Patterning of diverse mammalian cell types in serum free medium with photoablation

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer‐by‐layer self‐assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short‐term stability and low‐protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell‐selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108‐15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The ASPP interaction network: electrostatic differentiation between pro‐ and anti‐apoptotic proteins

The ASPP proteins are apoptosis regulators: ASPP1 and ASPP2 promote, while iASPP inhibits, apoptosis. The mechanism by which these different outcomes are achieved is still unknown. The C‐terminal ankyrin repeats and SH3 domain (ANK‐SH3) mediate the interactions of the ASPP proteins with major apoptosis regulators such as p53, Bcl‐2, and NFκB. The structure of the complex between ASPP2_ANK‐SH3 and the core domain of p53 (p53CD) was previously determined. We have recently characterized the individual interactions of ASPP2_ANK‐SH3 with Bcl‐2 and NFκB, as well as a regulatory intramolecular interaction with the proline rich domain of ASPP2. Here we compared the ASPP interactions at two levels: ASPP2_ANK‐SH3 with different proteins, and different ASPP family members with each protein partner. We found that the binding sites of ASPP2 to p53CD, Bcl‐2, and NFκB are different, yet lie on the same face of ASPP2_ANK‐SH3. The intramolecular binding site to the proline rich domain overlaps the three intermolecular binding sites. To reveal the basis of functional diversity in the ASPP family, we compared their protein‐binding domains. A subset of surface‐exposed residues differentiates ASPP1 and ASPP2 from iASPP: ASPP1/2 are more negatively charged in specific residues that contact positively charged residues of p53CD, Bcl‐2, and NFκB. We also found a gain of positive charge at the non‐protein binding face of ASPP1/2, suggesting a role in electrostatic direction towards the negatively charged protein binding face. The electrostatic differences in binding interfaces between the ASPP proteins may be one of the causes for their different function. Copyright © 2010 John Wiley & Sons, Ltd.     

Protein adsorption onto auto-assembled polyelectrolyte films

Surface modification by deposition of ordered protein systems constitutes one of the major objectives of bio-related chemistry and biotechnology. In this respect a concept has recently been reported aimed at fabricating multilayers by the consecutive adsorption of positively and negatively charged polyelectrolytes. We investigate the adsorption processes between polyelectrolyte multilayers and a series of positively and negatively charged proteins. The film buildup and adsorption experiments were followed by Scanning Angle Reflectometry (SAR). We find that proteins strongly interact with the polyelectrolyte film whatever the sign of the charge of both the multilayer and the protein. When charges of the multilayer and the protein are similar, one usually observes the formation of protein monolayers, which can become dense. We also show that when the protein and the multilayer become oppositely charged, the adsorbed amounts are usually larger and the formation of thick protein layers extending up to several times the largest dimension of the protein can be observed. Our results confirm that electrostatic interactions dominate protein/polyelectrolyte multilayer interactions.     

Electrostatic interactions play an essential role in the binding of oleic acid with α‐lactalbumin in the HAMLET‐like complex: A study using charge‐specific chemical modifications

H uman α ‐lactalbumin m ade le thal to t umor cells (HAMLET) and its analogs are partially unfolded protein‐oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge‐specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively‐charged lysine residues to negatively‐charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild‐type α‐lactalbumin‐oleic acid complex. With the addition of OA, the wild‐type and guanidinated α‐lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α‐lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively‐charged basic groups on α‐lactalbumin and the negatively‐charged carboxylate groups on OA molecules play an essential role in the binding of OA to α‐lactalbumin and that these interactions appear to be as important as hydrophobic interactions. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Differential interaction of PRMT1 with RGG‐boxes of the FET family proteins EWS and TAF15

The FET sub‐family (F US/T LS, E WS, T AF15) of RNA‐binding proteins have remarkably similar overall structure but diverse biological and pathological roles. The molecular basis for FET protein specialization is largely unknown. Gly‐Arg‐Rich regions (RGG‐boxes) within FET proteins are targets for methylation by Protein‐Arginine‐Methyl‐Transferase‐1 (PRMT1) and substrate capture is thought to involve electrostatic attraction between positively charged polyRGG substrates and negatively charged surface channels of PRMT1. Unlike FUS and EWS, a high proportion of TAF15 RGG‐boxes are embedded within neutrally charged YGGDR(S/G)G repeats, suggesting that they might not bind well to PRMT1. This notion runs contrary however to a report that YGGDR(S/G)G repeats are methylated by PRMT1. Using peptide‐based polyRGG substrates and a novel 2‐hybrid binding assay, we find that the Asp residue in YGGDR(S/G)G repeats confers poor binding to PRMT1. Our results therefore indicate that YGGDR(S/G)G repeats may contribute to TAF15 specialization by enabling differential interactions with PRMT1 and reduced overall levels of TAF15 methylation compared with other FET proteins. By analogy with molecular recognition of other disordered polyvalent ligands by globular protein partners, we also propose a dynamic polyelectrostatic model for substrate capture by PRMT1.     

Intracellular monitoring of target protein production in Staphylococcus aureus by peptide tag‐induced reporter fluorescence

An intracellular approach for monitoring protein production in Staphylococcus aureus is described. mCherry, fused to the dodecapeptide Tip, was capable of inducing tetracycline repressor (TetR). Time‐ and concentration‐dependent production of mCherry could be correlated to TetR‐controlled GFPmut2 activity. This approach can potentially be extended to native S. aureus proteins.     

An ionic–chemical–mechanical model for muscle contraction

The dynamic process underlying muscle contraction is the parallel sliding of thin actin filaments along an immobile thick myosin fiber powered by oar‐like movements of protruding myosin cross bridges (myosin heads). The free energy for functioning of the myosin nanomotor comes from the hydrolysis of ATP bound to the myosin heads. The unit step of translational movement is based on a mechanical‐chemical cycle involving ATP binding to myosin, hydrolysis of the bound ATP with ultimate release of the hydrolysis products, stress‐generating conformational changes in the myosin cross bridge, and relief of built‐up stress in the myosin power stroke. The cycle is regulated by a transition between weak and strong actin–myosin binding affinities. The dissociation of the weakly bound complex by addition of salt indicates the electrostatic basis for the weak affinity, while structural studies demonstrate that electrostatic interactions among negatively charged amino acid residues of actin and positively charged residues of myosin are involved in the strong binding interface. We therefore conjecture that intermediate states of increasing actin–myosin engagement during the weak‐to‐strong binding transition also involve electrostatic interactions. Methods of polymer solution physics have shown that the thin actin filament can be regarded in some of its aspects as a net negatively charged polyelectrolyte. Here we employ polyelectrolyte theory to suggest how actin–myosin electrostatic interactions might be of significance in the intermediate stages of binding, ensuring an engaged power stroke of the myosin motor that transmits force to the actin filament, and preventing the motor from getting stuck in a metastable pre‐power stroke state. We provide electrostatic force estimates that are in the pN range known to operate in the cycle.     

In planta chemical cross‐linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis

Site‐specific chemical cross‐linking in combination with mass spectrometry analysis has emerged as a powerful proteomic approach for studying the three‐dimensional structure of protein complexes and in mapping protein–protein interactions (PPIs). Building on the success of MS analysis of in vitro cross‐linked proteins, which has been widely used to investigate specific interactions of bait proteins and their targets in various organisms, we report a workflow for in vivo chemical cross‐linking and MS analysis in a multicellular eukaryote. This approach optimizes the in vivo protein cross‐linking conditions in Arabidopsis thaliana, establishes a MudPIT procedure for the enrichment of cross‐linked peptides, and develops an integrated software program, exhaustive cross‐linked peptides identification tool (ECL), to identify the MS spectra of in planta chemical cross‐linked peptides. In total, two pairs of in vivo cross‐linked peptides of high confidence have been identified from two independent biological replicates. This work demarks the beginning of an alternative proteomic approach in the study of in vivo protein tertiary structure and PPIs in multicellular eukaryotes.     

Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

Post‐translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM‐dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross‐linking‐assisted and stable isotope labeling in cell culture‐based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation‐dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine‐9 (H3K9Me₃)‐dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation‐dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine‐3 (H3T3‐Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3‐Phos‐binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3‐Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me₃). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs.     

Voltage regulation of single green fluorescent protein mutants

We report the analysis of the fluorescence intensity fluctuations of single proteins of a GFP mutant, GFPmut2, embedded in a polyelectrolyte nanocapsule adsorbed on thin conductive layers. Our results, based on single molecule fluorescence spectroscopy, indicate that the fluorescence blinking dynamics of GFP is strongly dependent on the bulk conductivity of the metal layer substrate, on the distance from the conductive surfaces and on the amplitude of the voltage applied to the poly-electrolyte layers. These findings suggest that fluorescence blinking itself might be employed as a reporter signal in nano-bio-technology applications.     

Dynamics of green fluorescent protein mutant2 in solution, on spin-coated glasses, and encapsulated in wet silica gels

Single-molecule experiments are performed by investigating spectroscopic properties of molecules either diffusing in and out of the observation volume or fixed in space by different immobilization procedures. To evaluate the effect of immobilization methods on the structural and dynamic properties of proteins, a highly fluorescent mutant of the green fluorescent protein, GFPmut2, was spectroscopically characterized in bulk solutions, dispersed on etched glasses, and encapsulated in wet, nanoporous silica gels. The emission spectrum, the fluorescence lifetimes, the anisotropy, and the rotational correlation time of GFPmut2, encapsulated in silica gels, are very similar to those obtained in solution. This finding indicates that the gel matrix does not alter the protein conformation and dynamics. In contrast, the fluorescence lifetimes of GFPmut2 on glasses are two-to fourfold higher and the fluorescence anisotropy decays yield almost no phase shifts. This indicates that the interaction of the protein with the bare glass surface induces a significant structural perturbation and severely restricts the rotational motion. Single molecules of GFPmut2 on glasses or in silica gels, identified by confocal image analysis, show a significant stability to illumination with bleaching times of the order of 90 and 60 sec, respectively. Overall, these data indicate that silica gels represent an ideal matrix for following biologically relevant events at a single molecule level.     

Development of constant-pH simulation methods in implicit solvent and applications in biomolecular systems

pH is a critical parameter for biological and technological systems directly related with electrical charges. It can give rise to peculiar electrostatic phenomena, which also makes them more challenging. Due to the quantum nature of the process, involving the forming and breaking of chemical bonds, quantum methods should ideally by employed. Nevertheless, due to the very large number of ionizable sites, different macromolecular conformations, salt conditions, and all other charged species, the CPU time cost simply becomes prohibitive for computer simulations, making this a quite complex problem. Simplified methods based on Monte Carlo sampling have been devised and will be reviewed here, highlighting the updated state-of-the-art of this field, advantages, and limitations of different theoretical protocols for biomolecular systems (proteins and nucleic acids). Following a historical perspective, the discussion will be associated with the applications to protein interactions with other proteins, polyelectrolytes, and nanoparticles.     

Gene delivery using biodegradable polyelectrolyte microcapsules prepared through the layer-by-layer technique

Biodegradable and non‐biodegradable microcapsules were prepared via the layer‐by‐layer (LbL) technique consisting of the polyelectrolyte pairs of dextran sulphate/poly‐L ‐arginine and poly(styrene sulfonate)/poly(allylamine hydrochloride), respectively, in an attempt to encapsulate plasmid DNA (pDNA) for efficient transfection into NIH 3T3 cells. Results indicated the retention of bioactivity in the encased pDNA, as well as a correlation between the level of in vitro gene expression and biodegradability properties of polyelectrolyte. Furthermore, the incorporation of iron oxide nanoparticles within the polyelectrolyte layers significantly improved the in vitro transfection efficiency of the microcapsules. As a novel pDNA delivery system, the reported biodegradable microcapsules provide useful insight into plasmid‐based vaccination and where there is a prerequisite to deliver genes into cells capable of phagocytosis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1088–1094, 2012     

Pressure perturbation calorimetry of apolipoproteins in solution and in model lipoproteins

High‐density lipoproteins (HDLs) are complexes of lipids and proteins (termed apolipoproteins) that remove cell cholesterol and protect from atherosclerosis. Apolipoproteins contain amphipathic α‐helices that have high content (≥1/3) and distinct distribution of charged and apolar residues, adopt molten globule‐like conformations in solution, and bind to lipid surfaces. We report the first pressure perturbation calorimetry (PPC) study of apolipoproteins. In solution, the main HDL protein, apoA‐I, shows relatively large volume contraction, ΔV_unf = ?0.33%, and an apparent reduction in thermal expansivity upon unfolding, Δα_unf ≤ 0, which has not been observed in other proteins. We propose that these values are dominated by increased charged residue hydration upon α‐helical unfolding, which may result from disruption of multiple salt bridges. At 5°C, apoA‐I shows large thermal expansion coefficient, α(5°) = 15·10^?4 K^?1, that rapidly declines upon heating from 5 to 40°C, α(40°) ? α(5°) = ?4·10^?4 K^?1; apolipoprotein C‐I shows similar values of α(5°) and α(40°). These values are larger than in globular proteins. They indicate dominant effect of charged residue hydration, which may modulate functional apolipoprotein interactions with a broad range of their protein and lipid ligands. The first PPC analysis of a protein–lipid complex is reported, which focuses on the chain melting transition in model HDL containing apoA‐I or apoC‐I, dimyristoyl phosphatidylcholine, and 0–20% cholesterol. The results may provide new insights into volumetric properties of HDL that modulate metabolic lipoprotein remodeling during cholesterol transport. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Non‐interacting surface solvation and dynamics in protein–protein interactions

Protein–protein interactions control a plethora of cellular processes, including cell proliferation, differentiation, apoptosis, and signal transduction. Understanding how and why proteins interact will inevitably lead to novel structure‐based drug design methods, as well as design of de novo binders with preferred interaction properties. At a structural and molecular level, interface and rim regions are not enough to fully account for the energetics of protein–protein binding, even for simple lock‐and‐key rigid binders. As we have recently shown, properties of the global surface might also play a role in protein–protein interactions. Here, we report on molecular dynamics simulations performed to understand solvent effects on protein–protein surfaces. We compare properties of the interface, rim, and non‐interacting surface regions for five different complexes and their free components. Interface and rim residues become, as expected, less mobile upon complexation. However, non‐interacting surface appears more flexible in the complex. Fluctuations of polar residues are always lower compared with charged ones, independent of the protein state. Further, stable water molecules are often observed around polar residues, in contrast to charged ones. Our analysis reveals that (a) upon complexation, the non‐interacting surface can have a direct entropic compensation for the lower interface and rim entropy and (b) the mobility of the first hydration layer, which is linked to the stability of the protein–protein complex, is influenced by the local chemical properties of the surface. These findings corroborate previous hypotheses on the role of the hydration layer in shielding protein–protein complexes from unintended protein–protein interactions. Proteins 2015; 83:445–458. © 2014 Wiley Periodicals, Inc.     

Inclusion of proteins into polyelectrolyte microparticles by alternative adsorption of polyelectrolytes on protein aggregates

A new method of protein immobilization into polyelectrolyte microparticles by alternative adsorption of the oppositely charged polyelectrolytes on the aggregates obtained by salting out of protein is proposed. The model protein -chymotrypsin (ChT) was included in the polyelectrolyte microparticles obtained by various number of polyelectrolyte adsorption steps (from 1 to 11). The main parameters of ChT inclusion into microparticles were calculated. Scanning electron and optical microscopy were used for characterization of morphology and determination of particle size which was from 1 to 10 m in most cases. It was shown that the size and shape of protein-containing particles and protein aggregates used as a matrix were similar. Change in ChT enzymatic activity during entrapment into polyelectrolyte particles and activity of released protein were studied. The effect of pH on release of incorporated proteins was investigated; it was shown that change in pH and the number of polyelectrolyte adsorption steps allows protein release to be manipulated.     

Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic,charge transfer,and cis‐diol interactions

Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis‐diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation‐exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge‐transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins.     

Polyelectrolytic aspects of the titration curve. The semi-flexible model

Careful examination of the concentration range where both intrinsic viscosity and light scattering show a polyelectrolyte effect, even for singly charged halato-telechelic ionomers in DMF, together with the neutron scattering results at higher concentration show that weakly charged polymers may be a very useful tool to understand the complicated effects of coulombic interaction in polyelectrolyte solutions. A theoretical framework is given for a systematic study of such weakly charged polymers. The current state of understanding is presented of the properties of solutions of strong polyelectrolytes and of charged rigid particles. Finally it is shown how the transposition to weak polyelectrolytes solutions sheds light on the respective contributions of intra- and intermolecular interactions.     

Ion-exchange resins greatly facilitate refolding of like-charged proteins at high concentrations

Protein refolding is a crucial step for the production of therapeutic proteins expressed in bacteria as inclusion bodies. In vitro protein refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high‐efficiency protein refolding. We have herein found that electrostatic repulsion between like‐charged protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery. This finding is extensively demonstrated with three different proteins and four kinds of ion‐exchange resins when the protein and ion‐exchange gel are either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect is significant at very high protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast protein refolding is realized at high protein concentrations. It was not realistic by any other approaches. The working mechanism of the like‐charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the inhibition of protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating protein folding. The refolding method with like‐charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous protein refolding and purification is possible. All the characters are desirable for preparative refolding of therapeutic proteins expressed in bacteria as inclusion bodies. Bioeng. 2011; 108:1068–1077. © 2010 Wiley Periodicals, Inc.