Binding of alcohols to the peptide CO-group of poly-L-proline in the I and II conformation. I. Demonstration of the binding by infrared spectroscopy and optical rotatory dispersion

The binding of n-butanol, benzyl alcohol, and trifluoroethanol to polyproline I (all peptide bonds in cis configuration) and II (all peptide bonds in trans configuration) has been demonstrated. It was found by infrared spectroscopy that hydrogen bonds are formed between the CO groups of the polymer and the OH groups of the alcohols. Their strength increases from n-butanol via benzyl alcohol to trifluoroethanol. The binding of benzyl alcohol could also be followed by optical rotatory dispersion due to the fact that the electronic transitions of the benzyl ring are rendered optically active by the binding to the polymer.     

Helical conformation of amylose in aqueous solution. I. Viscosity measurements

Two amylose samples, amylose V (DP_w = 2300) and amylose HE 15 (a low-substituted hydroxyethylamylose, DP_W = 1600) were studied. The intrinsic viscosity of the polymers in aqueous solution was measured with regard to its dependence on the alkalinity (0 to 5MNaOH), the ionic strength (0 to 5M), and the temperature (0 to 75°C). Additionally, the temperature dependence of the viscosity of the amylose-iodine complex was measured. It was found that the two amylose samples show the same dependence on the studied parameters. Therefore, it was concluded that the conformation is unchanged by the hydroxyethylation in the present case. In a discussion, steric, hydrodynamic, and thermodynamic data on amylose in solution are compared with the corresponding data on cellulose and dextran. The comparison leads to the conclusion that amylose in a neutral solution must have a helical conformation, corresponding to the well-accepted rod conformation of cellulose. The helical conformation also explains several viritual anomalies in the behavior of amylose. The results of the experiments support the helix model for amylose. The conclusion of the whole work, therefore, is that the amylose molecule in neutral aqueous solution can be regarded as a random coil, built up by helical segments. The average number of monomers per segment exceeds 100. This value decreases with increasing alkalinity.     

An infrared study on the conformation of poly-L-proline in concentrated aqueous salt solutions

The infrared absorption of poly-L -proline in concentrated aqueous salt solutions was measured in the fundamental region. Of primary interest were the carbonyl absorption of the peptide linkage and the methylene C–H bending absorption of the pyrrolidine ring. These spectral regions each show an additional component in the concentrated salt solutions. Using the position of the absorptions of poly-L -proline I (cis) and II (trans) as models, we conclude that both cis–trans linkages are present in the peptide in salt solutions. Increasing the temperature shifts the equilibrium slightly in favor of cis.     

Microcalorimetric study of polypeptides in the conformation of the left helix of the poly-L-proline II type

Microcalorimetry shows that polypeptide partial heat capacity changes linearly with temperature in the region of M-conformation existence and that there is a peculiarity in the transition region where this structure is exhausted, without cooperative heat absorption.     

Left-handed helix conformation of poly-L-proline II type in globular proteins. Statistics of incidence and a role of sequence]

Regions of left-handed polyproline II type conformation in globular proteins were studied throughout the PDB bank. The length and sequence of corresponding fragments were analyzed. It was found that a lot of tetrapeptides (from combinatorial possible ones) show the tendency to be included in the left-handed helices. Much more tetrapeptides do not occur in this structure type.     

NMR studies of the solution conformation of the sex peptide from Drosophila melanogaster

The insect sex peptide (SP) elicits a variety of biological responses upon transfer to the mated female. SP contains 36 amino acids, including a tryptophan-rich N-terminal region, a central region containing five hydroxyproline (Hyp) residues, and a C-terminal region enclosed by a disulfide bridge. The solution structure of SP, studied here using NMR spectroscopy, includes a motif WPWN that adopts a type I β-turn in the N-terminal Trp-rich region. This turn region is connected to the central Hyp-rich region, which adopts extended and/or PPII-like conformations. The C-terminal disulfide-bonded loop populates helical turns or nascent helical structure. Overall, the results reveal a rather flexible peptide that lacks a compact folded structure in solution.     

Helical conformation of amylose in aqueous solution. II. Electron spin resonance,fluorescence depolarization,and sedimentation measurements

In the previous study [Elmgren, H. (1984) Biopolymers 23, 2525–2541] concerning the conformation of amylose in aqueous solution, it was stated that amylose in a neutral aqueous solution is a random coil consisting of helical segments. In terms of Kuhn statistics, each segment contains more than 100 monomers. The number of monomers per segment decreases by alkali addition. In an attempt to verify these statements, a combined electron spin resonance (esr) and ultracentrifugation (uc) study of a weakly hydroxyethylated amylose sample in water and alkaline solvents was performed. This combination of measuring techniques makes it possible to estimate the Brownian motion, and thus the mass of the polymer segments. As a control for the obtained esr data, fluorescence depolarization (fdp) measurements were performed on the polymer sample in a bicarbonate buffer at pH 10. The result of the study confirms that the amylose segments are very heavy in water. In strong alkaline solvents, the segment mass corresponds to that of a few monomers. Our findings thus support the statements made in the preceding article, and the data obtained by others. [Kitamura S., Yunokawa H., & Kuge T., (1982) Polym. J. 14, 85–91; Kitamura S., Yunokawa H., Mitsu'ie S., & Kuge T., (1982) Polym J. 14, 93–99].     

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe. Beta-turn conformation of a sequence related to an active chemotactic peptide analog

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and 1H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II beta-turn in the solid state with Met and Aib as the corner residues (phi Met = -51.8 degrees, psi Met = 139.5 degrees, phi Aib = 58.1 degrees, psi Aib = 37.0 degrees). A single, weak 4----1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N---O 3.25 A, N-H---O 128.4 degrees). 1H n.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagnetic radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCl3 and (CD3)2SO. Nuclear Overhauser effects observed between Met C alpha H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II beta-turns in these solvents.     

Binding constants of Li+, K+, and Tl+ in the gramicidin channel determined from water permeability measurements.

In an open circuit there can be no net cation flux through membranes containing only cation-selective channels, because electroneutrality must be maintained. If the channels are so narrow that water and cations cannot pass by each other, then the net water flux through those "single-file" channels that contain a cation is zero. It is therefore possible to determine the cation binding constants from the decrease in the average water permeability per channel as the cation concentration in the solution is increased. Three different methods were used to determine the osmotic water permeability of gramicidin channels in lipid bilayer membranes. The osmotic water permeability coefficient per gramicidin channel in the absence of cations was found to be 6 x 10(-14) cm3/s. As the cation concentration was raised, the water permeability decreased and a binding constant was determined from a quantitative fit to the data. When the data were fitted assuming a maximum of one ion per channel, the dissociation constant was 115 mM for Li+, 69 mM for K+, and 2 mM for Tl+.     

Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces--modulation of conformation and activity,and lipid-protein interaction

Sticholysins I and II (St I and St II) are water-soluble toxins produced by the sea anemone Stichodactyla helianthus. St I and St II bind to biological and model membranes containing sphingomyelin (SM), forming oligomeric pores that lead to leakage of internal contents. Here we describe functional and structural studies of the toxins aiming at the understanding at a molecular level of their mechanism of binding, as well as their effects on membrane permeabilization. St I and St II caused potassium leakage from red blood cells and temperature-dependent hemolysis, the activation energy of the process being lower for the latter toxin. Protein intrinsic fluorescence measurements provided evidence for toxin binding to model membranes composed of 1:1 (mol:mol) egg phosphatidyl choline (ePC):SM. The fluorescence intensity increased and the maximum emission wavelength decreased as a result of binding. The changes were quantitatively different for both toxins. Circular dichroism spectra showed that both St I and St II exhibit a high content of beta-sheet structure and that binding to model membranes did not alter the toxin's conformation to a large extent. Changing the lipid composition by adding 5 mol% of negatively charged phosphatidic acid (PA) or phosphatidyl glycerol (PG) had small, but detectable, effects on protein conformation. The influence of lipid composition on toxin-induced membrane permeabilization was assessed by means of fluorescence measurements of calcein leakage. The effect was larger for ePC:SM bilayers containing 5 mol% of negative curvature-inducing lipids. Electron paramagnetic resonance (EPR) spectra of intercalated fatty acid spin probes carrying the nitroxide moiety at different carbons (5, 7, 12, and 16) evidenced the occurrence of lipid-protein interaction. Upon addition of the toxins, two-component spectra were observed for the probe labeled at C-12. The broader component, corresponding to a population of strongly immobilized spin probes, was ascribed to boundary lipid. The contribution of this component to the total spectrum was larger for St II than for St I. Moreover, it was clearly detectable for the C-12-labeled probe, but it was absent when the label was at C-16, indicating a lack of lipid-protein interaction close to the lipid terminal methyl group. This effect could be either due to the fact that the toxins do not span the whole bilayer thickness or to the formation of a toroidal pore leading to the preferential interaction with acyl chain carbons closer to the phospholipids head groups.     

Binding of ATP to human DNA topoisomerase I resulting in an alteration of the conformation of the enzyme.

It has been known for some time that ATP inhibits the DNA relaxation activity of human DNA topoisomerase I. However, the underlying mechanism of this inhibitory effect remains largely unknown. Using filter binding assays, the binding of human DNA topoisomerase I to DNA was decreased in the presence of ATP. This result suggests that the inhibition of DNA relaxation activity of human DNA topoisomerase I by ATP is at the binding step rather than at the nicking or resealing step. DNA topoisomerase I cleavage assay further supports this notion. ATP-agarose binding and UV cross-linking assays also demonstrate that ATP directly and specifically binds human DNA topoisomerase I. To address whether the ATP binding results in conformational changes in human DNA topoisomerase I, various proteases were employed for detecting potential protein conformational changes. Our results indicated that the proteolytic susceptibilities of trypsin and chymotrypsin were altered in the presence of ATP. The result suggests that the conformation of human DNA topoisomerase I was altered upon ATP binding. In addition, the binding between ATP and human DNA topoisomerase I was also reduced by increasing concentrations of DNA. Our data suggests that human DNA topoisomerase I exhibits at least two incompatible conformations. One conformation is in the form of a topoisomerase I-ATP complex, which inhibits DNA relaxation activity of human DNA topoisomerase I, and the other, a topoisomerase I-DNA complex, which exerts DNA relaxation activity. Our studies identify the role of ATP in the regulation of human DNA topoisomerase I and provide a substantial implication of how human DNA topoisomerase I compromises its versatile functions.     

The solution behavior of poly-L-proline: I. Light scattering studies in water and concentrated aqueous neutral salt solutions

The solution bebavior of poly-L -proline Form II has been studied in water and aqueous salt solutions by both elastic and quasi-elastic light -scattering techniques. The results of this study suggest that polyproline Form II can exist in water at 24 °C as an associated polymer complex and that certain salts which do not appear to affect the helix integrity, e.g., guanidinium-HCl, resutl in dissociation of the aggregate. Other neutral salts, of the variety effective in mediating unfolding of the Form II helix (e.g., 4M NaClO₄) also induce aggregate dissociation, but 4M CaCl₂ results in enhanced aggregation of polyproline. Kinetic experiments indicate that a time of 20 hours is necessary for the completion of the “large” to “small” transformation (at 22°C) which is induced by the addition of 4M NaClO₄. Thus it appears that neutral salts additives in aqueous solutions of polyproline influence both the state of aggregation and the conformation of this polymer.     

The solution conformation of poly(L-lysine). A Raman and infrared spectroscopic study.

The Raman and infrared spectra of poly(L -lysine) and poly(DL -lysine) in solution are reported and the effects of various salts are investigated. The results demonstrate that α-helix formation in solution is induced by specific salts and the spectral data support the hypothesis of regions of local order for poly(L -lysine) in aqueous solutions of low ionic strength.     

Carboxyl group involvement in the meta I and meta II stages in rhodopsin bleaching. A Fourier transform infrared spectroscopic study

Structural changes due to photoreceptor membrane bleaching can be studied by Fourier transform infrared difference spectroscopy [1,2]. In this paper we focus on the differences between rhodopsin and metarhodopsin I or II. Peaks in the 1700-1770 cm-1 region are observed, which may be produced by carbonyl groups in either carboxyl (COOH) or ester carbonyl (COOC) groups, the latter being found exclusively in membrane lipids. In order to distinguish between these two types of carbonyl groups, we have studied reconstituted membranes of rhodopsin in a synthetic phosphatidylcholine that lacks ester carbonyl groups. On this basis, we conclude that the major changes in this region are due to rhodopsin carboxyls which undergo either a change in local environment or a protonation/deprotonation reaction. Additional small changes in this region may reflect a direct involvement of phospholipids in the metarhodopsin I-to-II transition. One or more groups responsible for peaks near 1727 and 1702 cm-1 are inaccessible to the outside medium according to hydrogen/deuterium exchange. In contrast, carboxyl group(s) producing peaks near 1710, 1745 and 1768 cm-1 exchange freely with the outside medium and are therefore likely to be located near the membrane surface. Removal of a portion of the C-terminal tail region using proteinase K demonstrates that the carboxyl groups in the C-terminal sequence 248-348 are not involved directly in the rhodopsin to metarhodopsin II transition. At the meta I stage, only carboxyl peaks associated with buried groups appear, suggesting that the initial bleaching events, leading to the formation of this intermediate, produce structural rearrangements in the interior region of rhodopsin. These changes then spread to the peripheral surface regions during the metarhodopsin I-to-II transition.     

Dinucleoside monophosphates. I. Optical properties and conformation in solution with one base charged.

A least squares analysis of the titration properties of several dinucleoside monophosphates enables calculation of the pK's for protonation. These pK's are used to resolve the spectral properties of dinucleoside monophosphates with one base charged from the apparent spectral properties of a dinucleoside monophosphate in aqueous solution. This method is applied to dinucleoside monophosphates containing adenosine and/or cytidine. Results of CD, nmr, and CD-temperature dependence measurements are presented. The results indicate that singly protonated dimers of these nucleosides stack as do their unprotonated analogs. It is suggested that this is true for all dimers with one base charged.     

The solution behavior of poly-L-proline: II. Fluorescence behavior of labeled polyproline in water and conentrated aqueous neutral salt solytions

Fluorescence depolarization experiments performed on labaled poly-L -proline Forme II suggest the occurrence of aggrgation in water while 6M guanidinium-HCl induces dissociation. The solvent 4M CaCl₂ results in a reduction of polymer structural orgganization. These findings corroborate suggestion of polyproline aggregation and solution behavior in aqueous neutral salt solytion (see preceding article).