Synthesis of and NMR studies on the four diastereomeric 1-deoxy-d-ketohexoses

The four 1-deoxy-d-ketohexoses—1-deoxy-d-psicose, 1-deoxy-d-fructose, 1-deoxy-d-sorbose and 1-deoxy-d-tagatose—were synthesised by methyl lithium addition to suitably protected and readily available pentonolactones. The 1-deoxy-l-ketohexoses are available from the enantiomeric lactones. The NMR studies on aqueous solutions of each diastereomer show that the relative amounts of open chain ketones, α- and β-pyranoses, and α- and β-furanoses vary considerably; at least four different species are identifiable from each equilibrium.     

α-Synuclein Spontaneously Adopts Stable and Reversible α-Helical Structure in Water-Less Environment

A highly stable, spontaneous, and reversible α-helical-structure formation in recombinant and chemically modified α-synuclein protein is demonstrated for the first time in a water-less (1.5 % w/w H₂O) polymer surfactant environment. Using a combination of circular dichroism and ATR-FTIR spectroscopy, we show that whilst native α-synuclein in aqueous solution shows a predominant unordered conformation (≈64 %), mixing with polyethylene glycol based anionic polymer surfactant (PS) and removing water reveals a 25 % unordered, 25 % α-helical, and 27 % β-sheet structure. Interestingly, bioconjugation of native α-synuclein with a diamine molecule, to increase the positive charge on the protein chain, and subsequent electrostatic coupling with the PS forms a conjugate with a retained unordered structure. Removal of water from this system provides a highly stable α-helical (≈74 %) water-less liquid system. Surprisingly, the α-helical-to-unordered state transition is completely reversible and is achieved at ≈25–30 w/w% of water in the system. Moreover, the α-helix shows an extraordinary temporal stability (>6 months) in a waterless environment.     

Differential ordering of the protein backbone and side chains during protein folding revealed by site-specific recombinant infrared probes

The time scale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and ordering of the side chains is particularly important for the formation of β-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/β structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone ((13)C═(18)O) using recombinant protein expression. Temperature-jump time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side-chain dynamics with single residue resolution. We have found that side-chain ordering in a key region of the β-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely as a result of the transient formation of non-native side-chain interactions.     

Frustration and hydrophobicity interplay in protein folding and protein evolution

A lattice model is used to study mutations and compacting effects on protein folding rates and folding temperature. In the context of protein evolution, we address the question regarding the best scenario for a polypeptide chain to fold: either a fast nonspecific collapse followed by a slow rearrangement to form the native structure or a specific collapse from the unfolded state with the simultaneous formation of the native state. This question is investigated for optimized sequences, whose native state has no frustrated contacts between monomers, and also for mutated sequences, whose native state has some degree of frustration. It is found that the best scenario for folding may depend on the amount of frustration of the native structure. The implication of this result on protein evolution is discussed.     

A convenient synthesis of α-amino-β-lactams

A safe and convenient method is described for the synthesis of α-amido-β-lactams starting with glycine and an azomethine. The amino group of glycine is protected by reaction with a β-dicarbonyl compound following the method of Dane etal. and the carboxyl group is activated through the formation of a mixed anhydride or an active ester. Condensation between these glycine derivatives and acyclic or cyclic imino compounds (including thioimidates) in presence of triethylamine leads to stereospecific synthesis of 3-(β-carbonyl-vinylamino)-2-azetidinones in 40–60% yield. The vinylamino side chain can be hydrolyzed under mild acid conditions to form 3-amino-2-azetidinones which can be acylated to α-amido-β-lactams. Alternatively, the vinylamino side chain can be converted to an amido side chain by ozonolysis. The molecular parameters of a 3-(β-carbonyl-vinylamino)-2-azetidinone were determined by X-ray crystallography. Usefulness of this α-amido-β-lactam synthesis is illustrated by the preparation of isotopelabeled β-lactams and intermediates for some β-lactam antibiotics.     

The influence of the binding of low molecular weight surfactants on the thermal stability and secondary structure of IgG

The effect of low molecular weight surfactants on the thermal stability of immunoglobulin G is studied by differential scanning calorimetry. The corresponding change in the secondary structure is investigated using circular dichroism spectroscopy and the rate of aggregate formation, both in the presence and absence of surfactant, is monitored by dynamic light scattering. At low surfactant concentrations (SDS/Tween 20 mixture) the thermal stability of the protein was not affected. With increasing surfactant concentration the protein structure is perturbed, most probably due to hydrophobic interaction with the surfactant, leading to a lower thermal stability. At even higher concentrations the surfactant molecules encapsulate the protein molecules, so that the unfolded state is strongly suppressed due to restricted conformational freedom in a confined volume. Interaction with the surfactant mixture at intermediate concentration influences the secondary structure of IgG strongly, i.e. α-helix and random coil conformations are promoted and the amounts of β-sheets and β-turns are reduced.     

Protonation/deprotonation effects on the stability of the Trp-cage miniprotein

The Trp-cage miniprotein is a 20 amino acid peptide that exhibits many of the properties of globular proteins. In this protein, the hydrophobic core is formed by a buried Trp side chain. The folded state is stabilized by an ion pair between aspartic acid and an arginine side chain. The effect of protonating the aspartic acid on the Trp-cage miniprotein folding/unfolding equilibrium is studied by explicit solvent molecular dynamics simulations of the protein in the charged and protonated Asp9 states. Unbiased Replica Exchange Molecular Dynamics (REMD) simulations, spanning a wide temperature range, are carried out to the microsecond time scale, using the AMBER99SB forcefield in explicit TIP3P water. The protein structural ensembles are studied in terms of various order parameters that differentiate the folded and unfolded states. We observe that in the folded state the root mean square distance (rmsd) from the backbone of the NMR structure shows two highly populated basins close to the native state with peaks at 0.06 nm and 0.16 nm, which are consistent with previous simulations using the same forcefield. The fraction of folded replicas shows a drastic decrease because of the absence of the salt bridge. However, significant populations of conformations with the arginine side chain exposed to the solvent, but within the folded basin, are found. This shows the possibility to reach the folded state without formation of the ion pair. We also characterize changes in the unfolded state. The equilibrium populations of the folded and unfolded states are used to characterize the thermodynamics of the system. We find that the change in free energy difference due to the protonation of the Asp amino acid is 3 kJ mol(-1) at 297 K, favoring the charged state, and resulting in ΔpK(1) = 0.5 units for Asp9. We also study the differences in the unfolded state ensembles for the two charge states and find significant changes at low temperature, where the protonated Asp side chain makes multiple hydrogen bonds to the protein backbone.     

Protein stability at a carbon nanotube interface

The interactions of proteins with solid surfaces occur in a variety of situations. Motivated by the many nanoengineering applications of protein-carbon nanotube hybrids, we investigate the conformational transitions of hen egg white lysozyme adsorbed on a carbon nanotube. Using a C(α) structure-based model and replica exchange molecular dynamics, we show how the folding/unfolding equilibrium of the adsorbed protein varies with the strength of its coupling to the surface. The stability of the native state depends on the balance between the favorable entropy and unfavorable enthalpy change on adsorption. In the case of a weakly attractive surface when the former dominates, the protein is stabilized. In this regime, the protein can fold and unfold while maintaining the same binding fraction. With increasing surface attraction, the unfavorable enthalpic effect dominates, the native state is destabilized, and the protein has to extensively unbind before changing states from unfolded to folded. At the highest surface coupling, the entropic penalty of folding vanishes, and a folding intermediate is strongly stabilized. In this intermediate state, the α-domain of lysozyme is disrupted, while the β-sheet remains fully structured. We rationalize the relative stability of the two domains on the basis of the residue contact order.     

Carboxymethylated cyclodextrin derivatives as chiral NMR discriminating agents

Procedures to prepare cyclodextrins with carboxymethyl groups incorporated selectively at the primary (6-position) or secondary (2-position) are described. Complexation properties of the primary and secondary carboxymethylated derivatives of α-, β-, and γ-cyclodextrins are compared to native cyclodextrins and indiscriminately substituted carboxymethylated cyclodextrins, using pheniramine, chlorpheniramine, and brompheniramine as substrates. The stoichiometry of association of these substrates with the α-cyclodextrins is 1:1, whereas with the γ-cyclodextrins, a 2:1 substrate:cyclodextrin complex forms. Data for the β-cyclodextrins suggest that there is a mix of 1:1 and 2:1 substrate–cyclodextrin complexes. The position of the carboxymethyl groups on the cyclodextrin does not appear to alter the geometry of substrate–cyclodextrin association. The effectiveness of the carboxymethylated cyclodextrins as chiral NMR discriminating agents is compared with the native cyclodextrins. In all cases, the indiscriminately substituted α-, β-, and γ-cyclodextrins are more effective at enantiodistinction with the cationic substrates than native cyclodextrins or the derivatives with carboxymethyl groups at the primary or secondary positions. Among α-, β-, and γ-indiscriminately substituted cyclodextrins, there was no clearly optimal candidate for chiral NMR discrimination studies. The indiscriminately substituted carboxymethyl cyclodextrins are effective water-soluble chiral NMR discrimination reagents for cationic substrates.     

Complexation of zearalenone and zearalenols with native and modified β-cyclodextrins

The complexation of the oestrogenic mycotoxin zearalenone (ZEN) and its metabolites α- and β-zearalenol (ZOLs) with native β-cyclodextrins (β-CD) and modified hydroxypropyl-β-CD and dimethyl-β-CD was studied by fluorescence spectroscopy, nuclear magnetic resonance spectroscopy and electrospray-mass spectrometry. The formation of the inclusion complex was confirmed by NMR studies of zearalenone:β-CD solution. NMR, ESI-MS and fluorescence data are in agreement with the formation of a 1:1 complex between zearalenone and β-CD, characterized by the deep insertion of the phenolic moiety inside the cavity of the CD from its secondary side. The complexes formed between the guests and native β-CD are characterized by high stability constants (>10⁴), as measured by fluorescence titrations.     

Dynamic control of protein conformation transition in chromatographic separation based on hydrophobic interactions: Molecular dynamics simulation

Conformational transitions of a protein in hydrophobic interaction based chromatography, including hydrophobic interaction chromatography (HIC) and reversed-phase liquid chromatography (RPLC), and their impact on the separation process and performance were probed by molecular dynamics simulation of a 46-bead β-barrel coarse-grained model protein in a confined pore, which represents the porous adsorbent. The transition of the adsorbed protein from the native conformation to an unfolded one occurred as a result of strong hydrophobic interactions with the pore surface, which reduced the formation of protein aggregates. The conformational transition was also displayed in the simulation once an elution buffer characterized by weaker hydrophobicity was introduced to strip protein from pore surface. The discharged proteins that underwent conformational transition were prone to aggregation; thus, an unsatisfactory yield of the native protein was obtained. An orthogonal experiment revealed that in addition to the strengths of the protein–protein and protein–adsorbent hydrophobic interactions, the elution time required to reduce the above-mentioned interactions also determined the yield of native protein by HIC and RPLC. Stepwise elution, characterized by sequential reduction of the hydrophobic interactions between the protein and adsorbent, was presented as a dynamic strategy for tuning conformational transitions to favor the native conformation and reduce the formation of protein aggregates during the elution process. The yield of the native protein obtained by this dynamic operation strategy was higher than that obtained by steady-state elution. The simulation study qualitatively reproduced the experimental observations and provided molecular insight that would be helpful for designing and optimizing HIC and RPLC separation of proteins.     

DSC Study of Crystallinity Changes of Naproxen in Ground Mixtures with Linear Maltooligomers

Differential scanning calorimetry was applied for an analysis of the cogrinding-induced crystallinity changes of naproxen in binary mixtures with linear maltooligomers. Factors found to play a role in the amorphization process were the mixture composition, the duration of mechanical treatment and the degree of polymerization of the carrier. Maltopentaose was about as active as amorphous hydroxypropyl α- and β-cyclodextrin MS 0.6, while maltotetraose displayed practically the same amorphizing capacity as those of native α- and β-cyclodextrin. The melting peak temperature of naproxen was substantially unaltered by cogrinding with maltooligomers, whereas it was considerably lower in coground mixtures with cyclodextrin derivatives. This might be due to the formation of a true inclusion complex in the solid state.     

α-Distonic ions as transient species in the reactions of metastable alkyl benzoate radical cations

The key intermediates to the fragmentation of metastable methyl and ethyl benzoate radical cations are α- and β-distonic isomers of the molecular ions. The α-distocic isomers are also formed by fragmentation of longer chain alkyl benzoates, but may not be long-lived, stable species. Rearrangement of the α-distonic ions prior to fragmentation can take place, but (re)formation of the benzoate molecular ions does not occur.     

Native conformational tendencies in unfolded polypeptides: development of a novel method to assess native conformational tendencies in the reduced forms of multiple disulfide-bonded proteins

Oxidative folding is the concomitant formation of the native disulfide bonds and the native tertiary structure from the reduced and unfolded polypeptide. Of interest is the inherent conformational tendency (bias) present in the reduced polypeptide to dictate the formation of the full set of native disulfide bonds. Here, by application of a novel tool, we have been able to assess this "native conformational tendency" present in reduced and unfolded bovine pancreatic ribonuclease A (RNase A). The essence of this method lies in the ability of the oxidant [Pt(en)(2)Cl(2)](2+) (where "en" is ethylenediamine) to oxidize disulfide bonds under conditions in which both reduction and disulfide reshuffling, which are essential for rearranging non-native disulfide bonds, are extremely slow. When applied to RNase A, the method revealed little or no bias toward formation of the full native set of disulfide bonds in the fully reduced protein.     

Vibrational spectroscopy of solid state molecular dimers

The IR and Raman spectra of α- and β-perylene crystal vibrations are investigated, β-perylene is monomeric and α-perylene has face-to-face dimers. Frequencies and forms of the lattice vibrations as well as vibron relaxation and localization are discussed. Polarization ratios in α crystals are perturbed by coupling to dimer vibrations.     

Phase diagram of polypeptide chains

We use a coarse grained protein model that enables us to determine the equilibrium phase diagram of natively folded α-helical and unfolded β-sheet forming peptides. The phase diagram shows that there are only two thermodynamically stable peptide phases, the peptide solution and the bulk fibrillar phase. In addition, it reveals the existence of various metastable peptide phases. The liquidlike oligomeric phases are metastable with respect to the fibrillar phases, and there is a hierarchy of metastability. The presented phase diagram provides a solid basis for understanding the assembly of polypeptide chains into the phases formed in their natively folded and unfolded conformations.     

Control of Intrachain Morphology in the Formation of Polyfluorene Aggregates on the Single-Molecule Level

Controlling the morphology of π-conjugated polymers for organic optoelectronic devices has long been a goal in the field of materials science. Since the morphology of a polymer chain is closely intertwined with its photophysical properties, it is desirable to be able to change the arrangement of the polymers at will. We investigate the π-conjugated polymer poly(9,9-dioctylfluorene) (PFO), which can exist in three distinctly different structural phases: the α-, β-, and γ-phase. Every phase has a different chain structure and a unique photoluminescence (PL) spectrum. Due to its unique properties and the pronounced spectral structure-property relations, PFO can be used as a model system to study the morphology of π-conjugated polymers. To avoid ensemble averaging, we examine the PL spectrum of single PFO chains embedded in a non-fluorescent matrix. With single-molecule spectroscopy the structural phase of every single chain can be determined, and changes can be monitored very easily. To manipulate the morphology, solvent vapor annealing (SVA) was applied, which leads to a diffusion of the polymer chains in the matrix. The β- and γ-phases appear during the self-assembly of single α-phase PFO chains into mesoscopic aggregates. The extent of β- and γ-phase formation is directed by the solvent-swelling protocol used for aggregation. Aggregation unequivocally promotes formation of the more planar β- and γ-phases. Once these lower-energy more ordered structural phases are formed, SVA cannot return the polymer chain to the less ordered phase by aggregate swelling.     

Temperature dependent equilibrium native to unfolded protein dynamics and properties observed with IR absorption and 2D IR vibrational echo experiments

Dynamic and structural properties of carbonmonoxy (CO)-coordinated cytochrome c(552) from Hydrogenobacter thermophilus (Ht-M61A) at different temperatures under thermal equilibrium conditions were studied with infrared absorption spectroscopy and ultrafast two-dimensional infrared (2D IR) vibrational echo experiments using the heme-bound CO as the vibrational probe. Depending on the temperature, the stretching mode of CO shows two distinct bands corresponding to the native and unfolded proteins. As the temperature is increased from low temperature, a new absorption band for the unfolded protein grows in and the native band decreases in amplitude. Both the temperature-dependent circular dichroism and the IR absorption area ratio R(A)(T), defined as the ratio of the area under the unfolded band to the sum of the areas of the native and unfolded bands, suggest a two-state transition from the native to the unfolded protein. However, it is found that the absorption spectrum of the unfolded protein increases its inhomogeneous line width and the center frequency shifts as the temperature is increased. The changes in line width and center frequency demonstrate that the unfolding does not follow simple two-state behavior. The temperature-dependent 2D IR vibrational echo experiments show that the fast dynamics of the native protein are virtually temperature independent. In contrast, the fast dynamics of the unfolded protein are slower than those of the native protein, and the unfolded protein fast dynamics and at least a portion of the slower dynamics of the unfolded protein change significantly, becoming faster as the temperature is raised. The temperature dependence of the absorption spectrum and the changes in dynamics measured with the 2D IR experiments confirm that the unfolded ensemble of conformers continuously changes its nature as unfolding proceeds, in contrast to the native state, which displays a temperature-independent distribution of structures.     

α-Haloenolates: their preparation and synthetic applications

α-Haloenolates have been prepared by halogen—metal or hydrogen—metal interchange. Rearrangement of β-oxido carbenoids offers another useful route, whereas β-alkoxycarbenoids decompose via α- or β-elimination. Cu^I promotes the preparation of α-halomagnesium enolates. These organometallics undergo useful reactions as nucleophiles, but can also behave as electrophiles.     

The study of Turnip Mosaic virus coat protein by surface enhanced Raman spectroscopy

Using Turnip Mosaic virus (TuMV) coat protein as material, the secondary structure has been studied by both normal Raman spectroscopy (NRS) and surface enhanced Raman spectroscopy (SERS). The NRS of TuMV coat protein under certain conditions showed the α-helix, β-sheet and random coil structure. The CSSC comformations are trans—gauche—gauche and gauche—gauche—gauche. The SERS spectrum of TuMV coat protein under certain conditions reveals the α-helix structure. By studying SERS at different adsorbing times, we have observed the amide III vibration of α-helix, β-sheet and random coil structure. The CSSC conformations drawn from the SERS spectra are trans—gauche—gauche and trans—gauche—trans. Besides the amide I, amide III and CSSC bands, the CαCN band, aromatic amino acid bands and some other bands can also be seen in the SERS spectra.