Molecular origin of the self-assembly of lanreotide into nanotubes: a mutational approach

Lanreotide, a synthetic, therapeutic octapeptide analog of somatostatin, self-assembles in water into perfectly hollow and monodisperse (24-nm wide) nanotubes. Lanreotide is a cyclic octapeptide that contains three aromatic residues. The molecular packing of the peptide in the walls of a nanotube has recently been characterized, indicating four hierarchical levels of organization. This is a fascinating example of spontaneous self-organization, very similar to the formation of the gas vesicle walls of Halobacterium halobium. However, this unique peptide self-assembly raises important questions about its molecular origin. We adopted a directed mutation approach to determine the molecular parameters driving the formation of such a remarkable peptide architecture. We have modified the conformation by opening the cycle and by changing the conformation of a Lys residue, and we have also mutated the aromatic side chains of the peptide. We show that three parameters are essential for the formation of lanreotide nanotubes: i), the specificity of two of the three aromatic side chains, ii), the spatial arrangement of the hydrophilic and hydrophobic residues, and iii), the aromatic side chain in the β-turn of the molecule. When these molecular characteristics are modified, either the peptides lose their self-assembling capability or they form less-ordered architectures, such as amyloid fibers and curved lamellae. Thus we have determined key elements of the molecular origins of lanreotide nanotube formation.     

Ranking the affinity of aromatic residues for carbon nanotubes by using designed surfactant peptides.

A series of surfactant peptides were created to evaluate the affinity of aromatic AAs for single-walled carbon nanotubes in the absence of complications from peptide folding or self-association. Each surfactant peptide has a lipidlike architecture, with two Lys residues at the C-terminus as a hydrophilic head, five Val residues to form a hydrophobic tail, and the testing AA at the N-terminus. Raman and CD spectroscopic studies reveal that the surfactant peptides have a large unordered structural component which is independent of peptide concentration, suggesting that the peptides undergo minimal association under experimental conditions, thus removing this interference from interpretation of the peptide/carbon nanotube interactions. A lack of peptide self-association is also indicated by sedimentation equilibrium ultracentrifugation results. Optical spectroscopy of the peptide/carbon nanotube dispersions indicate that among the three aromatic AAs, tryptophan has the highest affinity for carbon nanotubes (both bundled and individual states) when incorporated into a surfactant peptide, while the Tyr-containing peptide is more selective for individual carbon nanotubes. Phe has the lowest overall affinity for carbon nanotubes. Raman spectra of dispersions made with SPF, SPY and SPW display similar types of nanotubes dispersed, although differences in the relative nanotube populations are observed by optical spectroscopy.     

Tuning β-sheet peptide self-assembly and hydrogelation behavior by modification of sequence hydrophobicity and aromaticity

Peptide self-assembly leading to cross-β amyloid structures is a widely studied phenomenon because of its role in amyloid pathology and the exploitation of amyloid as a functional biomaterial. The self-assembly process is governed by hydrogen bonding, hydrophobic, aromatic π-π, and electrostatic Coulombic interactions. A role for aromatic π-π interactions in peptide self-assembly leading to amyloid has been proposed, but the relative contributions of π-π versus general hydrophobic interactions in these processes are poorly understood. The Ac-(XKXK)(2)-NH(2) peptide was used to study the contributions of aromatic and hydrophobic interactions to peptide self-assembly. Position X was globally replaced by valine (Val), isoleucine (Ile), phenylalanine (Phe), pentafluorophenylalanine (F(5)-Phe), and cyclohexylalanine (Cha). At low pH, these peptides remain monomeric because of repulsion of charged lysine (Lys) residues. Increasing the solvent ionic strength to shield repulsive charge-charge interactions between protonated Lys residues facilitated cross-β fibril formation. It was generally found that as peptide hydrophobicity increased, the required ionic strength to induce self-assembly decreased. At [NaCl] ranging from 0 to 1000 mM, the Val sequence failed to assemble. Assembly of the Phe sequence commenced at 700 mM NaCl and at 300 mM NaCl for the less hydrophobic Ile variant, even though it displayed a mixture of random coil and β-sheet secondary structures over all NaCl concentrations. β-Sheet formation for F(5)-Phe and Cha sequences was observed at only 20 and 60 mM NaCl, respectively. Whereas self-assembly propensity generally correlated to peptide hydrophobicity and not aromatic character the presence of aromatic amino acids imparted unique properties to fibrils derived from these peptides. Nonaromatic peptides formed fibrils of 3-15 nm in diameter, whereas aromatic peptides formed nanotape or nanoribbon architectures of 3-7 nm widths. In addition, all peptides formed fibrillar hydrogels at sufficient peptide concentrations, but nonaromatic peptides formed weak gels, whereas aromatic peptides formed rigid gels. These findings clarify the influence of aromatic amino acids on peptide self-assembly processes and illuminate design principles for the inclusion of aromatic amino acids in amyloid-derived biomaterials.     

A spectrophotometric assay for the characterization of the S'' subsite specificity of alpha-chymotrypsin

Pig and horse colipases contain three tyrosine residues. In addition, horse colipase possesses a tryptophan residue. Some of the tyrosine residues are involved in the association of colipase and a bile salt micelle. The present report demonstrates that the aromatic residues responsible for colipase fluorescence are in an aqueous environment. In the presence of bile salt micelles, changes in colipase fluorescence properties indicate that the intrinsic fluorophores are located in a more hydrophobic environment upon colipase-micelle complex formation. In addition, the fluorescence of an NBD group fixed on lysine 60, which is very close to the aromatic region in the pig colipase, is also altered in the presence of micelles. These results show that the micelle binding site is not limited to the tyrosine residues but may be broadened to adjacent residues such as lysine 60 and also tryptophan 52 in horse colipase.     

Probing the role of aromatic residues in the self-assembly of Aβ(16–22) in fluorinated alcohols and their aqueous mixtures

The Aβ(16–22) sequence KLVFFAE spans the hydrophobic core of the Aβ peptide and plays an important role in its self-assembly. Apart from forming amyloid fibrils, Aβ(16–22) can self-associate into highly ordered nanotubes and ribbon-like structures depending on the composition of solvent used for dissolution. The Aβ(16–22) sequence which has FF at the 19th and 20th positions would be a good model to investigate peptide self-assembly in the context of aromatic interactions. In this study, self-assembly of Aβ(16–22) and its aromatic analogs obtained by replacement of F19, F20 or both by Y or W was examined after dissolution in fluorinated alcohols and their aqueous mixtures in solvent cluster forming conditions. The results indicate that the presence of aromatic residues Y and W and their position in the sequence plays an important role in self-assembly. We observe the formation of amyloid fibrils and other self-assembled structures such as spheres, rings and beads. Our results indicate that 20% HFIP is more favourable for amyloid fibril formation as compared to 20% TFE, when F is replaced with Y or W. The dissolution of peptides in DMSO followed by evaporation of solvent and dissolution in water appears to greatly influence peptide conformation, morphology and cross-β content of self-assembled structures. Our study shows that positioning of aromatic residues F, Y and W have an important role in directing self-assembly of the peptides.     

Chemical, photochemical and spectroscopic characterization of an alkaline proteinase from Bacillus subtilis variant DY

Circular-dichroism and fluorescence studies indicate that the 5-dimethylaminonaphthalene-1-sulphonyl and phenylmethanesulphonyl derivatives of subtilisin DY have three-dimensional structure closely similar to that of native enzyme. The single tryptophan residue is largely accessible to the aqueous solvent, and is not directly involved in the enzyme-substrate interactions, since its photochemical modification causes only a partial inhibition of the enzyme activity. It appears very likely that the location of the single tryptophan residue in the three-dimensional structure of subtilisin DY is similar to that of the single tryptophan residue in subtilisin Carlsberg. Fluorescence-quenching experiments further indicate that the 14 tyrosine residues are also largely accessible to the aqueous solvent, and probably interact with hydrated peptide carbonyl groups. The charge environment for tryptophan and tyrosine residues in subtilisin DY, as deduced by quenching experiments with ionic species, is also discussed. In general, subtilisin DY displays strong similarities to subtilisin Carlsberg, as suggested by a comparative analysis of the amino acid composition and fluorescence properties.     

The structure and orientation of class-A amphipathic peptides on a phospholipid bilayer surface

The amphipathic α-helix is a recognised structural motif that is shared by membrane-associating proteins and peptides of diverse function. The aim of this paper is to determine the orientation of an α-helical amphipathic peptide on the bilayer surface. We use five amphipathic 18-residue peptide analogues of a class A amphipathic peptide that is known to associate with a bilayer surface. Tyrosine and tryptophan are used as spectroscopic probes to sense local environments in the peptide in solution and when bound to the surface of unilamellar phosphatidylcholine vesicles. In a series of peptides, tryptophan is moved progressively along the sequence from the nonpolar face (positions 3, 7, 4) to the polar face of the peptide (positions 2, 12). The local environment of the tryptophan residue at each position is determined using fluorescence spectroscopy employing quantum yield, and the wavelength of the emission maximum as indicators of micropolarity. The exposure of the tryptophan residues at each site is assessed by acrylamide quenching. On association with vesicles, the tryptophan residues at positions 3, 7 and 14 are in nonpolar water-shielded environments, and the tryptophan at position 12 is in an exposed polar environment. The tryptophan at position 2, which is located near the bilayer-water interface, exhibits intermediate behaviour. Analysis of the second-derivative absorption spectrum confirmed that the tyrosine residue at position 7 is in a nonpolar water-shielded environment in the peptide-lipid complex. We conclude that these class A amphipathic peptides lie parallel to the lipid surface and penetrate no deeper than the ester linkages of the phospholipids. Received: 8 April 1998 / Revised version: 6 July 1998 / Accepted: 7 August 1998     

Low concentration structural dynamics of lanreotide and somatostatin‐14

Lanreotide, a synthetic cyclic octapeptide, analogue of the peptide hormone somatostatin‐14 (SST‐14), is routinely used as a long‐acting medication in the management of neuroendocrine tumors. Despite its therapeutic importance, low concentration structural data is still lacking for lanreotide. In fact, the major part of the previous structural investigations were focused on the remarkable aggregation properties of this peptide, appearing at high concentrations (>5 mM). Here, we have applied three optical spectroscopic techniques, i.e. fluorescence, circular dichroism and Raman scattering, for analyzing the structural dynamics at the concentrations below 5 mM, where lanreotide exists either in a monomer state or at the first stages of aggregation. The obtained data from lanreotide were discussed through their comparison with those collected from SST‐14, leading us to the following conclusions: (i) The central D‐Trp residue, forming with its adjacent Lys the main receptor interacting part of lanreotide, keeps a constant high rotational freedom whatever the environment (water, water/methanol, methanol). (ii) A solvent‐dependent tight β‐turn, belonging to the type‐II' family, is revealed in lanreotide. (iii) Raman data analyzed by band decomposition in the amide (I and III) regions allowed estimation of different secondary structural elements within the millimolar range. Interestingly, the applied protocol shows a perfect agreement between the structural features provided by the amide I and amide III Raman markers. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1019–1028, 2014.     

A study of subtilisin types Novo and Carlsberg by circular polarization of fluorescence.

The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.     

The structure of human acidic fibroblast growth factor and its interaction with heparin.

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.     

Role of amino acid hydrophobicity, aromaticity, and molecular volume on IAPP(20-29) amyloid self-assembly

Aromatic amino acids strongly promote cross-β amyloid formation; whether the amyloidogenicity of aromatic residues is due to high hydrophobicity and β-sheet propensity or formation of stabilizing π-π interactions has been debated. To clarify the role of aromatic residues on amyloid formation, the islet amyloid polypeptide 20-29 fragment [IAPP(20-29)], which contains a single aromatic residue (Phe 23), was adopted as a model. The side chain of residue 23 does not self-associate in cross-β fibrils of IAPP(20-29) (Nielsen et al., Angew Chem Int Ed 2009;48:2118-2121), allowing investigation of the amyloidogenicity of aromatic amino acids in a context where direct π-π interactions do not occur. We prepared variants of IAPP(20-29) in which Tyr, Leu, Phe, pentafluorophenylalanine (F5-Phe), Trp, cyclohexylalanine (Cha), α-naphthylalanine (1-Nap), or β-naphthylalanine (2-Nap) (in order of increasing peptide hydrophobicity) were incorporated at position 23 (SNNXGAILSS-NH2), and the kinetic and thermodynamic effects of these mutations on cross-β self-assembly were assessed. The Tyr, Leu, and Trp 23 variants failed to readily self-assemble at concentrations up to 1.5 mM, while the Cha 23 mutant fibrillized with attenuated kinetics and similar thermodynamic stability relative to the wild-type Phe 23 peptide. Conversely, the F5-Phe, 1-Nap, and 2-Nap 23 variants self-assembled at enhanced rates, forming fibrils with greater thermodynamic stability than the wild-type peptide. These results indicate that the high amyloidogenicity of aromatic amino acids is a function of hydrophobicity, β-sheet propensity, and planar geometry and not the ability to form stabilizing or directing π-π bonds.     

Self-assembly of a peptide sequence,EKKE, composed of exclusively charged amino acids: Role of charge in morphology and lead binding

The self-assembly of peptides is influenced by their amino acid sequence and other factors including pH, charge, temperature, and solvent. Herein, we explore whether a four-residue sequence, EKKE, consisting of exclusively charged amino acids shows the propensity to form self-assembled ordered nanostructures and whether the overall charge plays any role in morphological and functional properties. From a combination of experimental data provided by Thioflavin T fluorescence, Congo red absorbance, circular dichroism spectroscopy, dynamic light scattering, field emission-scanning electron microscopy, atomic force microscopy, and confocal microscopy, it is clear that the all-polar peptide and charged EKKE sequence shows a pH-dependent tendency to form amyloid-like structures, and the self-assembled entities under acidic, basic and neutral conditions exhibit morphological variation. Additionally, the ability of the self-assembled amyloid nanostructures to bind to the toxic metal, lead (Pb²⁺), was demonstrated from the analysis of the ultraviolet absorbance and X-ray photoelectron spectroscopy data. The modulation at the sequence level for the amyloid-forming EKKE scaffold can further extend its potential role not only in the remediation of other toxic metals but also towards biomedical applications.     

Melittin as model system for probing interactions between proteins and cyclodextrins

Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. This enables cyclodextrins to solubilize hydrophobic molecules in aqueous media. Cyclodextrins may inhibit aggregation by intercalating surface aromatic residues and competing with interprotein aromatic clusters (pi-pi interactions). In order to investigate this concept, the interaction of hydroxypropyl-beta-cyclodextrin (HPBCD) with melittin is studied with steady-state and time-resolved fluorescence, fluorescence polarization, circular dichroism, and IR spectroscopy. HPBCD inhibits the aggregation of melittin. This inhibition and the spectroscopic results are consistent with the lone aromatic tryptophan of the peptide being intercalated within HPBCD.     

Intrinsic fluorescence of binding-site fragments of the nicotinic acetylcholine receptor: perturbations produced upon binding alpha-bungarotoxin

Synthetic peptides corresponding to sequences contained within residues 173-204 of the alpha-subunit in the nicotinic acetylcholine receptor (nAChR) of Torpedo californica bind the competitive antagonist alpha-bungarotoxin (BGTX) with relative high affinity. Since the synthetic peptide fragments of the receptor and BGTX each contain a small number of aromatic residues, intrinsic fluorescence studies were used to investigate their interaction. We examined a number of receptor-derived peptide fragments of increasing length (4-32 amino acids). Changes in the lambda max and quantum yield with increasing polypeptide chain length suggest an increase in the hydrophobicity of the tryptophan environment. When selective excitation and subtraction were used to reveal the tyrosine fluorescence of the peptides, a significant red shift in emission was observed and was found to be due to an excited-state tyrosinate. The binding of BGTX to the receptor-derived peptide fragments resulted in a large increase in fluorescence. In addition, at equilibrium, the lambda max of tryptophan fluorescence was shifted to shorter wavelengths. The. fluorescence enhancement, which was saturable with either peptide or BGTX, was used to determine the dissociation constants for the complexes. At pH 7.4, the apparent Kd for a dodecameric peptide (alpha 185-196), consisting of residues 185-196 in the alpha-subunit of the nAChR from Torpedo californica, was 1.4 microM. The Kd for an 18-mer (alpha 181-198), consisting of residues 181-198 of the Torpedo alpha-subunit, was 0.3 microM. No binding or enhanced fluorescence was observed with an irrelevant synthetic peptide of comparable composition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies of the tyrosine residues of human plasma apolipoprotein A-II

Human apolipoprotein A-II (apo A-II) in solution and associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was investigated by a combination of absorbance and fluorescence methods. Each apo A-II polypeptide chain contains four tyrosine residues but no tryptophan residues. Two and three tyrosine residues, respectively, appear to be buried for apo A-II in aqueous solution and in the lipid-associated protein. The spectroscopic properties of the tyrosine residues of lipid-associated apo A-II were also investigated. Plots of fluorescence intensity against temperature revealed a discontinuity in the region of the phase transition; however, over the same temperature range, there was no change in the exposure of tyrosine residues to the aqueous environment or in their mobility as measured by fluorescence polarization. Near-ultraviolet circular dichroic measurements demonstrated that the environments of the tyrosine residues of lipid-associated apo A-II and nitrated apo A-II were different from that of the apo A-II in solution or in a denatured state. Similar measurements also revealed that the microenvironments around tyrosines of apo A-II bound to DMPC in the gel phase are different from those observed in the liquid crystalline phase. Using environmentally sensitive fluorescence lipid probes, we have previously demonstrated that the polarity of the lipid/water interface of DMPC changes through a phase transition. The observations presented here indicate that these environmental changes also occur at the lipid/protein interface.     

The aromatic and heme chromophores of rabbit hemopexin. Difference absorption and fluorescence spectra.

Spectrophotometric and fluorimetric techniques were employed to charcterize the environment of the heme chromophore of rabbit hemopexin and to monitor changes in the environment of aromatic amino acid residues induced by the interaction of hemopexin with porphyrins and metalloporphyrins. Difference spectra showed maxima at 292 and 285 nm when hemopexin binds heme or deuteroheme but not deuteroporphyrin. These maxima are attributed to alterations in the local environment of tryptophan and tyrosine residues. Spectro-photometric titrations of the tyrosine residues of hemopexin, heme-hemopexin and hemopexin in 8 M urea showed apparent pK values at 11.4, 11.7, and 10.9 respectively. Perturbation difference spectra produced by 20% v/v ethylene glycol are consistent with the exposure of 6-8 of the 14 tyrosine residues and 6-8 of the 15 tryptophan residues of rabbit hemopexin to this perturbant. Only small differences were found between the perturbation spectra of apo- and heme-hemopexin near 290 nm, suggesting that slight or compensating changes in the exposure to solvent of tryptophan chromophores occur. In the Soret spectral region, the exposure of heme in the heme-hemopexin complex to ethylene glycol was 0.7, relative to the fully exposed heme peptide of cytochrome c. The fluorescence quantum yields of rabbit apo- and heme-hemopexin were estimated to be 0.06 and 0.03, respectively, compared to a yield of 0.13 for L-tryptophan. Iodide quenched 50% of the fluorescence of the deuteroheme-hemopexin complex. Cesium was not an effective quencher. Modification of approximately, 4 tryptophan residues with N-bromosuccinimide also decreased the relative fluorescence of apo-hemopexin by 50% and concomitantly reduced the heme-binding ability of the protein by 70%. The existence of sterically unhindered tryptophan residues in either apo- heme-hemopexin is unlikely since no charge transfer compelxes between these proteins and N-methylnicotinamide were detected.     

Circular dichroism and fluorescence of a tyrosine side-chain residue monitors the concentration-dependent equilibrium between U-shaped and coiled-coil conformations of a peptide derived from the catalytic core of HIV-1 integrase

The peptide denoted K159 (30 residues) derives from the catalytic core (CC) sequence of HIV-1 integrase (IN, residues 147-175). In the crystal structure of CC, the corresponding segment belongs to the alpha4 helix (residues 148-168, including residues Glu 152, Lys 156 and Lys 159, crucial for enzyme activity and DNA recognition), a loop (residues 169-171) and a part of the alpha5 helix (171-175), involved in enzyme dimerization. We used the fluorescence and the circular dichroism (CD) properties in the near-UV of the aromatic side chain of a tyrosine residue added at the C-terminal end of K159 in order to analyze the behavior of the concentrated and diluted peptide in aqueous trifluoroethanol (TFE), in an attempt to connect the information obtainable at high (NMR), medium (CD) and low (fluorescence) concentrations of the peptide. Altogether, the C-terminal tyrosine residue provided indirect information on the global conformation of K159 and on the local orientation and environment of the residue. The propensity of TFE to stabilize alpha-helical conformations in peptides was confirmed in CD and fluorescence experiments at relatively high (20-160 microM) and low (2-16 microM) concentrations, respectively. At relatively high concentration, stabilization of the peptide into alpha-helical conformation favored its auto-association likely in parallel coiled-coil dimers, as pointed out in our previous work [Eur. J. Biochem. 253 (1998) 236]. This was further confirmed by ANS (1-anilinonaphtalene-8-sulfonic acid) analysis and fluorescence temperature coefficient measurement. With diluted K159, a Stern-Volmer analysis with positively and negatively charged quenchers indicated that, when the intermolecular interactions were absent, the tyrosine was in a positively charged environment, as if the peptide folded into a U-shaped conformation similar to that present in the crystal structure of the enzyme.     

Ultraviolet resonance Raman studies reveal the environment of tryptophan and tyrosine residues in the native and partially folded states of the E colicin-binding immunity protein Im7

Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.     

Evidence of π‐stacking interactions in the self‐assembly of hIAPP22‐29

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self‐assembly of human islet amyloid polypeptide (hIAPP)_22‐29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe‐23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe‐23 virtually abolish the ability of hIAPP_22‐29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β‐sheet secondary structure. Transmission electron microscopy images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the ? C?C? ring mode (ca. 1600 cm^?1) upon aggregation that has previously been shown to be indicative of π‐stacking. While previous work has demonstrated that π‐stacking is not an absolute requirement for fibrillization, our findings indicate that Phe‐23 also contributes to fibril formation through π‐stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self‐assembly of hIAPP_22‐29. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

The effect of cleaving the reactive-site peptide bond Lys-15--Ala-16 on the conformation of bovine trypsin-kallikrein inhibitor (K unitz) as revealed by solvent-perturbation spectra, circular dichroism and fluorescence.

Spectroscopic measurements of virgin bovine trypsin-kallikrein inhibitor and its modified species (in which the reactive-site peptide bond Lys-15--Ala-16 is split) indicate a conformational difference between both proteins. The inhibitor contains four tyrosines but no tryptophan. In the modified inhibitor a tyrosyl blue shift is seen in the difference absorption spectrum of modified against virgin inhibitor. The solvent perturbation spectra show an increase of the fraction of exposed tyrosyls from 0.45 in the virgin inhibitor to 0.59 in the modified form. Comparison of the circular dichroism spectra of the modified and virgin inhibitors reveals a decrease of the mean residue ellipticity in the tyrosine and peptide bond region of the modified inhibitor. In the fluorescence spectra a 50% increase in the quantum yield of the tyrosine fluorescence is observed in the modified inhibitor. All these spectroscopic data support the idea, which is also evidenced by the X-ray crystallographic model, that in the modified inhibitor up to five residues from Ala-16 to Arg-20 gain rotational freedom.