Probing the conformational features of a phage display polypeptide sequence directed against single-walled carbon nanohorn surfaces

Single-walled carbon nanohorns (SWNHs) are interesting carbon nanostructures that have applications to science and technology. Using M13 phage display technology, polypeptides directed again SWNHs surfaces have been created for a number of nanotechnology and pharmaceutical purposes, yet the molecular mechanism of polypeptide sequence interaction and binding to SWNHs surfaces is not known. Recently, we identified a linear 12-AA M13 phage pIII sequence, NH-12-5-2 (DYFSSPYYEQLF), that binds with high affinity to SWNHs surfaces. To probe the structure of this pIII tail polypeptide further, we investigated the conformation of a model peptide representing the 12 AA NH-12-5-2 sequence. At neutral pH, the NH-12-5-2 model polypeptide is conformationally labile and exhibits two-state conformational exchange involving the D1-S5 N-terminal segment. Simultaneous with this conformational exchange process is the observation that the P6 residue exhibits imido ring conformational variation. In the presence of the structure-stabilizing solvent, TFE, or at pH 2.5, both the exchange process and Pro ring motion phenomena disappear, indicating that the structure of this peptide sequence can be stabilized by extrinsic factors. Interestingly, we observe NMR parameters (ROEs, (3)J coupling constants) for NH-12-5-2 in 90% v/v TFE that are consistent with the presence of a partial helical structure, similar to what was observed at low pH in our earlier CD experiments. We conclude that the NH-12-5-2 model polypeptide sequence possesses an inherent conformational instability that involves the D1-S5 sequence segment and the P6 residue but that this instability can be offset by extrinsic factors (e.g., charge neutralization, imido ring interconversion, and hydrophobic-hydrophobic interactions). These nonbonding interactions may play a role in the recognition and binding of this phage sequence region to SWNHs surfaces.     

On-line monitoring of enzymatic conversion of adenosine triphosphate to adenosine diphosphate by micellar electrokinetic chromatography

Capillary electrophoresis can be a valuable tool for the on-line monitoring of bioprocesses. The enzymatic conversion of nucleotide adenosine triphosphate (ATP) to adenosine diphosphate (ADP) by hexokinase (HK) was monitored in the bioreactor interfaced by a laboratory-built microsampler to a capillary electrophoresis unit. The use of this specially designed sampling device enabled rapid consecutive injections to be performed without high-voltage (HV) interruptions. No additional sample preparation was required. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow (EOF) by cationic surfactant and reversed polarity mode provided a good resolution and short analysis time of less than 5 min. The samples were injected electrokinetically, using -25 kV voltage for 3 s and detected by their UV absorbance at 254 nm. The analytes were detected at a microg/ml level with a reproducibility of about 7%. To demonstrate the potential of CE in understanding the processes of biological interest, such as nucleotide degradation and metabolism, the investigation of the efficiency and the time course of the enzymatic transformation was carried out.     

Synthesis of CuCorePtShell Nanoparticles as Model Structures for Core–Shell Electrocatalysts by Direct Platinum Electrodeposition on Copper

The synthesis of Cu(core)Pt(shell) model catalysts by the direct electrochemical deposition of Pt on Cu particles is presented. Cu particles with an average diameter of 200 nm have been deposited on glassy‐carbon electrodes by double pulse electrodeposition from a copper sulfate solution. Subsequent deposition from a platinum nitrate solution under potential control allows for a high selectivity of the Pt deposition towards Cu. Using a combination of cyclic voltammetry, XPS and sputtering, the structure of the generated particles has been analyzed and their core–shell configuration proven. It is shown that the electrocatalytic activity for the oxygen reduction is similar to that of other PtCu catalyst systems. The synthesized structures could allow for the analysis of structure–activity relations of core–shell catalysts on the way to the simple and controlled synthesis of supported Cu(core)Pt(shell) nanoparticles as oxygen reduction catalysts.     

Fine structure in proton emission from 145Tm discovered with digital signal processing

Fine structure in proton emission from the 3.1(3) mus activity of 145Tm was discovered by using a novel technique of digital processing of overlapping recoil implantation and decay signals. Proton transitions to the ground state of 144Er and to its first excited 2(+) state at 0.33(1) MeV with a branching ratio I(p)(2(+))=9.6+/-1.5% were observed. The structure of the 145Tm wave function and the emission process were analyzed by using particle-core vibration coupling models.     

Structural properties of an artificial protein that regulates the nucleation of inorganic and organic crystals

Technological advances have facilitated the generation of artificial proteins that possess the capabilities of recognizing and binding to inorganic solids and/or controlling nucleation processes that form inorganic solids. However, very little is known regarding the structure of these interesting polypeptides and how their structure contributes to functionality. To address this deficiency, we report structural investigations of an artificial protein, p288, that self-assembles and controls the nucleation of simple salts and organic compounds into dendrite-like crystals. Under aqueous conditions at low pH and in the presence of high salt, p288 is conformationally labile and exists primarily as a random coil conformer in equilibrium with other undefined secondary structures, including polyproline type II and beta turn. We note that p288 can fold into either a partial beta strand (at neutral pH) or a predominantly alpha helical (in the presence of TFE) conformation. Solid-state 13C-15N NMR experiments also reveal that p288 in the lyophilized, hydrated state possesses some degree of nonrandom coil structure. These results indicate that p288 is conformationally labile but can undergo conformational transitions to a more stable structure when water solvent loss/displacement occurs and protein concentrations increase. We believe that conformational instability and the ability to adopt different structures as a function of different environmental conditions represent important molecular features that impact p288 supramolecular assembly and crystal nucleation processes.     

CE separation of various analytes of biological origin using polyether ether ketone capillaries and contactless conductivity detection

The development of efficient and sensitive analytical methods for the separation, identification and quantification of complex biological samples is continuously a topic of high interest in biological science. In the present study, the possibility of using a polyether ether ketone (PEEK) capillary for the CE separation of peptides, proteins and other biological samples was examined. The performance of the tubing was compared with that of traditional silica capillaries. The CE analysis was performed using contactless conductivity detection (C⁴D), which eliminated any need for the detection window and was suitable for the detection of optically inactive compounds. In the PEEK capillary the cathodic EOF was low and of excellent stability even at extremes pH. In view of this fast biological anions were analyzed using an opposite end injection technique without compromising separation. A comparison of the performances of fused‐silica and polymer capillaries during the separation of model sample mixtures demonstrated the efficiency and separation resolution of the latter to be higher and the reproducibility of the migration times and peak areas is better. Furthermore, PEEK capillaries allowed using simple experimental conditions without any complicated modification of the capillary surface or use of an intricate buffer composition. The PEEK capillaries are considered as an attractive alternative to the traditional fused‐silica capillaries and may be used for the analysis of complex biological mixtures as well as for developing portable devices.     

Engineering a β‐Helical d,l‐Peptide for Folding in Polar Media

β Helices—helices formed by alternating d,l ‐peptides and stabilized by β‐sheet hydrogen bonding—are found naturally in only a handful of highly hydrophobic peptides. This paper explores the scope of β‐helical structure by presenting the first design and biophysical characterization of a hydrophilic d,l ‐peptide, 1 , that forms a β helix in methanol. The design of 1 is based on the β‐hairpin/β helix—a new supersecondary that had been characterized previously only for hydrophobic peptides in nonpolar solvents. Incorporating polar residues in 1 provided solubility in methanol, in which the peptide adopts the expected β‐hairpin/β‐helical structure, as evidenced by CD, analytical ultracentrifugation (AUC), NMR spectroscopy, and NMR‐based structure calculations. Upon titration with water (at constant peptide concentration), the structure in methanol ( 1 m ) transitions cooperatively to an extended conformation ( 1 w ) resembling a cyclic β‐hairpin; observation of an isodichroic point in the solvent‐dependent CD spectra indicates that this transition is a two‐state process. In contrast, neither 1 m nor 1 w show cooperative thermal melting; instead, their structures appear intact at temperatures as high as 65 °C; this observation suggests that steric constraint is dominant in stabilizing these structures. Finally, the ¹H NMR CαH spectroscopic resonances of 1 m are downfield‐shifted with respect to random‐coil values, a hitherto unreported property for β helices that appears to be a general feature of these structures. These results show for the first time that an appropriately designed β‐helical peptide can fold stably in a polar solvent; furthermore, the structural and spectroscopic data reported should prove useful in the future design and characterization of water‐soluble β helices.     

Epitaxial electrodeposition of ZnO on Au(111) from alkaline solution: exploiting amphoterism in Zn(II)

The amphoteric nature of ZnO is used to produce the material from strongly alkaline solution. The solution pH is lowered globally to produce ZnO powder, and it is lowered locally at a Au(111) surface to produce epitaxial films. ZnO powder is precipitated from a solution of 10 mM Zn(II) in 0.25 M NaOH by simply adding 1 M HNO3 to the solution. For the film electrodeposition, the local pH at the electrode surface is decreased by electrochemically oxidizing the ascorbate dianion. The chemically precipitated ZnO powder grows with a sea urchin-like nanostructure, whereas the electrodeposited films have a columnar structure. ZnO electrodeposited onto a Au(111) single crystal has a ZnO(0001)[1011]//Au(111)[110] orientation relationship.     

In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography

A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin-permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed-polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast-twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.