Protein adsorption dynamics to polymer surfaces revisited-A multisystems approach.

Performance and safety of materials in contact with living matter are determined by sequential and competitive protein adsorption. However, cause and consequences of these processes remain hard to be generalized and predicted. In a new attempt to address that challenge, the authors compared and analyzed the protein adsorption and displacement on various thoroughly characterized polymer substrates using a combination of surface-sensitive techniques. A multiple linear regression approach was applied to model the dependence of protein adsorption, desorption, and exchange dynamics on protein and surface characteristics. While the analysis confirmed that protein properties primarily govern the observed adsorption and retention phenomena and hydrophobicity as well as surface charge are the most relevant polymer surface properties, the authors have identified several protein-surface combinations that deviate from these patterns and deserve further investigation.

Mixtures of cationic copolymers and oppositely charged surfactants: effect of polymer charge density and ionic strength on the adsorption behavior at the silica-aqueous interface.

This study addresses polymer-surfactant interactions at solid-liquid interfaces and how these can be manipulated by modulating the association between ionic surfactant and oppositely charged polymer, with a particular focus on electrostatic interactions. For this purpose, the interaction of a series of cationic copolymers of vinylpyrrolidone and quaternized vinylimidazol with sodium dodecyl sulfate (SDS) at the silica-aqueous interface was followed by in situ ellipsometry. To reveal the nature of the interaction, we performed measurements for different copolyion charge densities, in the absence and presence of added salt. The path-dependence of the interaction was studied by comparing the adsorption under two different conditions, adsorption from premixed solutions and sequential addition of surfactant to the polymer solution, but the same end state. The reversibility of the adsorption process was studied by following the effect of dilution on the adsorbed layer. All copolyions adsorbed to both silica and hydrophobized silica, revealing the importance of both hydrophobic and electrostatic attractive interactions. On both types of surface, an increase in adsorbed amount was found on lowering the fraction of charged units. An increased ionic strength gave an increased adsorbed amount in all cases, but especially on hydrophobic surfaces. The adsorbed amount on silica from mixtures of the copolyions with SDS peaked at an SDS concentration corresponding closely to the concentration of cationic charges of the different polyions. Around the region of charge equivalence, there was also a phase separation in the bulk. At higher concentrations of SDS, a redissolution in the bulk, and a decrease in adsorbed amount, occurred as a result of excess SDS binding to the complexes. For the most highly charged polyions, we observed a decrease in adsorbed amount, and a shift in the adsorption maxima to lower SDS concentrations, with increasing ionic strength.

A novel AFM based method for force measurements between individual hair strands.

Interactions between hairs and other natural fibers are of broad interest for both applications and fundamental understanding of biological interfaces. We present a novel method, that allows force measurements between individual hair strands. Hair fragments can be laser-cut without altering their surface chemistry. Subsequently, they are glued onto Atomic force microscopy (AFM) cantilevers. This allows carrying out measurements between the hair fragment and surface immobilized hair in a well-defined crossed-cylinder geometry. Both force-distance and friction measurements are feasible. Measurements in air with controlled humidity and in aqueous environment show clear differences which can be explained by the dominating role of capillary interactions in air. Friction is found to be anisotropic, reflecting the fine structure of hair cuticula. While the investigations are focused on the particular example of human hair, we expect that the approach can be extended to other animal/plant fibers and thus offers perspectives for broad spectrum systems.

Nanostructured films of amphiphilic fluorinated block copolymers for fouling release application.

New amphiphilic block copolymers S nSz m consisting of blocks with varied degrees of polymerization, n and m, of polystyrene, S, and polystyrene carrying an amphiphilic polyoxyethylene-polytetrafluoroethylene chain side-group, Sz, were prepared by controlled atom transfer radical polymerization (ATRP). The block copolymers, either alone or in a blend with commercial SEBS (10 wt% SEBS), were spin-coated in thinner films (200-400 nm) on glass and spray-coated in thicker films ( approximately 500 nm) on a SEBS underlayer (150-200 microm). Angle-resolved X-ray photoelectron spectroscopy (XPS) measurements proved that at any photoemission angle, varphi, the atomic ratio F/C was larger than that expected from the known stoichiometry. Consistent with the enrichment of the outer film surface (3-10 nm) in F content, the measured contact angles, theta, with water (theta w > or = 107 degrees ) and n-hexadecane (theta h > or = 64 degrees ) pointed to the simultaneous hydrophobic and lipophobic character of the films. The film surface tension gamma S calculated from the theta values was in the range 13-15 mN/m. However, the XPS measurements on the "wet" films after immersion in water demonstrated that the film surface underwent reconstruction owing to its amphiphilic nature, thereby giving rise to a more chemically heterogeneous structure. The atomic force microscopy (AFM) images (tapping mode/AC mode) revealed well-defined morphological features of the nanostructured films. Depending on the chemical composition of the block copolymers, spherical (ca. 20 nm diameter) and lying cylindrical (24-29 nm periodicity) nanodomains of the S discrete phase were segregated from the Sz continuous matrix (root-mean-square, rms, roughness approximately 1 nm). After immersion in water, the underwater AFM patterns evidenced a transformation to a mixed surface structure, in which the nanoscale heterogeneity and topography (rms = 1-6 nm) were increased. The coatings were subjected to laboratory bioassays to explore their intrinsic ability to resist the settlement and reduce the adhesion strength of two marine algae, viz., the macroalga (seaweed) Ulva linza and the unicellular diatom Navicula perminuta. The amphiphilic nature of the copolymer coatings resulted in distinctly different performances against these two organisms. Ulva adhered less strongly to the coatings richer in the amphiphilic polystyrene component, percentage removal being maximal at intermediate weight contents. In contrast, Navicula cells adhered less strongly to coatings with a lower weight percentage of the amphiphilic side chains. The results are discussed in terms of the changes in surface structure caused by immersion and the effects such changes may have on the adhesion of the test organisms.

Covalent and physical cross-linking of photonic crystals with 10-fold-enhanced chemomechanical stability.

We report opal photonic crystals that are self-assembled from functional polymer particles. We randomly copolymerized functional side-chain monomers containing motifs that form homodimers or heterobridges. These include ether or methylene bridges, hydrazone bridges, acids for anhydride formation, low- T g copolymers or physical cross-links by hydrogen bonds and/or polarity. To generate particles that are monodisperse, spherical, and functionalized, we combined emulsifier-free synthesis with swelling synthesis steps. Laser diffraction from centimeter-sized beams, white-light interferometry, and atomic force microscopy demonstrates symmetry and homogeneity across the entire crystal without the loss of interstitial volume. Compared to the stability of nonfunctional particles, the stability of the crystal against immersion in water and isopropanol was enhanced from 10 to a perfect 100%. One of the successful approaches (methylene bridges from N-methylolmethacrylamid) is triggered by thermal activation, but as shown, this is operative far from the trivial regime of sintering. We demonstrate successful infiltration with and solvation of a laser-polymerizable resin, thus enabling the processing of 3D photonic waveguide structures.

Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy.

Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future.

Effector-stimulated single molecule protein-DNA interactions of a quorum-sensing system in Sinorhizobium meliloti.

Intercellular communication by means of small signal molecules coordinates gene expression among bacteria. This population density-dependent regulation is known as quorum sensing. The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti Rm1021 possesses the Sin quorum sensing system based on N-acyl homoserine lactones (AHL) as signal molecules. Here, we demonstrate that the LuxR-type regulator ExpR binds specifically to a target sequence in the sinRI locus in the presence of different AHLs with acyl side chains from 8 to 20 carbons. Dynamic force spectroscopy based on the atomic force microscope provided detailed information about the molecular mechanism of binding upon activation by six different AHLs. These single molecule experiments revealed that the mean lifetime of the bound protein-DNA complex varies depending on the specific effector molecule. The small differences between individual AHLs also had a pronounced influence on the structure of protein-DNA interaction: The reaction length of dissociation varied from 2.6 to 5.8 A. In addition, dynamic force spectroscopy experiments indicate that N-heptanoyl-DL-homoserine lactone binds to ExpR but is not able to stimulate protein-DNA interaction.

Mechano-optical octave-tunable elastic colloidal crystals made from core-shell polymer beads with self-assembly techniques.

Elastic colloidal crystals, even without a full photonic band gap, hold promise for fascinating applications and for easy large-scale fabrication by self-assembly. However, high mechanical robustness is required for optical, decorative, or security applications, such as tunable optical modulators/filters or optical tension indicators. Here, we present brilliantly colored filled-pore colloidal crystals that withstand elongation by 100%, i.e., one optical octave. We employ a variety of vertical deposition techniques to self-assemble monodisperse core-shell polymer beads with a film-forming shell and flexible core. We find a good theoretical description of crystal thickness for all techniques. The crystals have centimeter-sized macroscopic order, and their orientation is fully controlled by the substrate plane and meniscus line.

Probing DNA-peptide interaction forces at the single-molecule level.

The versatility of chemical peptide synthesis combined with the high sensitivity of AFM single-molecule force spectroscopy allows us to investigate, quantify, and control molecular recognition processes (molecular nanotechnology), offering a tremendous potential in chemical biology.Single-molecule force spectroscopy experiments are able to detect fast intermediate transition states, details of the energy landscape, and structural changes, while avoiding multiple binding events that can occur under ensemble conditions. Dynamic force spectroscopy (DFS) is even able to provide data on the complex lifetime. This minireview outlines the biophysical methodology, discusses different experimental set-ups, and presents representative results in the form of two case studies, both dealing with DNA-binding peptides. They may serve as model systems, e.g., for transcription factors or gene transfection agents.

Detailed studies of the binding mechanism of the Sinorhizobium meliloti transcriptional activator ExpG to DNA.

The exopolysaccharide galactoglucan promotes the establishment of symbiosis between the nitrogen-fixing Gram-negative soil bacterium Sinorhizobium meliloti 2011 and its host plant alfalfa. The transcriptional regulator ExpG activates expression of galactoglucan biosynthesis genes by direct binding to the expA1, expG/expD1 and expE1 promoter regions. ExpG is a member of the MarR family of regulatory proteins. Analysis of target sequences of an ExpG(His)(6) fusion protein in the exp promoter regions resulted in the identification of a binding site composed of a conserved palindromic region and two associated sequence motifs. Association and dissociation kinetics of the specific binding of ExpG(His)(6) to this binding site were characterized by standard biochemical methods and by single-molecule spectroscopy based on the atomic force microscope (AFM). Dynamic force spectroscopy indicated a distinct difference in the kinetics between the wild-type binding sequence and two mutated binding sites, leading to a closer understanding of the ExpG-DNA interaction.

Single molecule force spectroscopy on ligand-DNA complexes: from molecular binding mechanisms to biosensor applications.

Recent developments in single molecule force spectroscopy (SMFS) allow direct observation and measurements of forces that hold protein-DNA complexes together. Furthermore, the mechanics of double-stranded (ds) DNA molecules in the presence of small binding ligands can be detected. The results elucidate molecular binding mechanisms and open the way for ultra sensitive and powerful biosensor applications.

Classification of hyper-variable Corynebacterium glutamicum surface-layer proteins by sequence analyses and atomic force microscopy.

The structural S-layer proteins of 28 different Corynebacterium glutamicum isolates have been analyzed systematically. Treatment of whole C. glutamicum cells with detergents resulted in the isolation of S-layer proteins with different apparent molecular masses, ranging in size from 55 to 66 kDa. The S-layer genes analyzed were characterized by coding regions ranging from 1,473 to 1,533 nucleotides coding for S-layer proteins with a size of 490-510 amino acids. Using PCR techniques, the corresponding S-layer genes of the 28 C. glutamicum isolates were all cloned and sequenced. The deduced amino acid sequences of the S-layer proteins showed identities between 69 and 98% and could be grouped into five phylogenetic classes. Furthermore, sequence analyses indicated that the S-layer proteins of the analyzed C. glutamicum isolates exhibit a mosaic structure of highly conserved and highly variable regions. Several conserved regions were assumed to play a key role in the formation of the C. glutamicum S-layers. Especially the N-terminal signal peptides and the C-terminal anchor sequences of the S-layer proteins showed a nearly perfect amino acid sequence conservation. Analyses by atomic force microscopy revealed a committed hexagonal structure. Morphological diversity of the C. glutamicum S-layers was observed in a class-specific unit cell dimension (ranging from 15.2 to 17.4 nm), which correlates with the sequence similarity-based classification. It could be demonstrated that differences in the primary structure of the S-layer proteins were reflected by the S-layer morphology.

Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti--a combined molecular biology and force spectroscopy investigation.

Specific protein-DNA interaction is fundamental for all aspects of gene transcription. We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis. ExpG is a transcriptional activator of exp gene expression. We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28 bp conserved region was found. AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165 pN in a logarithmic dependence from the loading rates of 70-79000 pN/s. Two different regimes of loading rate-dependent behaviour were identified. Thermal off-rates in the range of k(off)=(1.2+/-1.0) x 10(-3)s(-1) were derived from the lower loading rate regime for all promoter regions. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism.