Sulfated polysaccharide extracted of the green algae Caulerpa racemosa increase the enzymatic activity and paw edema induced by sPLA2 from Crotalus durissus terrificus venom

Sulfated polysaccharides derived from seaweed have shown great potential for use in the development of new drugs. In this study, we observed that a low-molecular-weight sulfated polysaccharide from Caulerpa racemosa, termed CrSP, could interact with secretory phospholipase A2 (sPLA2) isolated from Crotalus durissus terrificus venom. When native sPLA2 (14 kDa) was incubated with CrSP, they formed a molecular complex (sPLA2:CrSP) with a molecular mass of 32 kDa, approximately. Size exclusion chromatography experiments suggested that CrSP formed a stable complex with sPLA2. We belived that sPLA2 and SPCr are involved an ionic interaction between negatively charged CrSP and the positively charged basic amino acid residues of sPLA2, because this interaction induced significant changes in sPLA2 enzymatic and pharmacological activities. CrSP caused a significant increase in sPLA2 enzymatic and bactericidal activity and increased its edematogenic effect. A pharmacological assay showed that the myotoxic activity of sPLA2:CrSP is unrelated to its enzymatic activity and that sPLA2:CrSP may have a practical application as a natural antibacterial agent for use in humans and commercially raised animals.

Identification of folding intermediates of streblin, the most stable serine protease: biophysical analysis

Streblin, a serine proteinase from plant Streblus asper, has been used to investigate the conformational changes induced by pH, temperature, and chaotropes. The near/far UV circular dichroism activities under fluorescence emission spectroscopy and 8-aniline-1-naphthalene sulfonate (ANS) binding have been carried out to understand the unfolding of the protein in the presence of denaturants. Spectroscopic studies reveal that streblin belongs to the alpha+beta class of proteins and exhibits stability towards chemical denaturants, guanidine hydrochloride (GuHCl). The pH-induced transition of this protein is noncooperative for transition phases between pH 0.5 and 2.5 (midpoint, 1.5) and pH 2.5 and 10.0 (midpoint, 6.5). At pH 1.0 or lower, the protein unfolds to form acid-unfolded state, and for pH 7.5 and above, protein turns into an alkaline denatured state characterized by the absence of ANS binding. At pH 2.0 (1M GuHCl), streblin exists in a partially unfolded state with characteristics of amolten globule state. The protein is found to exhibit strong and predominant ANS binding. In total, six different intermediate states has been identified to show protein folding pathways.

Deconstructing the DGAT1 enzyme: Binding sites and substrate interactions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interaction of biologically-relevant peptides with membrane model systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Assembly and characterization of supramolecular architectures for biosensor applications

The research has included the efforts in designing, assembling and structurally and functionally characterizing supramolecular biofunctional architectures for optical biosensing applications. In the first part of the study, a class of interfaces based on the biotin-NeutrAvidin binding matrix for the quantitative control of enzyme surface coverage and activity was developed. Genetically modified ß-lactamase was chosen as a model enzyme and attached to five different types of NeutrAvidin-functionalized chip surfaces through a biotinylated spacer. All matrices are suitable for achieving a controlled enzyme surface density. Data obtained by SPR are in excellent agreement with those derived from optical waveguide measurements. Among the various protein-binding strategies investigated in this study, it was found that stiffness and order between alkanethiol-based SAMs and PEGylated surfaces are very important. Matrix D based on a Nb2O5 coating showed a satisfactory regeneration possibility. The surface-immobilized enzymes were found to be stable and sufficiently active enough for a catalytic activity assay. Many factors, such as the steric crowding effect of surface-attached enzymes, the electrostatic interaction between the negatively charged substrate (Nitrocefin) and the polycationic PLL-g-PEG/PEG-Biotin polymer, mass transport effect, and enzyme orientation, are shown to influence the kinetic parameters of catalytic analysis. Furthermore, a home-built Surface Plasmon Resonance Spectrometer of SPR and a commercial miniature Fiber Optic Absorbance Spectrometer (FOAS), served as a combination set-up for affinity and catalytic biosensor, respectively. The parallel measurements offer the opportunity of on-line activity detection of surface attached enzymes. The immobilized enzyme does not have to be in contact with the catalytic biosensor. The SPR chip can easily be cleaned and used for recycling. Additionally, with regard to the application of FOAS, the integrated SPR technique allows for the quantitative control of the surface density of the enzyme, which is highly relevant for the enzymatic activity. Finally, the miniaturized portable FOAS devices can easily be combined as an add-on device with many other in situ interfacial detection techniques, such as optical waveguide lightmode spectroscopy (OWLS), the quartz crystal microbalance (QCM) measurements, or impedance spectroscopy (IS). Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) allows for an absolute determination of intrinsic rate constants describing the true parameters that control interfacial hybridization. Thus it also allows for a study of the difference of the surface coupling influences between OMCVD gold particles and planar metal films presented in the second part. The multilayer growth process was found to proceed similarly to the way it occurs on planar metal substrates. In contrast to planar bulk metal surfaces, metal colloids exhibit a narrow UV-vis absorption band. This absorption band is observed if the incident photon frequency is resonant with the collective oscillation of the conduction electrons and is known as the localized surface plasmon resonance (LSPR). LSPR excitation results in extremely large molar extinction coefficients, which are due to a combination of both absorption and scattering. When considering metal-enhanced fluorescence we expect the absorption to cause quenching and the scattering to cause enhancement. Our further study will focus on the developing of a detection platform with larger gold particles, which will display a dominant scattering component and enhance the fluorescence signal. Furthermore, the results of sequence-specific detection of DNA hybridization based on OMCVD gold particles provide an excellent application potential for this kind of cheap, simple, and mild preparation protocol applied in this gold fabrication method. In the final chapter, SPFS was used for the in-depth characterizations of the conformational changes of commercial carboxymethyl dextran (CMD) substrate induced by pH and ionic strength variations were studied using surface plasmon resonance spectroscopy. The pH response of CMD is due to the changes in the electrostatics of the system between its protonated and deprotonated forms, while the ionic strength response is attributed from the charge screening effect of the cations that shield the charge of the carboxyl groups and prevent an efficient electrostatic repulsion. Additional studies were performed using SPFS with the aim of fluorophore labeling the carboxymethyl groups. CMD matrices showed typical pH and ionic strength responses, such as high pH and low ionic strength swelling. Furthermore, the effects of the surface charge and the crosslink density of the CMD matrix on the extent of stimuli responses were investigated. The swelling/collapse ratio decreased with decreasing surface concentration of the carboxyl groups and increasing crosslink density. The study of the CMD responses to external and internal variables will provide valuable background information for practical applications.

Visualization, manipulation and force spectroscopy of cylindrical polymer brushes

The Ph.D. thesis deals with the conformational study of individual cylindrical polymer brush molecules using atomic force microscopy (AFM). Imaging combined with single molecule manipulation has been used to unravel questions concerning conformational changes, desorption behavior and mechanical properties of individual macromolecules and supramolecular structures. In the first part of the thesis (chapter 5) molecular conformations of cylindrical polymer brushes with poly-(N-isopropylacrylamide) (PNIPAM) side chains were studied in various environmental conditions. Also micelle formation of cylindrical brush-coil blockcopolymers with polyacrylic acid side chains and polystyrene coil have been visualized. In chapter 6 the mechanical properties of single cylindrical polymer brushes with (PNIPAM) side chains were investigated. Assuming that the brushes adopt equilibrium conformation on the surface, an average persistence length of lp= (29 ± 3) nm was determined by the end-to-end distance vs. contour length analysis in terms of the wormlike chain (WLC) model. Stretching experiments suggest that an exact determination of the persistence length using force extension curves is impeded by the contribution of the side chains. Modeling the stretching of the bottle brush molecule as extension of a dual spring (side chain and main chain) explains the frequently observed very low persistence length arising from a dominant contribution of the side chain elasticity at small overall contour lengths. It has been shown that it is possible to estimate the “true” persistence length of the bottle brush molecule from the intercept of a linear extrapolation of the inverse square root of the apparent persistence length vs. the inverse contour length plot. By virtue of this procedure a “true” persistence length of 140 nm for the PNIPAM brush molecules is predicted. Chapter 7 and 8 deal with the force-extension behavior of PNIPAM cylindrical brushes studied in poor solvent conditions. The behavior is shown to be qualitatively different from that in a good solvent. Force induced globule-cylinder conformational changes are monitored using “molecule specific force spectroscopy” which is a combined AFM imaging and SMFS technique. An interesting behavior of the unfolding-folding transitions of single collapsed PNIPAM brush molecules has been observed by force spectroscopy using the so called “fly-fishing” mode. A plateau force is observed upon unfolding the collapsed molecule, which is attributed to a phase transition from a collapsed brush to a stretched conformation. Chapter 9 describes the desorption behavior of single cylindrical polyelectrolyte brushes with poly-L-lysine side chains deposited on a mica surface using the “molecule specific force spectroscopy” technique to resolve statistical discrepancies usually observed in SMFS experiments. Imaging of the brushes and inferring the persistence length from a end-to-end distance vs. contour length analysis results in an average persistence length of lp = (25 ± 5) nm assuming that the chains adopt their equilibrium conformation on the surface. Stretching experiments carried out on individual poly-L-lysine brush molecules by force spectroscopy using the “fly-fishing” mode provide a persistence length in the range of 7-23 nm in reasonable accordance with the imaging results. In chapter 10 the conformational behavior of cylindrical poly-L-lysine brush-sodium dodecyl sulfate complexes was studied using AFM imaging. Surfactant induced cylinder to helix like to globule conformational transitions were observed.

Designed functional defects in colloidal photonic crystals: switching, biomonitoring and modified photoluminescence

Es werden zwei komplementäre "bottom-up" Methoden präsentiert, die den kontrollierten Einbau von "intelligenten" planaren Defekten in selbstorganisierte kolloidale photonische Kristalle (KPKs) ermöglichen. Die Defektschicht basiert auf einem funktionellen, nanometer-skalierten dünnen Film, der entweder durch schichtweise ("layer-by-layer") Selbstorganisation und Mikrokontakttransferübertragung oder durch Aufschleudern und einer KPK-Opferfüllung hergestellt wird. Die entwickelten Techniken gestatten die Integration von maßgeschneiderten dünnen Defektfilmen bestehend aus einer enorm großen Vielfalt an Materialien; sie sind kostengünstig und können im größeren Maßstab angewendet werden. Optische Untersuchungen zeigen einen engen, durch den Defekt hervorgerufenen Transmissionszustand in der photonischen Bandlücke. Die Defektwellenlänge hängt von der optischen Dicke der Defektschicht ab. Aktives Schalten der Defektwellenlänge wird erreicht, indem Defektschichten aus Makromolekülen hergestellt werden, die über externe Erreger wie Licht, Temperatur, Redoxzyklen und mechanischen Druck adressiert werden können. Die Ergebnisse der Untersuchungen sind im Einklang mit separat durchgeführten Ellipsometrie-Messungen und theoretischen "scalar wave approximation"-Berechnungen. Darüber hinaus werden KPKs mit funktionellen biomolekularen Defekten vorgestellt. Über Verschiebungen der Defektmode können DNA-Konformationsänderungen, die enantioselektive Einlagerung eines chiralen Antitumormedikaments sowie Enzymaktivitäten optisch beobachtet werden. Die Einlagerung von fluoreszierenden Farbstoffen und Quantenpunkten in Defekt-KPKs führt zu einer eindeutigen, durch die photonische Bandlücke und den Defektzustand hervorgerufenen Modifizierung der Photolumineszenz (PL)-Spektren. Schaltbare PL-Modifizierungen werden detektiert, wenn adressierbare Defekt-KPKs verwendet werden.

Fluctuation spectroscopy of single plasmonic nanostructures

Plasmonic nanoparticles are great candidates for sensing applications with optical read-out. Plasmon sensing is based on the interaction of the nanoparticle with electromagnetic waves where the particle scatters light at its resonance wavelength. This wavelength depends on several intrinsic factors like material, shape and size of the nanoparticle as well as extrinsic factors like the refractive index of the surrounding medium. The latter allows the nanoparticle to be used as a sensor; changes in the proximate environment can be directly monitored by the wavelength of the emitted light. Due to their minuscule size and high sensitivity this allows individual nanoparticles to report on changes in particle coverage.rnrnTo use this single particle plasmon sensor for future sensing applications it has to meet the demand for detection of incidents on the single molecule level, such as single molecule sensing or even the detection of conformational changes of a single molecule. Therefore, time resolution and sensitivity have to be enhanced as today’s measurement methods for signal read-out are too slow and not sensitive enough to resolve these processes. This thesis presents a new experimental setup, the 'Plasmon Fluctuation Setup', that leads to tremendous improvements in time resolution and sensitivity. This is achieved by implementation of a stronger light source and a more sensitive detector. The new setup has a time resolution in the microsecond regime, an advancement of 4-6 orders of magnitude to previous setups. Its resonance wavelength stability of 0.03 nm, measured with an exposure time of 10 ms, is an improvement of a factor of 20 even though the exposure time is 3000 times shorter than in previous reports. Thus, previously unresolvable wavelength changes of the plasmon sensor induced by minor local environmental alteration can be monitored with extremely high temporal resolution.rnrnUsing the 'Plasmon Fluctuation Setup', I can resolve adsorption events of single unlabeled proteins on an individual nanorod. Additionally, I monitored the dynamic evolution of a single protein binding event on a millisecond time scale. This feasibility is of high interest as the role of certain domains in the protein can be probed by a study of modified analytes without the need for labels possibly introducing conformational or characteristic changes to the target. The technique also resolves equilibrium fluctuations in the coverage, opening a window into observing Brownian dynamics of unlabeled macromolecules. rnrnA further topic addressed in this thesis is the usability of the nanoruler, two nanospheres connected with a spacer molecule, as a stiffness sensor for the interparticle linker under strong illumination. Here, I discover a light induced collapse of the nanoruler. Furthermore, I exploit the sensing volume of a fixed nanorod to study unlabeled analytes diffusing around the nanorod at concentrations that are too high for fluorescence correlation spectroscopy but realistic for biological systems. Additionally, local pH sensing with nanoparticles is achieved.

A tool for the qualitative comparison of membrane-embedded and detergent-solubilized membrane protein structures in projection

The calculation of projection structures (PSs) from Protein Data Bank (PDB)-coordinate files of membrane proteins is not well-established. Reports on such attempts exist but are rare. In addition, the different procedures are barely described and thus difficult if not impossible to reproduce. Here we present a simple, fast and well-documented method for the calculation and visualization of PSs from PDB-coordinate files of membrane proteins: the projection structure visualization (PSV)-method. The PSV-method was successfully validated using the PS of aquaporin-1 (AQP1) from 2D crystals and cryo-transmission electron microscopy, and the PDB-coordinate file of AQP1 determined from 3D crystals and X-ray crystallography. Besides AQP1, which is a relatively rigid protein, we also studied a flexible membrane transport protein, i.e. the L-arginine/agmatine antiporter AdiC. Comparison of PSs calculated from the existing PDB-coordinate files of substrate-free and L-arginine-bound AdiC indicated that conformational changes are detected in projection. Importantly, structural differences were found between the PSV-method calculated PSs of the detergent-solubilized AdiC proteins and the PS from cryo-TEM of membrane-embedded AdiC. These differences are particularly exciting since they may reflect a different conformation of AdiC induced by the lateral pressure in the lipid bilayer.

Mutagenesis and computer modeling studies of a GPCR conserved residue W5.43(194) in ligand recognition and signal transduction for CB2 receptor

W5.43(194), a conserved tryptophan residue among G-protein coupled receptors (GPCRs) and cannabinoid receptors (CB), was examined in the present report for its significance in CB2 receptor ligand binding and adenylyl cyclase (AC) activity. Computer modeling postulates that this site in CB2 may be involved in the affinity of WIN55212-2 and SR144528 through aromatic contacts. In the present study, we reported that a CB2 receptor mutant, W5.43(194)Y, which had a tyrosine (Y) substitution for tryptophan (W), retained the binding affinity for CB agonist CP55940, but reduced binding affinity for CB2 agonist WIN55212-2 and inverse agonist SR144528 by 8-fold and 5-fold, respectively; the CB2 W5.43(194)F and W5.43(194)A mutations significantly affect the binding activities of CP55940, WIN55212-2 and SR144528. Furthermore, we found that agonist-mediated inhibition of the forskolin-induced cAMP production was dramatically diminished in the CB2 mutant W5.43(194)Y, whereas W5.43(194)F and W5.43(194)A mutants resulted in complete elimination of downstream signaling, suggesting that W5.43(194) was essential for the full activation of CB2. These results indicate that both aromatic interaction and hydrogen bonding are involved in ligand binding for the residue W5.43(194), and the mutations of this tryptophan site may affect the conformation of the ligand binding pocket and therefore control the active conformation of the wild type CB2 receptor. W5.43(194)Y/F/A mutations also displayed noticeable enhancement of the constitutive activation probably attributed to the receptor conformational changes resulted from the mutations.

Norepinephrine-induced hypertension dilates vasospastic basilar artery after subarachnoid haemorrhage in rabbits

BACKGROUND: Vasopressor-induced hypertension is routinely indicated for prevention and treatment of cerebral vasospasm (CVS) after subarachnoid haemorrhage (SAH). Mechanisms underlying patients' clinical improvement during vasopressor-induced hypertension remain incompletely understood. The aim of this study was to evaluate angiographic effects of normovolaemic Norepinephrine (NE)-induced hypertension therapy on the rabbit basilar artery (BA) after SAH. METHODS: Cerebral vasospasm was induced using the one-haemorrhage rabbit model; sham-operated animals served as controls. Five days later the animals underwent follow-up angiography prior to and during NE-induced hypertension. Changes in diameter of the BA were digitally calculated in mean microm +/- SEM (standard error of mean). FINDINGS: Significant CVS of 14.2% was documented in the BA of the SAH animals on day 5 compared to the baseline angiogram on day 0 (n = 12, p < 0.01), whereas the BA of the control animals remained statistically unchanged (n = 12, p > 0.05). During systemic administration of NE, mean arterial pressure increased from 70.0 +/- 1.9 mmHg to 136.0 +/- 2.1 mmHg in the SAH group (n = 12, p < 0.001) and from 72.0 +/- 3.1 to 137.8 +/- 1.3 in the control group (n = 12, p < 0.001). On day 5 after SAH, a significant dilatation of the BA in response to norepinephrine could be demonstrated in both groups. The diameter of the BA in the SAH group increased from 640.5 +/- 17.5 microm to 722.5 +/- 23.7 microm (n = 12, p < 0.05; ). In the control group the diameter increased from 716.8 +/- 15.5 microm to 779.9 +/- 24.1 microm (n = 12, p < 0.05). CONCLUSION: This study demonstrated that NE-induced hypertension causes angiographic dilatation of the BA in the SAH rabbit model. Based on these observations, it can be hypothesised that clinical improvement during vasopressor-induced hypertension therapy after SAH might be explained with cerebral vasodilatation mechanisms that lead to improvement of cerebral blood flow.

Glucocorticoid induced impairment of lung structure assessed by digital image analysis

Glucocorticoids (GC) are successfully applied in neonatology to improve lung maturation in preterm born babies. Animal studies show that GC can also impair lung development. In this investigation, we used a new approach based on digital image analysis. Microscopic images of lung parenchyma were skeletonised and the geometrical properties of the septal network characterised by analysing the 'skeletal' parameters. Inhibition of the process of alveolarisation after extensive administration of small doses of GC in newborn rats was confirmed by significant changes in the 'skeletal' parameters. The induced structural changes in the lung parenchyma were still present after 60 days in adult rats, clearly indicating a long lasting or even definitive impairment of lung development and maturation caused by GC. Conclusion: digital image analysis and skeletonisation proved to be a highly suited approach to assess structural changes in lung parenchyma.

Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.

Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

Molecular mechanisms of Ca$\sp{2+}$ signal transduction from cardiac troponin C to troponin I

$\rm Ca\sp{2+}$-dependent exposure of an N-terminal hydrophobic region in troponin C (TnC) is thought to be important for the regulation of contraction in striated muscle. To study these conformational changes in cardiac troponin (cTnC), the $\varepsilon$C and $\varepsilon$H chemical shifts for all 10 Met residues in cTnC were sequence-specific assigned on NMR spectra using a combination of two dimensional NMR techniques and site-directed mutagenesis. The assigned methyl-Met chemical shifts were used as structural markers to monitor conformational changes induced by $\rm Ca\sp{2+}.$ The results showed that binding of $\rm Ca\sp{2+}$ to the regulatory site in the N-domain induced large changes in the $\varepsilon$H and $\varepsilon$C chemical shifts of Met 45, Met 80, Met 81 in the predicted N-terminal hydrophobic region, but had no effect on the chemical shifts of Met residues located in the C-domain. These results suggest that the $\rm Ca\sp{2+}$-dependent functions of cTnC are mainly through N-terminal domain of cTnC.^ To further define the molecular mechanism by which TnC regulates muscle contraction, single Cys residues were engineered at positions 45, 81, 84 or 85 in the N-terminal hydrophobic region of cTnC to provide sites for attachment of specific blocking groups. Blocking groups were coupled to these Cys residues in cTnC mutants and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity resulted when the peptide or biotin was conjugated to residue 81 in cTnC(C81), while less inhibition resulted from covalent modification of cTnC(C84) or cTnC(C85). The results suggest that limited sites of the N-terminal hydrophobic region in cTnC are important for transducing the $\rm Ca\sp{2+}$ signal to troponin I (TnI) and are sensitive to modification, while other regions are less important or can adapt to steric hindrances introduced by bulky blocking groups.^ Although the exposed TnI interaction site in the N-terminal hydrophobic region of TnC is crucial for function of TnC, other regions in the N-domain of TnC may also participate in transducing the $\rm Ca\sp{2+}$ signal and conferring the maximal activation of actomyosin ATPase. The interactions between the B-/C-helices of cTnC and cTnI were characterized using a combination of site-directed mutagenesis, fluorescence and covalent modification. The results suggest that the $\rm Ca\sp{2+}$-dependent interactions of the B-/C-helices of cTnC with TnI may be required for the maximal activation of muscle contraction. ^