Friction force spectroscopy of β- and κ-casein monolayers

Friction force spectroscopy (FFS) has been applied to study the tribological properties of β- and κ-casein layers on hydrophobic substrates in aqueous solutions. Nanometer-sized imaging tips were employed. This allowed exerting and determining the high pressures needed to remove the layers and registering the topographic evolution during this process. Both β- and κ-casein layers showed similar and not particularly high initial frictional responses (friction coefficient of ～1 when measured with a silicon nitride tip). The pressures needed to remove the layers were of the same order of magnitude for both proteins, ～10(8) Pa, but slightly higher for those composed of β-casein. The technique has also shown to be useful in studying the two-dimensional lateral diffusion of the proteins and the wear on the layers they form.     

Synthesis and structural study of novel dimethylcyclobutyl β-peptides

The synthesis of some representative compounds of a new class of cyclobutane-containing β-peptides starting from (?)-verbenone as a chiral precursor is presented. In these products, the cyclobutane moiety is not a part of the peptide backbone but a bulky substituent at the β³-position. These compounds have been carefully characterized and studied on the basis of the combined use of several experimental techniques together with molecular modeling by means of theoretical calculations. In the solid state, the non-cyclic β-peptides adopt a hairpin-like molecular folding ruled by intermolecular hydrogen bonds in the crystal packing.     

Predicting three-dimensional structures of transmembrane domains of β-barrel membrane proteins

β-Barrel membrane proteins are found in the outer membrane of gram-negative bacteria, mitochondria, and chloroplasts. They are important for pore formation, membrane anchoring, and enzyme activity. These proteins are also often responsible for bacterial virulence. Due to difficulties in experimental structure determination, they are sparsely represented in the protein structure databank. We have developed a computational method for predicting structures of the transmembrane (TM) domains of β-barrel membrane proteins. Based on physical principles, our method can predict structures of the TM domain of β-barrel membrane proteins of novel topology, including those from eukaryotic mitochondria. Our method is based on a model of physical interactions, a discrete conformational state space, an empirical potential function, as well as a model to account for interstrand loop entropy. We are able to construct three-dimensional atomic structure of the TM domains from sequences for a set of 23 nonhomologous proteins (resolution 1.8-3.0 ?). The median rmsd of TM domains containing 75-222 residues between predicted and measured structures is 3.9 ? for main chain atoms. In addition, stability determinants and protein-protein interaction sites can be predicted. Such predictions on eukaryotic mitochondria outer membrane protein Tom40 and VDAC are confirmed by independent mutagenesis and chemical cross-linking studies. These results suggest that our model captures key components of the organization principles of β-barrel membrane protein assembly.     

Positional screening and NMR structure determination of side-chain-to-side-chain cyclized β3-peptides

Many β-peptides fold in a 14-helical secondary structure in organic solvents, but similar 14-helix formation in water requires additional stabilizing elements. Especially the 14-helix stabilization of short β-peptides in aqueous solution is critical, due to the limited freedom for incorporating stabilizing elements. Here we show how a single lactam bridge, connecting two β-amino acid side-chains, can lead to high 14-helix character in short β(3)-peptides in water. A comparative study, using CD and NMR spectroscopy and structure calculations, revealed the strong 14-helix inducing power of a side-chain-to-side-chain cyclization and its optimal position on the β(3)-peptide scaffold with respect to pH and ionic strength effects. The lactam bridge is ideally incorporated in the N-terminal region of the β(3)-peptide, where it limits the conformational flexibility of the peptide backbone. The lactam bridge induces a 14-helical conformation in methanol and water to a similar extent. Based on the presented first high resolution NMR 3D structure of a lactam bridged β(3)-peptide, the fold shows a large degree of high order, both in the backbone and in the side-chains, leading to a highly compact and stable folded structure.     

Using one-step perturbation to predict the folding equilibrium of differently stereochemically substituted β-peptides

The one-step perturbation technique is used to predict the folding equilibria for 16 peptides with different stereochemical side-chain substitutions through one or two long-time simulations, one of an unphysical reference state and another of one of the 16 peptides for which many folding events can be sampled. The accuracy of the one-step perturbation results was investigated by comparing to results available from long-time MD simulations of particular peptides. Their folding free energies were reproduced within statistical accuracy. The one-step perturbation results show that an axial substitution at either the C(α) or the C(β) position destabilizes the 3(14)-helical conformation of the hepta-β-peptide, which is consistent with data inferred from experimental CD spectra. The methodology reduces the number of required separate simulations by an order of magnitude.     

Photoelectron spectroscopy and electronic structures of β-diketonate complexes of rare-earth elements

The electronic structures of lanthanide tris(β-diketonate) complexes and their adducts, being of interest as luminophores, were studied by UV photoelectron spectroscopy of vapors and X-ray photoelectron spectroscopy. The spectral regularities identified by the density functional theory revealed the influence of the substitution of the Me groups in the ligands by Bu^t, CF₃, and neutral ligand in the adducts on the electronic structure of the complexing agent from Pr to Lu.     

Characterization of collagen fibers in Bruch’s membrane using chemical force microscopy

Bruch’s membrane is a layer composed of collagen fibers located just beneath the retina. This study validates a strategy used to map the morphological and adhesion characteristics of collagen fibers in Bruch’s membrane. Atomic force microscopy tips were functionalized with different chemical groups and used to map the hydrophilic and hydrophobic regions on the surface of the eye tissue. The largest adhesion forces were observed when tips functionalized with NH₂ groups were used. The trend in the adhesion forces was rationalized based on the distribution of different functional groups in the triple-helical structure of the collagen fibers. The results of this study can be used to design more effective strategies to treat eye diseases such as age-related macular degeneration.     

Concurrent display of both α- and β-turns in a model peptide

This article describes a model peptide that concurrently displays both α- and β-turns, as demonstrated by structural investigations using single crystal X-ray crystallography and solution-state NMR studies. The motif reported herein has the potential for the design of novel conformationally ordered synthetic oligomers with structural architectures distinct from those classically observed.     

Computational design of a β-peptide that targets transmembrane helices

The design of β-peptide foldamers targeting the transmembrane (TM) domains of complex natural membrane proteins has been a formidable challenge. A series of β-peptides was designed to stably insert in TM orientations in phospholipid bilayers. Their secondary structures and orientation in the phospholipid bilayer was characterized using biophysical methods. Computational methods were then devised to design a β-peptide that targeted a TM helix of the integrin α(IIb)β(3). The designed peptide (β-CHAMP) interacts with the isolated target TM domain of the protein and activates the intact integrin in vitro.     

Synthesis and utility of β-selenol-phenylalanine for native chemical ligation-deselenization chemistry

An efficient synthetic route to a suitably protected β-selenol-phenylalanine derivative from commercially available Garner's aldehyde is described. The incorporation of this building block into peptides and its application in native chemical ligation reactions with peptide thioesters are demonstrated. Ligation products were chemoselectively deselenized (including in the presence of unprotected cysteine residues) to provide native peptides.     

Vibrational spectra of 1,1-dichloroethanes and βCH stretching interaction force constants

Infrared and Raman spectra are described and assignments are reported for CH₃CDCl₂ and CD₃CHCl₂, together with new i.r. data for CH₃CHCl₂ and CD₂HCHCl₂. Using previously obtained “isolated” CH stretching frequencies, the above data enable the trans and gauche stretch—stretch interaction force constants to be determined:f′_g = ±0.011, f′_t = ∓0.026 mdyn/Å. Similar values, f′_g = +0.016, f′_t = −0.029, may be obtained from the earlier spectra of CHD₂CHD₂ due to Van Riet.The relations between the fundamental deformation bands and their overtones and combinations are examined in order to estimate the accuracy of predictions of v_s (=v₃) for the CH₃ and CD₃ groups from the 4 × 4 refinement and frequency sum rule. The data can be interpreted so as to give good agreement on the values of v^O_s of 2920 cm⁻¹ (CH₃) and 2115 cm⁻¹ (CD₃), provided that there are significance resonance shifts on both 2δ_a and 2δ′_s levels. That this is the case seems to be true for the CD₃ group, but is uncertain for the CH₃ one.     

Influence of degree and patterns of substitution on the inclusion properties of ethylated β-cyclodextrins

Different batches of ethylated derivatives of -cyclodextrins (Et-CD) obtained by various synthetic routes were used as host molecules to prepare inclusion compounds of salbutamol. Previous results showed that these complexes were suitable to achieve variable sustained-release behaviour of salbutamol. The mechanism of sustained-release was related to the inclusion capacity and physicochemical properties of Et-CD. Among the various analytical techniques carried out to characterize them, high resolution NMR afforded relevant information in terms of degree and patterns of subtitution, which could explain the differences observed between CD derivatives.     

Design,synthesis and structural analysis of mixed α/β-peptides that adopt stable cyclic hairpin-like conformations

The strategic replacement of four α-amino acid residues of a cyclo-(ααααα)₂ peptide by β-, β²- or β³-amino acids residues provided a series of novel 2:1 α/β-mixed peptides that were designed to adopt cyclic hairpin-like structures. It was shown that conformationally stable cyclo-(αβαβα)₂ isomers can be obtained using both enantiomers of the central two basic α-amino acid residues, a known α-amino acid turn sequence and several combinations of facing β-amino acid residues with no side chain or a hydrophobic side chain having specific regio- and stereochemistry. The X-ray analysis of two derivatives provides molecular details of the intra-molecular hydrogen bonding interaction, dihedral angles of the backbone and side chain positioning of the novel cyclic hairpin-like structures. One of these isomers forms an unprecedented hexagon-shaped nano-channel assembly in the crystal structure. Well-defined cyclic hairpin-like structures as described here and derivatives that can be readily designed based on this research can be used as scaffolds onto which functional groups can be grafted in a spatially controlled manner and as β-hairpin mimics with specific biological properties.     

Antagonism of chemical genetic interaction networks resensitize MRSA to β-lactam antibiotics

Antibiotic drug resistance among hospital and community acquired methicillin resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current therapeutics. We describe a chemical genetic strategy using antisense interference to broadly identify new drug targets that potentiate the effects of existing antibiotics against both etiological classes of MRSA infection. Further, we describe the resulting chemical genetic interaction networks and highlight the prominent and overlapping target sets that restore MRSA susceptibility to penicillin, cephalosporins, and carbapenems. Pharmacological validation of this approach is the potent synergy between a known inhibitor to a member of this genetic potentiation network (GlmS) and a broad set of β-lactam antibiotics against methicillin resistant Staphylococci. Developing drug-like leads to these targets may serve as rational and effective combination agents when paired with existing β-lactam antibiotics to restore their efficacy against MRSA.     

2DIR spectroscopy of human amylin fibrils reflects stable β-sheet structure

The aggregation of human amylin to form amyloid contributes to islet β-cell dysfunction in type 2 diabetes. Studies of amyloid formation have been hindered by the low structural resolution or relatively modest time resolution of standard methods. Two-dimensional infrared (2DIR) spectroscopy, with its sensitivity to protein secondary structures and its intrinsic fast time resolution, is capable of capturing structural changes during the aggregation process. Moreover, isotope labeling enables the measurement of residue-specific information. The diagonal line widths of 2DIR spectra contain information about dynamics and structural heterogeneity of the system. We illustrate the power of a combined atomistic molecular dynamics simulation and theoretical and experimental 2DIR approach by analyzing the variation in diagonal line widths of individual amide I modes in a series of labeled samples of amylin amyloid fibrils. The theoretical and experimental 2DIR line widths suggest a "W" pattern, as a function of residue number. We show that large line widths result from substantial structural disorder and that this pattern is indicative of the stable secondary structure of the two β-sheet regions. This work provides a protocol for bridging MD simulation and 2DIR experiments for future aggregation studies.     

Synthesis and chemical properties of cyclic β-keto sulfones (review)

Published data on the synthesis and chemical properties of dihydrothiophen-3(2H)-one 1,1-dioxide, dihydro-2H-thiopyran-3(4H)-one 1,1-dioxide, 1-benzothiophen-3(2H)-one 1,1-dioxide, and 1H-isothio-chromen-4(3H)-one 2,2-dioxide are reviewed. The choice of subjects was based on the presence of identical structural fragments, carbonyl, active methylene, and sulfonyl groups.     

Configurations of anions of β-ketoaldehydes in solutions studied by i.r. spectroscopy

The i.r. spectra of the Li, Na and K salts of β-ketoaldehydes, RCOCH₂CHO (R = Me, Et, i-Pr and t-Bu) in DMSO, methanol and water were investigated. Assignments of bands to stretching modes of particular configurations of the enolate system were given. Influence of the cation radius on the Z,Z ⇌ E,E/Z,E equilibrium and of the substituent R on the E,E ⇌ Z,E equilibrium was observed. Shifts of the enolate bands in protic solvents were observed and hydrogen-bond formation accounted for this phenomenon.     

Development of sample preparation technique to characterize chemical structure of humin by synchrotron-radiation–based X-ray photoelectron spectroscopy

Carbon-binding state of humin (HM, a non-conductive insoluble organo-mineral humic substance) was successfully characterized for the first time by synchrotron-radiation–based X-ray photoelectron spectroscopy (XPS). Four sample preparation techniques—HM on double-sided carbon tape, indium sheet, copper mesh, and in pellet formed from the mixture of HM and copper powder (Cu) at different mixing ratios (1:1, 1:2, and 1:6 v/v)—were compared. The results show that HM samples prepared using the first three methods had significant charge buildup, which made the interpretation of the XPS spectra impossible because of the shifts in the binding energy of C 1s XPS spectra. Pellets of HM:Cu mixture enhanced the electrical conductivity and reduced charge buildup on the sample surface. Pellets prepared with HM:Cu ratio of 1:1 (v/v) provided the minimum charge buildup and high sensitivity with difference in C 1s spectra regardless of the observing position. The C 1s spectra, estimated by the subtraction of the carbon contamination in Cu, showed the resolution of CC (284.0 eV), C C/C H (285.1 eV), C O (286.3 eV), CO (287.3 eV), and OC O (288.3 eV) and three additional peaks of CF (289.3 eV), CF₂ (290.2 eV), and CF₃ (291.4 eV). Soft X-ray absorption spectroscopic (XAS) analysis further proved the existence of fluoride (F 1s) in HM structure. The detection of fluorinated carbon in HM showed a great advancement of XPS compared with other conventional analyses. X-ray with the incident angle of 0° provided the smallest (nearly negligible) energy shift in the C 1s spectra of HM and did not damage the surface of the sample.     

Macrocyclic β-sheet peptides that inhibit the aggregation of a tau-protein-derived hexapeptide

This paper describes studies of a series of macrocyclic β-sheet peptides 1 that inhibit the aggregation of a tau-protein-derived peptide. The macrocyclic β-sheet peptides comprise a pentapeptide "upper" strand, two δ-linked ornithine turn units, and a "lower" strand comprising two additional residues and the β-sheet peptidomimetic template "Hao". The tau-derived peptide Ac-VQIVYK-NH(2) (AcPHF6) aggregates in solution through β-sheet interactions to form straight and twisted filaments similar to those formed by tau protein in Alzheimer's neurofibrillary tangles. Macrocycles 1 containing the pentapeptide VQIVY in the "upper" strand delay and suppress the onset of aggregation of the AcPHF6 peptide. Inhibition is particularly pronounced in macrocycles 1a, 1d, and 1f, in which the two residues in the "lower" strand provide a pattern of hydrophobicity and hydrophilicity that matches that of the pentapeptide "upper" strand. Inhibition varies strongly with the concentration of these macrocycles, suggesting that it is cooperative. Macrocycle 1b containing the pentapeptide QIVYK shows little inhibition, suggesting the possibility of a preferred direction of growth of AcPHF6 β-sheets. On the basis of these studies, a model is proposed in which the AcPHF6 amyloid grows as a layered pair of β-sheets and in which growth is blocked by a pair of macrocycles that cap the growing paired hydrogen-bonding edges. This model provides a provocative and appealing target for future inhibitor design.