9.0, the SAXS curves are consistent with the PNA scattering in its crystallographic native homotetrameric structure, with monomers in a jelly roll fold, associated by non-covalent bonds resulting in an open structure. At pH 2.5, the results indicate that PNA tends to dissociate into smaller sub-units, as dimers and monomers, followed by a self-assembling into larger aggregates. Furthermore, the conformational stability under thermal denaturation follows the pH sequence 7.4>9.0>4.0>2.5. Such results are consistent with the conformational behavior found upon GndHCl influence. The presence of T-antigen does not affect the protein quaternary structure in all studied systems within the SAXS resolution.

Tuning Structural Changes in Glucose Oxidase for Enzyme Fuel Cell Applications

Stabilization and electrical contacting of redox enzymes with electrodes are fundamental requirements for bioelectronics devices, including biosensors and enzyme fuel cells (EFCs). In this study, we show increased glucose oxidase (GOx) stability by immobilization with Nafion. The immobilization process affected GOx conformation but was not detrimental to its activity, which was maintained for more than 120 days. The GOx/Nafion system was interfaced to a carbon cloth electrode and assembled in a prototypal EFC fed with glucose. Polarization and power density curves demonstrated that GOx/Nafion system was able to generate power, exploiting a Nafion-assisted electron transfer process to the electrode. Our findings are consistent with the onset of pH-dependent conformational equilibrium for the enzyme secondary structure and its active site. Significantly, the protective effect exerted by Nafion on the enzyme structure may be tuned by varying parameters such as the pH to fabricate durable EFCs with good electrocatalytic performance.

Structure-activity relationship of Trp-containing analogs of the antimicrobial peptide gomesin.

Gomesin (Gm) has a broad antimicrobial activity making it of great interest for development of drugs. In this study, we analyzed three Gm analogs, [Trp1]‐Gm, [Trp7]‐Gm, and [Trp9]‐Gm, in an attempt to gain insight into the contributions of different regions of the peptide sequence to its activity. The incorporation of the tryptophan residue in different positions has no effect on the antimicrobial and hemolytic activities of the Gm analogs in relation to Gm. Spectroscopic studies (circular dichroism, fluorescence and absorbance) of Gm and its analogs were performed in the presence of SDS, below and above its critical micelle concentration (CMC) (~8 mM), in order to monitor structural changes induced by the interaction with this anionic surfactant (0–15 mM). Interestingly, we found that the analogs interact more strongly with SDS at low concentrations (0.3‐6.0 mM) than close to or above its CMC. This suggests that SDS monomers are able to cover the whole peptide, forming large detergent‐peptide aggregates. On the other hand, the peptides interact differently with SDS micelles, inserting partially into the micelle core. Among the peptides, Trp in position 1 becomes more motionally‐restricted in the presence of SDS, probably because this residue is located at the N‐terminal region, which presents higher conformational freedom to interact stronger with SDS molecules. Trp residues in positions 7 and 9, close to and in the region of the turn of the molecule, respectively, induced a more constrained structure and the compounds cannot insert deeper into the micelle core or be completely buried by SDS monomers. Copyright