Binding of amphiphilic peptides to a carboxy-terminal tryptic fragment of calmodulin

Calmodulin (CaM) fragments 1-77 (CaM 1-77) and 78-148 (CaM 78-148) were prepared by tryptic cleavage of CaM. CaM 78-148 exhibited Ca2+-dependent binding to mastoparan X, Polistes mastoparan, and melittin with apparent dissociation constants less than 0.2 microM as judged from changes in the fluorescence spectrum and anisotropy of the single tryptophan residue of each of these cationic, amphiphilic peptides. This interaction was accompanied by a large spectral blue shift of the peptide fluorescence spectrum. These findings are consistent with earlier results [Malencik, D.A., & Anderson, S.R. (1984) Biochemistry 23, 2420-2428] on the binding of mastoparan X to CaM fragment 72-148. The binding of the peptide to CaM 78-148 also caused a significant loss of the accessibility of the peptide tryptophan to the fluorescence quencher acrylamide. The CaM 78-148 induced effects on the fluorescence spectra and tryptophan accessibility of the peptides were most pronounced for mastoparan X, a peptide with tryptophan on the apolar face of the putative amphiphilic helix. The data were comparable with results from parallel experiments on the Ca2+-dependent interaction of these peptides with intact CaM. Difference circular dichroic spectra suggested that binding to CaM 78-148 was associated with the induction of considerable degrees of helicity in the amphiphilic peptides, which by themselves have predominantly random coil structures in aqueous solution. This finding is also reminiscent of the interaction of these peptides with intact CaM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Band-selective 3D NOESY-TOCSY: Measurement of through-space correlations between aliphatic protons of membrane peptides and proteins in non-deuterated detergents

Summary Recently the use of band-selective excitation to obtain ¹H 2D NMR spectra of membrane peptides and proteins in non-deuterated detergents has been demonstrated [Seigneuret, M. and Levy, D. (1995) J. Biomol. NMR, 5, 345–352]. A limitation of the method was the inability to obtain through-space correlation between aliphatic protons. Here, a 3D F3-band-selective NOESY-TOCSY experiment is described that allows such correlations to be observed in the presence of an excess of non-deuterated detergent. Application to the measurement of proximities between aliphatic protons of the membrane peptide mastoparan X solubilized in non-deuterated n-octylglucoside is presented. With this additional experiment, it is now possible to obtain the same amount of structural constraints on membrane peptides and protein in non-deuterated detergent as in deuterated detergent and therefore to perform complete structural studies.     

Vibrational spectroscopic characteristics of secondary structure polypeptides in liquid water: constrained MD simulation studies

Using the constrained MD simulation method in combination with quantum chemistry calculation, Hessian matrix reconstruction, and fragmentation approximation methods, we established a computational scheme for numerical simulations of amide I IR absorption, vibrational circular dichroism (VCD), and 2D IR photon echo spectra of peptides in solution. Six different secondary structure peptides, i.e., alpha-helix, 3(10)-helix, pi-helix, antiparallel and parallel beta-sheets, and polyproline II (P(II)), are considered, and the vibrational characteristic features in their linear and nonlinear spectra in the amide I band region are discussed. Isotope-labeling effects on IR and VCD spectra are notable only for alpha- and pi-helical peptides due to the strong vibrational couplings between two nearest neighboring amide I local oscillators. The amplitudes of difference 2D IR spectra are shown to be strongly dependent on both the extent of mode delocalization and the relative orientation of local mode transition dipoles determined by secondary structure.     

NMR solution structure and position of transportan in neutral phospholipid bicelles

Transportan is a chimeric cell-penetrating peptide constructed from the peptides galanin and mastoparan, which has the ability to internalize living cells carrying a hydrophilic load. In this study, we have determined the NMR solution structure and investigated the position of transportan in neutral bicelles. The structure revealed a well-defined alpha-helix in the C-terminal mastoparan part of the peptide and a weaker tendency to form an alpha-helix in the N-terminal domain. The position of the peptide in relation to the membrane, as studied by adding paramagnetic probes, shows that the peptide lies parallel to, and in the head-group region of the membrane surface. This result is supported by amide proton secondary chemical shifts.     

Secondary structure and position of the cell-penetrating peptide transportan in SDS micelles as determined by NMR

Transportan is a 27-residue peptide (GWTLN SAGYL LGKIN LKALA ALAKK IL-amide) which has the ability to penetrate into living cells carrying a hydrophilic load. Transportan is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan in the C-terminus and a connecting lysine. Circular dichroism studies of transportan and mastoparan show that both peptides have close to random coil secondary structure in water. Sodium dodecyl sulfate (SDS) micelles induce 60% helix in transportan and 75% helix in mastoparan. The 600 MHz (1)H NMR studies of secondary structure in SDS micelles confirm the helix in mastoparan and show that in transportan the helix is localized to the mastoparan part. The less structured N-terminus of transportan has a secondary structure similar to that of the same sequence in galanin [Ohman, A., et al. (1998) Biochemistry 37, 9169-9178]. The position of mastoparan and transportan relative to the SDS micelle surface was studied by adding spin-labeled 5-doxyl- or 12-doxyl-stearic acid or Mn2+ to the peptide/micelle system. The combined results show that the peptides are for the most part buried in the SDS micelles. Only the C-terminal parts of both peptides and the central segment connecting the two parts of transportan are clearly surface exposed. For mastoparan, the secondary chemical shifts of the amide protons were found to vary periodically and display a pattern almost identical to those reported for mastoparan in phospholipid bicelles [Vold, R., et al. (1997) J. Biomol. NMR 9, 329-335], indicating similar structures and interactions in the two membrane-mimicking environments.     

A high-resolution 1H NMR approach for structure determination of membrane peptides and proteins in non-deuterated detergent: Application to mastoparan X solubilized in n-octylglucoside

Summary Application of ¹H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a ¹H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by ¹H 2D NMR in any type of non-deuterated detergent. The approach is based on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the ¹H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein ¹H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and -protons. Regioselective COSY spectra provide scalar coupling constants between amide and -protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants. It is shown that mastoparan adopts an -helical conformation when bound to nonionic detergent micelles. The present method is expected to increase the applicability of ¹H solution NMR methods to membrane proteins and peptides.Abbreviations 2D NMR two-dimensional NMR - COSY correlated spectroscopy - DANTE delays alternating nutations for tailored excitation - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy     

Conformational analysis of neuropeptide Y-[18-36] analogs in hydrophobic environments.

The interactive and conformational behavior of a series of neuropeptide Y-[18-36] (NPY-[18-36]) analogs in hydrophobic environments have been investigated using reversed-phase high-performance liquid chromatography (RP-HPLC) and circular dichroism (CD) spectroscopy. The peptides studied comprised a series of 16 analogs of NPY-[18-36], each containing a single D-amino acid substitution. The influence of these single L-->D substitutions on the alpha-helical conformation of the NPY-[18-36] analogs in different solvent environments was determined by CD spectroscopy. Retention parameters related to the hydrophobic contact area and the affinity of interaction were determined with an n-octadecyl (C18) adsorbent. Structural transitions for all peptides were manifested as significant changes in the hydrophobic binding domain and surface affinity between 4 degrees C and 37 degrees C. The results indicated that the central region of NPY-[18-36] (residues 23-33) is important for maintenance of the alpha-helical conformation. Moreover, L-->D amino acid residue substitutions within the N- and C-terminal regions, as well as Asn29 and Leu30, do not appear to affect the secondary structure of the peptide. These studies demonstrate that RP-HPLC provides a powerful adjunct for investigations into the induction of stabilized secondary structure in peptides upon their interaction with hydrophobic surfaces.     

Conformational analysis of a 12-residue analogue of mastoparan and of mastoparan X.

We have investigated the conformational properties of a truncated analogue of mastoparan and of mastoparan X, both peptides from wasp venom. The electrostatically driven Monte Carlo method was used to explore the conformational space of these short peptides. The initial conformations used in this study, mainly random ones, led to alpha-helical conformations. The alpha-helical conformations thus found exhibit an amphipathic character. These results are in accord with experimental data from NMR and CD spectroscopy.     

Two-dimensional infrared spectroscopy displays signatures of structural ordering in peptide aggregates

In the presence of lipid bilayers, the hexapeptide AcWL(5) forms membrane-bound aggregates dominated by beta-secondary structure and is thus a useful model for the onset of peptide aggregation in membrane environments. Two-dimensional infrared (2D IR) spectra in the amide I region for aggregates of AcWL(5) peptides with single isotopic labels provide new insight into the residue-level structural ordering of the aggregated peptides. Separation of spectral information along two axes provides clear indications of the band frequencies and relative intensities, which together are an indication of extended amide coupling networks across structural regions of a particular size. The lowered anharmonicities, relative to free peptide, and the narrow, homogeneous lineshapes of the 2D IR peaks indicate the delocalization of vibrational modes through an ordered structure whose flexibility varies with relative peptide concentration. Crosspeaks between delocalized transitions can be used to estimate the strength of the coupling interactions between neighboring residues. The sensitivity of 2D IR spectra to residue-level structural ordering shows that 2D IR spectroscopy is a powerful technique for probing structures formed during the onset of peptide aggregation.     

‘Random coil’ 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG

Summary Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D ¹H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain ‘random coil’ amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these ‘random coil’ peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K. (1979) Biopolymers, 18, 285–297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH₂ [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67–81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.     

Secondary structure of proteins associated in thin films

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L -lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy¹ and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all α-helical myoglobin, decreased the molar ellipticity of the α/β RNase A, and increased the molar ellipticity of all-β Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less α-helix and more β-sheet structure. The results suggest that the protein α-helices are less stable in films and that the secondary structure may rearrange into β-sheets at high temperature. Two heterooligomeric peptides and poly (L -lysine), all in solution at neutral pH with “random coil” conformation, formed films with variable degrees of their secondary structure in β-sheets or β-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a “random” structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable α-helix or β-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent. © 1993 John Wiley & Sons, Inc.     

A distinct utility of the amide III infrared band for secondary structure estimation of aqueous protein solutions using partial least squares methods

Fourier transform infrared spectroscopy is becoming an increasingly important method to study protein secondary structure. The amide I region of the protein infrared spectrum is the widely used region, whereas the amide III region has been comparatively neglected due to its low signal. Since there is no water interference in the amide III region and, more importantly, the different secondary structures of proteins have more resolved differences in their amide III spectra, it is quite promising to use the amide III region to determine protein secondary structure. In our current study, a partial least squares (PLS) method was used to predict protein secondary structures from the protein IR spectra. The IR spectra of aqueous solutions of 16 different proteins of known crystal structure have been recorded, and the amide I, the amide III, and the amide I combined with the amide III region of these proteins were used to set up the calibration set for the PLS algorithm. Our results correlate quite well with the data from X-ray studies, and the prediction from the amide III region is better than that from amide I or combined amide I and amide III regions.     

Temperature dependence of amino acid side chain IR absorptions in the amide I' region

Amide I' IR spectra are widely used for studies of structural changes in peptides and proteins as a function of temperature. Temperature dependent absorptions of amino acid side‐chains that overlap the amide I' may significantly complicate the structural analyses. While the side‐chain IR spectra have been investigated previously, thus far their dependence on temperature has not been reported. Here we present the study of the changes in the IR spectra with temperature for side‐chain groups of aspartate, glutamate, asparagine, glutamine, arginine, and tyrosine in the amide I' region (in D₂O). Band fitting analysis was employed to extract the temperature dependence of the individual spectral parameters, such as peak frequency, integrated intensity, band width, and shape. As expected, the side‐chain IR bands exhibit significant changes with temperature. The majority of the spectral parameters, particularly the frequency and intensity, show linear dependence on temperature, but the direction and magnitude vary depending on the particular side‐chain group. The exception is arginine, which exhibits a distinctly nonlinear frequency shift with temperature for its asymmetric CN₃H₅⁺ bending signal, although a linear fit can account for this change to within ~1/3 cm^‐1. The applicability of the determined spectral parameters for estimations of temperature‐dependent side‐chain absorptions in peptides and proteins are discussed. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 536–548, 2014.     

Botulinum neurotoxin type A: Structure and interaction with the micellar concentration of SDS determined by FT-IR spectroscopy

Secondary structures of botulinum neurotoxin type A have been determined using Fourier transform infrared spectroscopy in the amide I and amide III frequency regions. Using Fourier self-deconvolution, second derivatization, and curve-fit analysis, the amide I frequency contour was resolved into Gaussian bands at 1678, 1654, 1644, and 1634 cm^–1. In the amide III frequency region, several small bands were resolved between 1320 and 1225 cm^–1. Assignments of the bands in both amide I and amide III frequency regions to various types of secondary structures and the estimation of spectral band strengths by integrating areas under each band suggested that the neurotoxin contains 29% -helix, 45–49% -sheets and 22–26% random coils. These values agreed very well with those determined earlier from CD spectra. The neurotoxin was treated with a micellar concentration of sodium dodecyl sulfate to simulate interaction between the protein and the amphipathic molecules. Sodium dodecyl sulfate micelles induced significant alterations both in the spectral band positions, and their strengths suggest refolding of the neurotoxin polypeptides. However, these changes were not entirely reversible, which could implicate the role of the altered structures in the function of the neurotoxin.     

Temperature dependence of 1H chemical shifts in proteins

Temperature coefficients have been measured by 2D NMR methods forthe amide and CH proton chemical shifts in two globularproteins, bovine pancreatic trypsin inhibitor and hen egg-white lysozyme.The temperature-dependent changes in chemical shift are generally linear upto about 15° below the global denaturation temperature, and the derivedcoefficients span a range of roughly –16 to +2 ppb/K for amide protonsand –4 to +3 ppb/K for CH. The temperaturecoefficients can be rationalized by the assumption that heating causesincreases in thermal motion in the protein. Precise calculations oftemperature coefficients derived from protein coordinates are not possible,since chemical shifts are sensitive to small changes in atomic coordinates.Amide temperature coefficients correlate well with the location of hydrogenbonds as determined by crystallography. It is concluded that a combined useof both temperature coefficients and exchange rates produces a far morereliable indicator of hydrogen bonding than either alone. If an amide protonexchanges slowly and has a temperature coefficient more positive than–4.5 ppb/K, it is hydrogen bonded, while if it exchanges rapidly andhas a temperature coefficient more negative than –4.5 ppb/K, it is nothydrogen bonded. The previously observed unreliability of temperaturecoefficients as measures of hydrogen bonding in peptides may arise fromlosses of peptide secondary structure on heating.     

Temperature jump-induced secondary structural change of the membrane protein bacteriorhodopsin in the premelting temperature region: a nanosecond time-resolved Fourier transform infrared study

The secondary structural changes of the membrane protein, bacteriorhodopsin, are studied during the premelting reversible transition by using laser-induced temperature jump technique and nanosecond time-resolved Fourier transform infrared spectroscopy. The helical structural changes are triggered by using a 15 degrees C temperature jump induced from a preheated bacteriorhodopsin in D2O solution at a temperature of 72 degrees C. The structural transition from alphaII- to alphaI-helices is observed by following the change in the frequency of the amide I band from 1667 to 1651 cm-1 and the shift in the frequency of the amide II vibration from 1542 cm-1 to 1436 cm-1 upon H/D exchange. It is found that although the amide I band changes its frequency on a time scale of <100 ns, the H/D exchange shifts the frequency of the amide II band and causes a complex changes in the 1651-1600 cm-1 and 1530-1430 cm-1 frequency region on a longer time scale (>300 ns). Our result suggests that in this "premelting transition" temperature region of bacteriorhodopsin, an intrahelical conformation conversion of the alphaII to alphaI leads to the exposure of the hydrophobic region of the protein to the aqueous medium.     

Calmodulin and troponin C: a comparative study of the interaction of mastoparan and troponin I inhibitory peptide [104-115]

Recent studies using bee and wasp venom peptides have led to the hypothesis that proper complex formation with calmodulin (CaM) requires the presence of a basic amphiphilic helix on the surface of the target protein [Cox, J. A. (1984) Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 3000]. We have tested this hypothesis by examining CaM and troponin C (TnC) complex formation with two basic peptides, the wasp venom tetradecapeptide mastoparan and the physiologically relevant synthetic troponin I (TnI) inhibitory peptide [104-115], using far-ultraviolet circular dichroism as a secondary structure probe. Complex formation between mastoparan and either CaM or TnC results in an increase in helical content, whereas the helical content of TnI inhibitory peptide does not increase when bound to either protein. Significantly, mastoparan is 78% alpha-helical in a 50% solution of the helix-inducing solvent trifluoroethanol and has a high helix-forming potential according to the Chou-Fasman rules while TnI inhibitory peptide contains none and is not predicted to have any. We interpret these data as indicating that these peptides exhibit substantially different secondary structures upon binding to CaM or TnC. The ability of mastoparan to regulate the acto-subfragment 1-tropomyosin ATPase has also been examined. Mastoparan and TnI inhibitory peptide inhibited 31% and 45% of the activity, respectively. TnC and CaM promote differing degrees of Ca2+-sensitive release of inhibition by both peptides. Sequence comparison suggests that the basic residues present in both peptides are important for binding. However, we conclude that an alpha-helical structure is not a prerequisite for the binding of target proteins to CaM and TnC.     

Synthesis and Properties of the <Emphasis Type="Italic">retro</Emphasis>-Analogue of Myelopeptide MP-2

The bone marrow myelopeptide MP-2 (Leu-Val-Val-Tyr-Pro-Trp), exhibiting antitumor activity, and its retro-analogue (Trp-Pro-Tyr-Val-Val-Leu) were synthesized, and their properties were studied. The in vitro and in vivo activities of retro-MP-2 were comparable with those of MP-2. Both peptides equally restored the functional activity of T-lymphocytes inhibited by toxins released by HL-60 cells and inhibited by 70–82% the growth of various types of transplantable solid tumors: Ca-755 adenocarcinoma of the mammary gland, Lewis adenocarcinoma of the lung, and S180 sarcoma. The positions and intensities of the Cotton effects in CD spectra of the MP-2 peptide and its retro-analogue in various solvents are almost indistinguishable. The positions of extrema and integral intensities of the amide I and amide A bands in IR spectra of both peptides were practically identical.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 239–244.Original Russian Text Copyright © 2005 by Fonina, Ovchinnikov, Gur’yanov, Sychev, Belevskaya, Treshchalina.     

Temperature coefficients of peptides dissolved in hexafluoroisopropanol monitor distortions of helices

Summary Temperature coefficients are widely used as an indication of solvent accessibility to amide protons. Low temperature coefficients are related to low accessibility and are often interpreted as evidence for intramolecular hydrogen bonding. Conformational shifts, i.e. the difference between chemical shifts of a particular residue in a structured and in a random-coil conformation, provide information on secondary structure. In particular, negative CH^α conformational shifts are often used to delineate the extent of helical stretches. NH conformational shifts show large oscillations within a helix that have been interpreted as the result of helix distortions affecting hydrogen bond lengths. In the ocurse of the study of differnet peptides that adopt a helical structure in the presence of the structure-inducing solvent hexafluoroisopropanol (HFIP), we have found a strong correlation between temperature coefficients and amide conformational shifts. However, contrary to the initial expectations, lower temperature coefficients were associated to amide protons involved in longer, and presumably weaker, hydrogen bonds. The correlation can be explained, however, assuming that, in helical peptides dissolved in HFIP, temperature affects the chemical shift of amide protons mainly by changing the average length of intramolecular hydrogen bonds and changes in solvent accessibility play only a secondary role under these experimental conditions. The pattern of temperature coefficients in helical peptides can therefore be used to identify short or long hydragen bonds causing bending of the helix axis.     

Membranotropic effects of peptides from the V3 loop of HIV-1

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell–cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the pH and the of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an -helix/-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus–cell fusion.