Main-chain length control of conformation, membrane activity, and antibiotic properties of lipopeptaibol sequential analogues

To investigate the effect of backbone length and amphiphilicity on the 3D structure, membrane permeability, and antibacterial properties of trichogins, a subclass of lipopeptaibols, we prepared, by the segment condensation approach in solution and chemically characterized, a set of N(alpha)-1-octanoylated -X-(GLUG)(n)-I-L- ( X=G or U where U=Aib; n=1-4) sequential peptide esters. In parallel, the 12-mer (UGGL)(3) aneurism peptide, an analogue of the 11-mer sequential peptide (n=2) with an amino acid insertion was also synthesized and studied. By FT-IR absorption technique, we clearly showed that, in CDCl(3) solution, all peptides essentially populate intramolecularly H-bonded, helical conformations. Moreover, CD spectroscopy indicates that all peptides, with the single exception of the shortest oligomer (the heptamer), adopt mixed 3(10)-/alpha-helical structures, to an extent approximately correlating with main-chain length, in MeOH solution and sodium dodecylsulfate (SDS) micelles. Significant membrane permeability properties were found only for the three longest GLUG-based peptides, with the 15-mer oligomer (n=4) resulting the most active. The lack of activity exhibited by the aneurism peptide in this experiment strongly suggests a relevant role for the sequence amphiphilicity. In addition, antibacterial activity and selectivity were highlighted and demonstrated to be dependent on peptide main-chain length and amphiphilicity, in the sense that the two shortest GLUG-based homologues are active against Gram-positive strains, whereas the two longest homologues are able to penetrate the membranes of the Gram-negative strains, and the UGGL-based aneurism peptide is inactive.     

A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

Steady-state intrinsic tryptophan fluorescence spectroscopy is used as a rapid, robust and economic way for screening the thermal protein conformational stability in various formulations used during the early biotechnology development phase. The most important parameters affecting protein stability in a liquid formulation, e. g. during the initial purification steps or preformulation development, are the pH of the solution, ionic strength, presence of excipients and combinations thereof. A well-defined protocol is presented for the investigation of the thermal conformational stability of proteins. This allows the determination of the denaturation temperature as a function of solution conditions. Using intrinsic tryptophan fluorescence spectroscopy for monitoring the denaturation and folding of proteins, it is crucial to understand the influence of different formulation parameters on the intrinsic fluorescence probes of proteins. Therefore, we have re-evaluated and re-assessed the influence of temperature, pH, ionic strength, buffer composition on the emission spectra of tryptophan, phenylalanine and tyrosine to correctly analyse and evaluate the data obtained from thermal-induced protein denaturation as a function of the solution parameters mentioned above. The results of this study are a prerequisite for using this method as a screening assay for analysing the conformational stability of proteins in solution. The data obtained from intrinsic protein fluorescence spectroscopy are compared to data derived from calorimetry. The advantage, challenges and applicability using intrinsic tryptophan fluorescence spectroscopy as a routine development method in pharmaceutical biotechnology are discussed.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.     

Cisplatin differently affects amino terminal and carboxyl terminal domains of HSP90

The 90-kDa heat shock protein (HSP90) is a molecular chaperone that assists in the folding and assembly of proteins in the cytosol. We previously demonstrated that the antineoplastic reagent, cisplatin, inhibits the aggregation prevention activity of mammalian HSP90. We now show that cisplatin binds both the amino terminal and carboxyl terminal domains of the human HSP90 and differently affects these two domains. Cisplatin blocks the aggregation prevention activity of HSP90C, but not HSP90N. In contrast, cisplatin induces a conformational change in HSP90N, but not HSP90C. These results indicate that cisplatin modulates the HSP90 activities through two different mechanisms using the two distinct binding sites of the HSP90 molecule.     

A tool for the prediction of structures of complex sugars

In two recent back to back articles(Xia et al., J Chem Theory Comput 3:1620–1628 and 1629–1643, 2007a, b) we have started to address the problem of complex oligosaccharide conformation and folding. The scheme previously presented was based on exhaustive searches in configuration space in conjunction with Nuclear Overhauser Effect (NOE) calculations and the use of a complex rotameric library that takes branching into account. NOEs are extremely useful for structural determination but only provide information about short range interactions and ordering. Instead, the measurement of residual dipolar couplings (RDC), yields information about molecular ordering or folding that is long range in nature. In this article we show the results obtained by incorporation RDC calculations into our prediction scheme. Using this new approach we are able to accurately predict the structure of six human milk sugars: LNF-1, LND-1, LNF-2, LNF-3, LNnT and LNT. Our exhaustive search in dihedral configuration space combined with RDC and NOE calculations allows for highly accurate structural predictions that, because of the non-ergodic nature of these molecules on a time scale compatible with molecular dynamics simulations, are extremely hard to obtain otherwise (Almond et al., Biochemistry 43:5853–5863, 2004). Molecular dynamics simulations in explicit solvent using as initial configurations the structures predicted by our algorithm show that the histo-blood group epitopes in these sugars are relatively rigid and that the whole family of oligosaccharides derives its conformational variability almost exclusively from their common linkage (β-d-GlcNAc-(1→3)-β-d-Gal) which can exist in two distinct conformational states. A population analysis based on the conformational variability of this flexible glycosidic link indicates that the relative population of the two distinct states varies for different human milk oligosaccharides. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

NMR analysis of the closed conformation of syntaxin-1

The Sec1/Munc18 (SM) protein Munc18-1 and the SNAREs syntaxin-1, SNAP-25 and synaptobrevin form the core of the membrane fusion machinery that triggers neurotransmitter release. Munc18-1 binds to syntaxin-1 folded into a closed conformation and to the SNARE complex formed by the three SNAREs, which involves an open syntaxin-1 conformation. The former interaction is likely specialized for neurotransmitter release, whereas SM protein/SNARE complex interactions are likely key for all types of intracellular membrane fusion. It is currently unclear whether the closed conformation is highly or only marginally populated in isolated syntaxin-1, and whether Munc18-1 stabilizes the close conformation or helps to open it to facilitate SNARE complex formation. A detailed NMR analysis now suggests that the closed conformation is almost quantitatively populated in isolated syntaxin-1 in the absence of oligomerization, and indicates that its structure is very similar to that observed previously in the crystal structure of the Munc18-1/syntaxin-1 complex. Moreover, we demonstrate that Munc18-1 binding prevents opening of the syntaxin-1 closed conformation. These results support a model whereby the closed conformation constitutes a key intrinsic property of isolated syntaxin-1 and Munc18-1 binding stabilizes this conformation; in this model, Munc18-1 plays in addition an active role in downstream events after another factor(s) helps to open the syntaxin-1 conformation.     

Molecular structure of eight possible configurational isomers of 2,3- and 3,4-epimino derivatives of 1,6-anhydro-β-D-hexopyranoses: conformation analysis, intra- and inter-molecular hydrogen bonds

The crystal structures of a complete series of configurational isomers of 2,3-epimino and 3,4-epimino derivatives of 1,6-anhydro-β-d-hexopyranoses were determined by single-crystal X-ray analysis. The structures exhibited conformational rigidity within the series regardless of the position and orientation of the aziridine ring. Possible formation of intramolecular hydrogen bonds involving the NH group is discussed with respect to the results of IR spectroscopy and to the intermolecular hydrogen bonds found in the crystal packing.     

Synthesis and geometry of methyl (methyl 4-O-acetyl-3-azido-2,3-dideoxy-alpha/beta-D-arabino- and -alpha/beta-D-ribo-hexopyranosid)uronates

The synthesis of methyl (methyl 4-O-acetyl-3-azido-2,3-dideoxy-alpha/beta-D-arabino- and -alpha/beta-D-ribo-hexopyranosid)uronates is presented. High resolution (1)H and (13)C NMR spectral data for all diastereoisomers and single-crystal X-ray diffraction analysis for methyl (methyl 3-azido-2,3-dideoxy-beta-D-arabino-hexopyranosid)uronate are reported. The planarity of the 4-OAc and 5-COOMe groups as well as the orientations of the aglycone and azide groups in the crystal lattice is discussed. The influence of the 5-COOMe group on the pyranose ring conformation is considered.     

A modified approach to the measurement problem: objective reduction in the retinal molecule prior to conformational change

A new analysis of the measurement problem reveals the possibility that collapse of the wavefunction may now take place just before photoisomerization of the rhodopsin molecule in the retinal rods. It is known that when a photon is initially absorbed by the retinal molecule which, along with opsin comprises the rhodopsin molecule, an electron in the highest pi orbital is immediately excited to a pi* orbital. This means that a measurement or transfer of information takes place at the quantum level before the retinal molecule commences the conformational change from cis to trans. This could have profound implications for resolving some of the foundational issues confronting quantum mechanics.