Protein GB1 Folding and Assembly from Structural Elements

Folding of the Protein G B1 domain (PGB1) shifts with increasing salt concentration from a cooperative assembly of inherently unstructured subdomains to an assembly of partly pre-folded structures. The salt-dependence of pre-folding contributes to the stability minimum observed at physiological salt conditions. Our conclusions are based on a study in which the reconstitution of PGB1 from two fragments was studied as a function of salt concentrations and temperature using circular dichroism spectroscopy. Salt was found to induce an increase in β-hairpin structure for the C-terminal fragment (residues 41 – 56), whereas no major salt effect on structure was observed for the isolated N-terminal fragment (residues 1 – 41). In line with the increasing evidence on the interrelation between fragment complementation and stability of the corresponding intact protein, we also find that salt effects on reconstitution can be predicted from salt dependence of the stability of the intact protein. Our data show that our variant (which has the mutations T2Q, N8D, N37D and reconstitutes in a manner similar to the wild type) displays the lowest equilibrium association constant around physiological salt concentration, with higher affinity observed both at lower and higher salt concentration. This corroborates the salt effects on the stability towards denaturation of the intact protein, for which the stability at physiological salt is lower compared to both lower and higher salt concentrations. Hence we conclude that reconstitution reports on molecular factors that govern the native states of proteins

Cell-Nanoparticle Interactions at (Sub)-Nanometer Resolution Analyzed by Electron Microscopy and Correlative Coherent Anti-Stokes Raman Scattering

A wide variety of nanoparticles are playing an increasingly important role in drug delivery. Label-free imaging techniques are especially desirable to follow the cellular uptake and intracellular fate of nanoparticles. The combined correlative use of different techniques, each with unique advantages, facilitates more detailed investigation about such interactions. The synergistic use of correlative coherent anti-Stokes Raman scattering and electron microscopy (C-CARS-EM) imaging offers label-free, chemically-specific, and (sub)-nanometer spatial resolution for studying nanoparticle uptake into cells as demonstrated in the current study. Coherent anti-Stokes Raman scattering (CARS) microscopy offers chemically-specific (sub)micron spatial resolution imaging without fluorescent labels while transmission electron microscopy (TEM) offers (sub)-nanometer scale spatial resolution and thus visualization of precise nanoparticle localization at the sub-cellular level. This proof-of-concept imaging platform with unlabeled drug nanocrystals and macrophage cells revealed good colocalization between the CARS signal and electron dense nanocrystals in TEM images. The correlative TEM images revealed subcellular localization of nanocrystals inside membrane bound vesicles, showing multivesicular body (MVB)-like morphology typical for late endosomes (LEs), endolysosomes, and phagolysosomes. C-CARS-EM imaging has much potential to study the interactions between a wide range of nanoparticles and cells with high precision and confidence.Peer reviewe

Modeling the Time Evolution of the Nanoparticle-Protein Corona in a Body Fluid

Background: Nanoparticles in contact with biological fluids interact with proteins and other biomolecules, thus forming a dynamic corona whose composition varies over time due to continuous protein association and dissociation events. Eventually equilibrium is reached, at which point the continued exchange will not affect the composition of the corona. Results: We developed a simple and effective dynamic model of the nanoparticle protein corona in a body fluid, namely human plasma. The model predicts the time evolution and equilibrium composition of the corona based on affinities, stoichiometries and rate constants. An application to the interaction of human serum albumin, high density lipoprotein (HDL) and fibrinogen with 70 nm N-iso-propylacrylamide/N-tert-butylacrylamide copolymer nanoparticles is presented, including novel experimental data for HDL. Conclusions: The simple model presented here can easily be modified to mimic the interaction of the nanoparticle protein corona with a novel biological fluid or compartment once new data will be available, thus opening novel applications in nanotoxicity and nanomedicine

PoKe-kuntoutus - Ajanvietettä vai mahdollisuuksia? : PoKe-kuntoutuksen vaikutus autististen lasten vuorovaikutukseen ja haastavaan käyttäytymiseen työntekijöiden näkökulmasta.

Lindman, Tiia & Rossinen, Sara. PoKe-kuntoutus - Ajanvietettä vai mahdollisuuksia? PoKe-kuntoutuksen vaikutus autististen lasten vuorovaikutukseen ja haastavaan käyttäytymiseen työntekijöiden näkökulmasta. Pieksämäki, kevät 2012, 53 sivua, 3 liitettä Diakonia-ammattikorkeakoulu, Diak Itä, Pieksämäki. Sosiaalialan koulutusohjelma. sosionomi (AMK). Opinnäytetyön tavoitteena oli tutkia kokemuksia PoKe-kuntoutuksen vaikutuksista Vaalijalan kuntoutuskeskuksen yhden osaston työntekijöiden näkökulmasta. Tutkimuksen tavoitteena oli saada tietoa, miten PoKe-kuntoutusmenetelmä on vaikuttanut autististen lasten vuorovaikutuksen lisääntymiseen ja haastavan käyttäytymisen vähenemiseen. Tutkimus oli kvalitatiivinen eli laadullinen. Aineisto kerättiin yhden osaston henkilökunnalta helmi-maaliskuussa 2012. Aineistonkeruumenetelmänä oli avoin kyselylomake. Tutkimukseen osallistui 7 työntekijää. Tutkimustulokset osoittivat, että PoKe-kuntoutuksella on voinut olla vaikutusta vuorovaikutuksen lisääntymiseen ja haastavan käyttäytymisen vähenemiseen. Myös lapsen normaalilla kehityksellä, lääkityksellä ja muilla terapiamuodoilla oli vaikutusta asiaan. Tutkimuksen mukaan PoKe-kuntoutusmenetelmä koettiin hyödylliseksi, mutta perusteet harjoitteiden toteuttamiselle tulisi olla selkeämmät. Työntekijät toivoivat myös enemmän tietoa kuntoutusmenetelmästä PoKe-klinikalta. Avainsanat: autismi, PoKe-kuntoutus, haastava käyttäytyminen, kvalitatiivinen tutkimusLindman, Tiia & Rossinen, Sara. PoKe-rehabilitation - Entertainment or possibilities? The effects of the PoKe-rehabilitation on autistic children’s interaction and challenging behavior from the perspective of the employees. 53 p., 3 appendices. Language: Finnish. Pieksämäki, spring 2012. Diaconia University of Applied Sciences. Degree Programme in Social Services. Degree: Bachelor of Social Services. The aim of this thesis was to explore the experiences the effects of the PoKe-rehabilitation from the perspective of employees in the Vaalijala rehabilitation centre. The target was to get information on how the PoKe-method has increased the autistic children’s interaction and reduced their challenging behavior The study was qualitative. The study material was collected between February and March 2012 from the employees of one unit. The method of collecting material was an open questionnaire. There were 7 members of staff participating in the research. The results show that PoKe-rehabilitation may have contributed to increasing the autistic children’s interaction and reducing their challenging behavior. Also children’s normal development, medication and other forms of therapy had an effect on that thing. In conclusion it can be said that PoKe-rehabilitation were considered as useful, but the reasons for the implementation of the exercises should be clearer. The employees also hoped for more information about the rehabilitation method from the PoKe-clinic. Keywords: autism, PoKe-rehabilitation, challenging behavior, qualitative researc

Electrostatic contributions to residue-specific protonation equilibria and proton binding capacitance for a small protein

Charge-charge interactions in proteins are important in a host of biological processes. Here we use C-13 NMR chemical shift data for individual aspartate and glutamate side chain carboxylate groups to accurately detect site-specific protonation equilibria in a variant of the B1 domain of protein G (PGB1-QDD). Carbon chemical shifts are dominated by changes in the electron distribution within the side chain and therefore excellent reporters of the charge state of individual groups, and the data are of high precision. We demonstrate that it is possible to detect local charge interactions within this small protein domain that stretch and skew the chemical shift titration curves away from "ideal" behavior and introduce a framework for the analysis of such convoluted data to study local charge-charge interactions and electrostatic coupling. It is found that, due to changes in electrostatic potential, the proton binding affinity, K-a, of each carboxyl group changes throughout the titration process and results in a linearly pH dependent pK(a) value. This result could be readily explained by calculations of direct charge-charge interactions based on Coulomb's law. In addition, the slope of pK(a) versus pH was dependent on screening by salt, and this dependence allowed the selective study of charge-charge interactions. For PGB1-QDD, it was established that mainly differences in self-energy, and not direct charge-charge interactions, are responsible for shifted pK(a) values within the protein environment

Green fluorescence induced by EF-hand assembly in a split GFP system

The affinity between the 1–157 and 158–238 fragments of green fluorescent protein (GFP) is too low for spontaneous in vivo reassembly of the protein upon co-expression of the two fragments. This prevents chromophore maturation and the cells lack GFP fluorescence. We have utilized the very high affinity between the two EF-hands of calbindin D9k to facilitate GFP assembly from its fragments and to introduce a calcium dependent molecular switch. In GFPN-EF1, residues 1–157 of GFP are fused to residues 1–43 of calbindin, and in EF2-GFPC, residues 44–75 of calbindin are fused to residues 158–238 of GFP. When co-expressed, GFPN-EF1 and EF2-GFPC associate spontaneously and rapidly resulting in a folded reconstituted protein with bright GFP fluorescence. The high affinity of GFPN-EF1 for EF2-GFPC leads to brighter fluorescence of the cells compared to cells with a control constructs carrying leucine zippers (Wilson et al., Nature Methods 2004;3:255). The complex of GFPN-EF1 and EF2-GFPC was purified from cells using metal-ion chelate chromatography and the temperature dependence of GFP fluorescence was found to be calcium dependent. The GFPN-EF1 and EF2-GFPC fragments were separated by ion exchange chromatography. The assembly of the fragments was found to be reversible and the complex was regained upon mixing, as evidenced by surface plasmon resonance (SPR) data. The affinity between GFPN-EF1 and EF2-GFPC as well as rates of association and dissociation were found to be Ca2+-dependent

Oral delivery of all-trans retinoic acid mediated by liposome carriers

All-trans retinoic acid (ATRA) is a molecule that finds wide applications in medicine. Connection between cancer cell proliferation and ATRA is a well-established item. Driven by the potential applications of liposomes in stabilizing and protecting therapeutic compounds thus enabling effective delivery of encapsulated compounds, recent research efforts have been directed to understanding mechanisms of oral delivery through the gastrointestinal tract. The surface charge of the liposome bilayers can modify the interactions between the aggregates and the gastrointestinal fluids. Here, we investigated the ability of cationic and anionic liposomes to encapsulate, protect and deliver ATRA in an in-vitro digestion process as a different oral administration route. Stability and encapsulation efficiency of ATRA in negatively and positively charged liposomes enriched with α-tocopherol were investigated by means of UV–vis spectroscopy, dynamic light scattering and ζ-potential. The applicability of the carriers was tested by means of an in-vitro digestion procedure allowing for the measurement of the bioavailability of ATRA. From this study evidence was provided that the water insoluble molecules, ATRA and α-tocopherol are intercalated in liposome membranes regardless of the surface charge of the vesicle bilayers. Comparisons between cationic and anionic liposomes incorporating retinoic acid show differences in bioavailability. The cationic vesicles are preferable for a larger amount of ATRA bioavailability, which can be understood from electrostatic interactions. Thus ATRA is ionized in a wide range of pHs but protonated in anionic vesicles