Conformational study of melectin and antapin antimicrobial peptides in model membrane environments.

Antimicrobial peptides have long been considered as promising compounds against drug-resistant pathogens. In this work, we studied the secondary structure of antimicrobial peptides melectin and antapin using electronic (ECD) and vibrational circular dichroism (VCD) spectroscopies that are sensitive to peptide secondary structures. The results from quantitative ECD spectral evaluation by Dichroweb and CDNN program and from the qualitative evaluation of the VCD spectra were compared. The antimicrobial activity of the selected peptides depends on their ability to adopt an amphipathic α-helical conformation on the surface of the bacterial membrane. Hence, solutions of different zwitterionic and negatively charged liposomes and micelles were used to mimic the eukaryotic and bacterial biological membranes. The results show a significant content of α-helical conformation in the solutions of negatively charged liposomes mimicking the bacterial membrane, thus correlating with the antimicrobial activity of the studied peptides. On the other hand in the solutions of zwitterionic liposomes used as models of the eukaryotic membranes, the fraction of α-helical conformation was lower, which corresponds with their moderate hemolytic activity.

Isoquinoline Alkaloids from Fumaria officinalis L. and Their Biological Activities Related to Alzheimer's Disease.

Two new isoquinoline alkaloids, named fumaranine (2) and fumarostrejdine (10), along with 18 known alkaloids were isolated from aerial parts of Fumaria officinalis. The structures of the isolated compounds were elucidated on the basis of spectroscopic analyses and by comparison with literature data. The absolute configuration of the new compound 2 was determined by comparing its circular dichroism spectra with those of known analogs. Compounds isolated in sufficient amounts were evaluated for their acetylcholinesterase, butyrylcholinesterase, prolyl oligopeptidase (POP), and glycogen synthase kinase-3ß inhibitory activities. Parfumidine (8) and sinactine (15) exhibited potent POP inhibition activities (IC50 99±5 and 53±2 µM, resp.).

A Solid Phase Vibrational Circular Dichroism Study of Polypeptide-Surfactant Interaction.

We studied the interaction of poly-l-lysine (PLL) and poly-l-arginine (PLAG) with sodium dodecyl sulfate (SDS) surfactant and the interaction of poly-l-glutamic acid (PLGA) and poly-l-aspartic acid (PLAA) with tetradecyltrimethylammonium bromide (TTAB) surfactant using vibrational circular dichroism (VCD) spectroscopy in the region of C-H stretching vibration and in the Amide I region both in solution and in mulls. A chirality transfer from polypeptides to achiral surfactants was observed in the C-H stretching region, where measurements in solution were impossible. This observation was enabled by a special sample treatment technique using lyophilization and the preparation of mulls. This technique demonstrated itself as an interesting and beneficial tool for VCD measurements. In addition, we observed that SDS changed the secondary structure of PLL to the ß-sheet and of PLAG to the α-helix. TTAB disrupted the PLGA and PLAA structure. These results were obtained in the mull but were confirmed by the VCD spectra measured in solution and by electronic circular dichroism. The chirality transfer from the polypeptides to SDS was caused by polypeptides ordered into a specific conformation during the interaction, while in the TTBA system it was induced primarily by the chirality of the amino acid residues.

Chiral recognition of bilirubin and biliverdin in liposomes and micelles.

The structural formula of biologically important chiral pigments bilirubin and biliverdin differs only by one double bond. We showed that this results in dissimilar interactions with two models of membranes: cationic liposomes composed of 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol and zwitterionic micelles from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). While the liposomes recognized the P-form of bilirubin, the micelles recognized its M-form. Both recognized the P-form of biliverdin. Our study also comprised ternary systems consisting of the pigments, model membranes and serum albumin (human and bovine). Bilirubin preferentially interacted with the albumins even in the presence of the liposomes. On the other hand, biliverdin preferred the liposomes. Remarkably, the presence of CHAPS completely changed the biliverdin binding to the protein. Because our study was oriented on different chiral interactions, a chiroptical method of electronic circular dichroism was chosen as the principal method to study our systems. As complementary methods, UV-vis absorption and fluorescence emission were used.

Bilirubin, model membranes and serum albumin interaction: The influence of fatty acids.

Electronic circular dichroism (ECD), absorption and fluorescence spectroscopy were used to study the enantioselective interactions which involved bilirubin (BR), liposomes, human serum albumin of two different purities, pure (HSA) and non-purified of fatty acids (FA-HSA), and individual fatty acids. The application of the ECD technique to such a complex problem provided a new perspective on the BR binding to liposomes. Our results demonstrated that in the presence of pure HSA, BR preferred to bind to the protein over the liposomes. However, in the presence of FA-HSA, BR significantly bound to the liposomes composed either of DMPC or of sphingomyelin and bound only moderately to the primary and secondary binding sites of FA-HSA even at high BR concentrations. For the DMPC liposomes, even a change of BR conformation upon binding to the primary binding site was observed. The individual saturated fatty acids influenced the BR binding to HSA and liposomes in a similar way as fatty acids from FA-HSA. The unsaturated fatty acids interacted with BR alone and prevented it from interacting with either 99-HSA or the liposomes. In the presence of arachidonic acid, BR interacted enantioselectively with the liposomes and only moderately with 99-HSA. Hence, our results show a substantial impact of the liposomes on the BR binding to HSA. As a consequence of the existence of fatty acids in the blood plasma and in the natural structure of HSA, BR may possibly bind to the cell membranes even though it is normally bound to HSA.

Functional helquats: helical cationic dyes with marked, switchable chiroptical properties in the visible region.

Helquat dyes are the first helicene-like cationic styryl dyes obtained as separate enantiomers. Their remarkable chiroptical properties are due to the unique combination of a cationic hemicyanine chromophore and a helicene-like motif. The magnitude of the ECD response and the pH switching along with their positioning in the visible region are unprecedented among helicenoids.

Cyclopeptides containing the DEKS motif as conformationally restricted collagen telopeptide analogues: synthesis and conformational analysis.

The collagen telopeptides play an important role for lysyl oxidase-mediated crosslinking, a process which is deregulated during tumour progression. The DEKS motif which is located within the N-terminal telopeptide of the α1 chain of type I collagen has been suggested to adopt a ßI-turn conformation upon docking to its triple-helical receptor domain, which seems to be critical for lysyl oxidase-catalysed deamination and subsequent crosslinking by Schiff-base formation. Herein, the design and synthesis of cyclic peptides which constrain the DEKS sequence in a ß-turn conformation will be described. Lysine-side chain attachment to 2-chlorotrityl chloride-modified polystyrene resin followed by microwave-assisted solid-phase peptide synthesis and on-resin cyclisation allowed for an efficient access to head-to-tail cyclised DEKS-derived cyclic penta- and hexapeptides. An N(ε)-(4-fluorobenzoyl)lysine residue was included in the cyclopeptides to allow their potential radiolabelling with fluorine-18 for PET imaging of lysyl oxidase. Conformational analysis by (1)H NMR and chiroptical (electronic and vibrational CD) spectroscopy together with MD simulations demonstrated that the concomitant incorporation of a D-proline and an additional lysine for potential radiolabel attachment accounts for a reliable induction of the desired ßI-turn structure in the DEKS motif in both DMSO and water as solvents. The stabilised conformation of the cyclohexapeptide is further reflected by its resistance to trypsin-mediated degradation. In addition, the deaminated analogue containing allysine in place of lysine has been synthesised via the corresponding ε-hydroxynorleucine containing cyclohexapeptide. Both ε-hydroxynorleucine and allysine containing cyclic hexapeptides have been subjected to conformational analysis in the same manner as the lysine-based parent structure. Thus, both a conformationally restricted lysyl oxidase substrate and product have been synthetically accessed, which will enable their potential use for molecular imaging of these important enzymes.

Insight into the structural and biological relevance of the T/R transition of the N-terminus of the B-chain in human insulin.

The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin-insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1-B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the "classical" T-state and that a substantial flexibility of the B1-B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin-IR interaction.

Mutual structural effect of bilirubin and model membranes by vibrational circular dichroism.

In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1×10(-4)M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes-a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15-70°C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.

Nonplanar tertiary amides in rigid chiral tricyclic dilactams. Peptide group distortions and vibrational optical activity.

We investigate amide nonplanarity in vibrational optical activity (VOA) spectra of tricyclic spirodilactams 5,8-diazatricyclo[6,3,0,0(1,5)]undecan-4,9-dione (I) and its 6,6',7,7'-tetradeuterio derivative (II). These rigid molecules constrain amide groups to nonplanar geometries with twisted pyramidal arrangements of bonds to amide nitrogen atoms. We have collected a full range vibrational circular dichroism (VCD) and Raman optical activity (ROA) spectra including signals of C-H and C-D stretching vibrations. We report normal-mode analysis and a comparison of calculated to experimental VCD and ROA. The data provide band-to-band assignment and offer a possibility to evaluate roles of constrained nonplanar tertiary amide groups and rigid chiral skeletons. Nonplanarity shows as single-signed VCD and ROA amide I signals, prevailing the couplets expected to arise from the amide-amide interaction. Amide-amide coupling dominates amide II (mainly C'-N stretching, modified in tertiary amides by the absence of a N-H bond) transitions (strong couplet in VCD, no significant ROA) probably due to the close proximity of amide nitrogen atoms. At lower wavenumbers, ROA spectra exhibit another likely manifestation of amide nonplanarity, showing signals of amide V (δ(oop)(N-C) at ~570 cm(-1)) and amide VI (δ(oop)(C'═O) at ~700 cm(-1) and ~650 cm(-1)) vibrations.

CH Stretching Region: Computational Modeling of Vibrational Optical Activity.

Most organic compounds provide vibrational spectra within the CH stretching region, yet the signal is difficult to interpret because of multiple difficulties in experiment and modeling. To better understand various factors involved, the ability of several harmonic and anharmonic computational approaches to describe these vibrations was explored for α-pinene, fenchone, and camphor as test compounds. Raman, Raman optical activity (ROA), infrared absorption (IR), and vibrational circular dichroism (VCD) spectra were measured and compared to quantum chemical computations. Surprisingly, the harmonic vibrational approach reasonably well reproduced the measured spectral patterns, including the vibrational optical activity (VOA). The CH stretching, however, appeared to be more sensitive to the basis set and solvent variations than lower-frequency vibrations. For a higher accuracy in frequencies and spectral shapes, anharmonic corrections were necessary. Accurate harmonic and anharmonic force fields were obtained with the mPW2PLYP double-hybrid functional. A limited vibrational configuration interaction (LVCI) where the CH stretching motion was decoupled from other vibrations provided the best simulated spectra. A balanced harmonic oscillator basis set had to be used, containing also states indirectly interacting with fundamental vibrations. A simpler second-order perturbational approach (PT2) appeared less useful. The modeling provided unprecedented agreement with experimental vibrational frequencies; spectral shapes were reproduced less faithfully. The possibility of ab initio interpretation of the CH spectral region for relatively large molecules further broadens the application span of vibrational spectroscopy.

Vibrational circular dichroism study of polypeptide model-membrane systems.

In this article, we describe the mutual structural effect of the interaction between the model membranes and polylysine and poly-l-arginine. Vibrational circular dichroism (VCD), a method exceptionally sensitive to the polypeptide structure that has not been established in such studies before, was the primary method of this study. A complementary technique, electronic circular dichroism, was applied to verify the newly obtained results and as a bridge to the previous studies. We used micelles composed of sodium dodecyl sulfate (SDS) as a monolayer membrane model and large unilamellar vesicles composed of phospholipids as a bilayer membrane model. We describe the conformational changes of the polypeptides caused by the interaction with the model membranes. Among others, the presence of the liposomes in the solution generated special conditions for the formation of the α-helical structure of poly-l-arginine; the presence of SDS induced the formation of the ß-structure of polylysine. From a methodological point of view, we emphasize the advantages of infrared spectroscopic techniques for the liposomic membrane studies as well as the preference of ultraviolet techniques for smaller micellar systems.

The C-terminal segment of yeast BMH proteins exhibits different structure compared to other 14-3-3 protein isoforms.

Yeast 14-3-3 protein isoforms BMH1 and BMH2 possess a distinctly variant C-terminal tail which differentiates them from the isoforms of higher eukaryotes. Their C-termini are longer and contain a polyglutamine stretch of unknown function. It is now well established that the C-terminal segment of 14-3-3 proteins plays an important regulatory role by functioning as an autoinhibitor which occupies the ligand binding groove and blocks the binding of inappropriate ligands. Whether the same holds true or not for the yeast isoforms is unclear. Therefore, we investigated the conformational behavior of the C-terminal segment of BMH proteins using various biophysical techniques. Dynamic light scattering, sedimentation velocity, time-resolved fluorescence anisotropy decay, and size exclusion chromatography measurements showed that the molecules of BMH proteins are significantly larger compared to the human 14-3-3zeta isoform. On the other hand, the sedimentation analysis confirmed that BMH proteins form dimers. Time-resolved tryptophan fluorescence experiments revealed no dramatic structural changes of the C-terminal segment upon the ligand binding. Taken together, the C-terminal segment of BMH proteins adopts a widely opened and extended conformation that makes difficult its folding into the ligand binding groove, thus increasing the apparent molecular size. It seems, therefore, that the C-terminal segment of BMH proteins does not function as an autoinhibitor.