ChemPhysChem

The cover picture displays the tentacles of the sea anemone Entacmaea quadricolor, a source of red fluorescent proteins. The 2-imino-5-(4-hydroxybenzylidene)-imidazolinone chromophore (upper right) resides in the center of an 11-stranded β-barrel (lower left). The spectral properties of the fluorescent proteins depend on the conformation of the chromophore and its interactions with the surrounding protein scaffold. In the animal, the proteins form tight dimers or tetramers. Protein engineering yielded monomeric variants with optimized performance as protein tags in live cell imaging applications. On page 1369, G. U. Nienhaus and J. Wiedenmann review the structural diversity of GFP-like proteins and discuss how structure and dynamics govern their optical properties and how they can be modified to create advanced marker proteins.

The investigation of native nuclear pore complexes (NPCs) with single-molecule force spectroscopy requires stable and non-destructive attachment of the nuclear envelope. Modified, micro-structured PDMS turns out to be perfectly suited for membrane preparation as it preserves structure and function of the entire NPC, as shown on p. 1553 by A. Ebner et al.

Microspectroscopic imaging: Surface-enhanced Raman scattering (SERS) microscopy is a novel method of vibrational microspectroscopic imaging for the selective detection of biomolecules in targeted research. This review summarizes current designs of nanoparticle-based SERS probes (see figure) and highlights first biomedical applications of SERS microscopy for protein localization ex and in vivo.

NMR spectroscopy for the molecular life sciences: Two areas of recent active research [fast methods and direct detection of low-γ nuclei (see schematic)] are reviewed herein. These are powerful new tools for structural characterization of short-lived molecules, large and intrinsically unstructured proteins, paramagnetic systems, as well as for the study of molecular kinetic processes at atomic resolution.

Glow in the dark: Fluorescent proteins of the GFP family (see picture) are key tools for life sciences research. Recent structure–dynamics–function studies have yielded new insights that aid in the rational development of advanced fluorescent marker proteins. These new markers should further extend the range of possible applications.

Molecular interfacial properties of colloidal soft matter systems (for example vesicle fusion, or particle synthesis) can be investigated without the need of planar model systems or fluorescent labelling. Exclusively observing chemical properties and changes in a molecular layer of a few molecular dimensions thick around a small particle adds a new dimension to the field of surface science (see figure).

Macromolecular dynamics: A technique for probing conformational dynamics of biomolecules based on contact-induced fluorescence quenching is discussed. Photoinduced electron transfer can be used to monitor structural variations with single-molecule sensitivity on sub-nanometre length scales and with nanosecond temporal resolution (see figure).

DNA molecules do the twist: The DNA double helix is a remarkably adaptable molecule that can undergo major conformational rearrangements without being irreversibly damaged. Indeed, DNA deformation is an intrinsic feature of many biological processes. In this Minireview, the authors summarize recent advances in the study of DNA deformation. The picture shows five different conformations of the double helix of DNA.

The transport of gaseous compounds across biological membranes (see picture) is essential in all forms of life. In this Minireview, the authors discuss recently reported violations of the well-established Meyer–Overton rule for small molecules, including carboxylic acids and gases, and show that Meyer and Overton continue to rule.

Intrinsically disordered proteins exist and function without well-defined three-dimensional structures and are common in proteomes where they carry out essential functions. Herein, evidence for their lack of structure and the major functional benefits that this confers, are surveyed. An example is Tβ4, a sequence that is disordered until it binds to G-actin, when it adopts an ordered conformation (see picture).

DNA photophysics: Femtosecond transient absorption experiments reveal that excited states produced by UV light in a duplex DNA oligonucleotide decay at essentially the same rate in B and Z helix conformers (see figure).

Low-energy electrons (LEEs) interact with a DNA model system to create a variety of excited states. In the gas phase, dissociative (σ*) states are accessible by LEEs with energy <4 eV (see picture) and cause facile strand breaks through a dissociative electron attachment mechanism. However, under solvation these dissociative (σ*) states are blue-shifted and are less accessible to LEEs.

Chiral spectroscopic study: The intensities of second harmonic generation chiral spectroscopy are obtained from the dispersion of the nonlinear optical susceptibility components, as a function of wavelength for helicenbisquinone thin films (see figure). A single formalism fits all the data simultaneously, and the findings constitute an important milestone towards the development of a new experimental technique.

Unusual strength: In the complex glycine–BH₄⁻, unconventional dihydrogen bonds are predicted. An ultrashort dihydrogen bond BH⋅⋅⋅HN and a very strong interaction of about −41 kcal mol⁻¹) are found in complex 3 (see structures), in which the zwitterionic glycine is stabilized with a big margin.

Stronger or weaker H-bonds? H-bonds in interlamellar water in partially hydrated lipid multibilayers are stronger with respect to bulk water. In contrast, the authors show by ATR–FTIR spectroscopy that the H-bonds are weaker in multibilayers in excess water (see spectra). This finding is of biological relevance for water-mediated phenomena in membranes.

Vibrational resonance plays a vital role in manifesting a broad infrared band (>300 cm⁻¹) and ultrafast vibrational energy relaxation (<100 femtoseconds) of the intermolecular NH stretching vibrations (see spectra).

Beyond measurements of equilibria: An α-hemolysin nanopore is used as a single-molecule sensor to follow the effects of different salts on the complexation reaction of γ-cyclodextrin and adamantane carboxylate (see picture). The kinetics underlying the dynamic equilibrium are studied to reveal qualitatively different dynamic actions of various cosolute salts.

Quantifying metal-binding by force: A quantitative single-molecule force-spectroscopy-based assay is developed to measure the binding affinity of metal ions to proteins. The method uses the unfolding force of a protein as a direct probe to distinguish the apo and metal-ion-bound forms of that protein and quantify the partitioning between the two forms (see figure).

At the end of the rainbow… The structure and dynamics of three- and four- way DNA junctions are studied using triple alternating laser excitation. Steroid binding distorts the structure of three-way junctions with correlated bending of all branches towards a steeper pyramid (see picture). In four-way junctions, the dynamics of three distances are visualized simultaneously.

Remote release: Triangular gold nanoprisms convert 1064 nm laser irradiation into heat selectively to allow the dehybridization of oligonucleotide conjugated to their surface (see scheme). These conjugates show unprecedented morphological stability under hours of irradiation. Released nucleic acids are unharmed by this process and can be repeatedly dehybridized and sequestered under spatiotemporal control.

True colors: High-quality InP and InP/ZnS quantum dots (QDs) are obtained by means of a simple one-pot method in the presence of polyethylene glycol (PEG). Rapid and size-controlled reactions lead to highly crystalline and nearly monodisperse QDs at relatively low temperatures. The particles emit from cyan blue to far-red, and are successfully used in cellular imaging (see figure).

The recombinant production of a novel chimeric polyprotein is described. The new protein contains either wild-type β₂-microglobulin (β₂m) or its truncated variant (ΔN6 β₂m) (see picture). Structural characterization is achieved by means of single-molecule force spectroscopy studies of specific β₂m regions which could be involved in amyloidogenesis.

Native-protein nanolithography is combined with topography and recognition imaging to synergistically use AFM tips to write and image nanoscale protein patterns on a surface (see picture). The approach is validated with different feedback modes, using surface-bound biotinylated bovine serum albumin (BSA) protein and AFM tips carrying streptavidin.

AFM topography data of non-covalently assembled biomolecule–nanoparticle hybrids is examined by statistical cluster analysis (see figure). As exemplified for fluorescent-protein-modified quantum dots and DNA/protein-conjugated gold nanoparticles, this analytical approach provides insights into the formation of bioconjugate populations.

Structure of immobilised enzymes: Circular dichroism, infrared and fluorescence spectroscopic methods are used to characterise in situ structural stability of enzymes immobilised on particles. The enzyme subtilisin Carlsberg exhibits a similar secondary structure in solution and in the immobilised state but on the surface of porous silica gel a rigid tertiary structure is preferred (see picture).

Chicken nugget: Simulations of the full chicken prion protein (see picture) and chirality analysis provide possible explanations for its resistance to proteases—and for the fold stability of the avian prion globular core. The three alpha helices exhibit different flexibilities and secondary structure propensities.

Molecular motors: By combining optical tweezers and high-speed particle tracking, individual steps of microtubule motor proteins transporting organelles can be detected under known force loads in living mammalian cells (see figure).

Sensing protein environment: The femtosecond fluorescence dynamics of a molecular probe attached to avidin and streptavidin (see figure) depends markedly on the location of the probe and on the protein. These differences reflect not only the protein primary and tertiary structures but also the effect of the surrounding water molecules.

Adhesion strategies: Open coil-like T4 pili use different adhesion strategies in the presence of external forces (see figure) compared to the helix-like P pili. When exposed to significant forces, bacteria expressing helix-like pili remain attached by distributing the external force among a multitude of pili, whereas bacteria expressing open coil-like pili sustain large forces primarily by their multitude of binding adhesins.

Detecting specific bond formation: The formation of specific bonds between a particle and a substrate results in an immobilization of the particle that occurs in discrete steps, even for nanometer-sized complexes. Each configuration of bonds corresponds to a characteristic distribution of the particle's thermal position fluctuations (see picture).

The detected conformational sub-states of an R-SNARE peptide inserted in a lipid bilayer (see figure) give insights into the membrane fusion mechanism. The simulations are in agreement with most experimental data on the SNARE system, but differ in some details that may have a functional interest. Comparing rat and yeast sequences shows some minor differences in their behaviour.

Stable anchorage: Micro-structured polydimethylsiloxane serves as an optimal specimen-support for immobilizing native nuclear membranes. It offers a solid basis for studying single-molecule interaction of proteins involved in nucleocytoplasmatic transport (see figure).

Unraveling the fast and complex processes involved in intracellular transport: Herein, the authors use a rolling-window analysis with very high temporal resolution to dissect the trajectory of an intracellular tracer and isolate transient active states (see graph). This is a signature of the activity which characterizes living materials.

A novel iodine peptide label for X-ray analysis of membrane-active peptide structures is applied to solid-phase peptide synthesis. The resulting pore-structured labeled peptide as well as a non-labeled reference were reconstituted in lipid bilayer stacks (see scheme). The results indicate the exhibition of a membrane-spanning β^5.6-double helical peptide structure and illustrate the quality of the new label.

Spot the dots: Spatial organization of cell surface proteins plays a key role in the process of transmembrane signalling. Receptor clustering and changes in their cell surface distribution are often determining factors in the final outcome of ligand–receptor interactions. Herein, the authors show the applicability of Ripley's K(t) with additional algorithms a tool for analyzing the cell surface receptor patterns (see picture).

Diffusional recruitment of proteins: Classical Brownian motion analysis describes the long-range movements of biomolecules on a spherical biological membrane. This protein diffusion process is prerequisite for the assembly of proteins which then cooperatively catalyze the polymerization of actin monomers to sustain the growth of actin tails (see picture).

Coming together: Surface diffusion of particles over a sphere and their clustering (see picture) may be adequately modelled by means of Brownian motion simulations or by using continuous Fick's diffusion law with a moving boundary in the low-concentration limit.

UV vibronic spectrum for cytosine has been calculated using high-level quantum chemistry. Coupled with a careful analysis of the available experimental spectra, it is suggested that the spectrum contains only two π–π* transitions; the other two, less resolved bands cannot be type π–π*, and are probably not even n–π* but rather vibrational subbands (see figure).

Two into one: Simultaneous absorption of two photons of different colors (2c2p) extends the applicable wavelength range of the Ti:Sa laser beyond that of conventional two-photon excitation (TPE) into the UV region (see schematic). The intrinsic fluorescence of bovine serum albumin during proteolytic cleavage by subtilisin (see plot) are monitored without exposing the protein to damaging UV radiation.

Keep that motor running: Type IV pili are among the strongest molecular motors characterized to date. Herein it is reported that pilus motors of the human pathogen Neisseria gonorrhoeae are very active for at least one day post-infection of epithelial cells. They generate force in the range on 70 pN and retract at a higher velocity as compared to abiotic environments (see picture).

The branching ratio between ammonia loss and NC_α bond cleavage of singly charged microsolvated peptides after electron capture from cesium depends on the solvent molecule attached. Density functional calculations reveal that for [GA+H]⁺(CE) (G=glycine, A=alanine, CE=crown ether), the singly occupied molecular orbital of the neutral radical is located mainly on the amide group (see picture).

The effects of polyelectrolyte on the interaction of RecA protein and DNA are investigated in vitro by agarose gel electrophoresis and atomic force microscopy (see figure). Through changing the concentration, the length, and the addition orders of the polyelectrolyte, its effects on the DNA–RecA filaments are investigated and the probable mechanism is deduced.

Spectral assignment of the IRMPD bands and identification of the vibrational signatures of deprotonated phosphorylated amino acids are attained. The H₂PO₄⁻ anion is used as a simple model of a free deprotonated phosphate group to identify the IR signatures of phosphorylation. The fragmentation efficiency is lower for [pSer-H]⁻ (left scale in figure) than for [pThr-H]⁻ and [pTyr-H]⁻ (right scale).

Active molecular environment: Oxidation of the cysteine residue by the superoxide radical (see picture) is studied in the gas phase and in aqueous solution using the integrated molecular orbital+molecular orbital. It is shown that the molecular environment plays an important role in the oxidation of biologically relevant thiol compounds by the superoxide radical.

Attracted to gold: The interaction of cytidine 5′-monophosphate (CMP) with gold surfaces is studied at the Au(111) | aqueous solution interface. In situ infrared spectroscopy studies show that cytidine 5′-monophosphate is chemisorbed on Au(111) through the N3 atom of the pyrimidine ring (see picture).

Targeting the hydrophobic core: Solid-state NMR rotational resonance width experiments are performed to measure ¹³C–¹³C distances between aromatic and aliphatic residues that make up the hydrophobic core of streptococcal protein G (see picture). Their value in structure calculations and the effect of chemical shift anisotropy at high magnetic field are discussed.

Charge-transfer probe (E)-3-(4-methylaminophenyl)acrylic acid methyl ester (MAPAME) exhibits a blue shift and enhanced intensity of its charge-transfer emission band on binding to bovine serum albumin (BSA; see picture). These spectral changes can be used to investigate the MAPAME–BSA binding interaction and to monitor the unfolding of BSA induced by denaturants, such as urea and surfactant micelles.

New avenues in pathogenesis research: Single-molecule measurements using AFM elucidate the specific binding forces between pathogen–host interactions. A bacterial adhesin (HBHA) on the AFM tip detects single HSPG receptors directly on living host cells (see figure). In vivo HBHA-HSPG binding forces are similar to those measured in vitro for HBHA-heparin complexes.

The bioelectrochemistry symposium at the 59th annual meeting of the International Society of Electrochemistry (ISE) covered significant developments in this area—from biomimetics, redox proteins, and enzymes, to biosensors and biofuel-cell applications. This conference report highlights some of the most important contributions (the picture shows a schematic representation of an amperometric enzyme electrode, courtesy of P.N. Bartlett).

Biophysics in Linz: In February biophysicists from across the world converged on Linz for two biophysical conferences. The city which is the Cultural Capital of Europe 2009, provided the perfect environment for fruitful discussions on single-molecule techniques in biophysics, bio-nanotechnology, cell biology, and drug discovery.