Ultraviolet Raman examination of the environmental dependence of bombolitin I and bombolitin III secondary structure.

Bombolitin I and III (BI and BIII) are small amphiphilic peptides isolated from bumblebee venom. Although they exist in predominately nonhelical conformations in dilute aqueous solutions, we demonstrate, using UV Raman spectroscopy, that they become predominately alpha-helical in solution at pH > 10, in high ionic strength solutions, and in the presence of trifluoroethanol (TFE) and dodecylphosphocholine (DPC) micelles. In this paper, we examine the effects of electrostatic and hydrophobic interactions that control folding of BI and BIII by systematically monitoring their secondary structures as a function of solution conditions. We determine the BI and BIII secondary structure contents by using the quantitative UV Raman methodology of Chi et al. (1998. Biochemistry. 37:2854-2864). Our findings suggest that the alpha-helix turn in BIII at neutral pH is stabilized by a salt bridge between residues Asp2 and Lys5. This initial alpha-helical turn results in different BI and BIII alpha-helical folding mechanisms observed in high pH and high salt concentrations: BIII folds from its single alpha-helix turn close to its N-terminal, whereas the BI alpha-helix probably nucleates within the C-terminal half. We also used quasielastic light scattering to demonstrate that the BI and BIII alpha-helix formation in 0.2 M Ca(ClO4)2 is accompanied by formation of trimers and hexamers, respectively.     

Changes in the abnormal alpha-subunit upon CO-binding to the normal beta-subunit of Hb M Boston: resonance Raman,EPR and CD study

Heme-heme interaction in Hb M Boston (His alpha 58-->Tyr) was investigated with visible and UV resonance Raman (RR), EPR, and CD spectroscopies. Although Hb M Boston has been believed to be frozen in the T quaternary state, oxygen binding exhibited appreciable co-operativity (n=1.4) and the near-UV CD spectrum indicated weakening of the T marker at pH 9.0. Binding of CO to the normal beta-subunit gave no change in the EPR and visible Raman spectra of the abnormal alpha-subunit at pH 7.5, but it caused an increase of EPR rhombicity and significant changes in the Raman coordination markers as well as the Fe(III)-tyrosine related bands of the alpha-subunit at pH 9.0. The UVRR spectra indicated appreciable changes of Trp but not of Tyr upon CO binding to the alpha-subunit at pH 9.0. Therefore, we conclude that the ligand binding to the beta heme induces quaternary structure change at pH 9.0 and is communicated to the alpha heme, presumably through His beta 92-->Trp beta 37-->His alpha 87.     

Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin

The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis. However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential. Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed. The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states. Similar changes followed cleavage of peptide or disulfide bonds. Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions.     

UV resonance Raman studies of alpha-nitrosyl hemoglobin derivatives: relation between the alpha 1-beta 2 subunit interface interactions and the Fe-histidine bonding of alpha heme.

Human alpha-nitrosyl beta-deoxy hemoglobin A, alpha(NO)beta(deoxy), is considered to have a T (tense) structure with the low O(2) affinity extreme and the Fe-histidine (His87) (Fe-His) bond of alpha heme cleaved. The Fe-His bonding of alpha heme and the intersubunit interactions at the alpha 1-beta 2 contact of alpha(NO)-Hbs have been examined under various conditions with EPR and UV resonance Raman (UVRR) spectra excited at 235 nm, respectively. NOHb at pH 6.7 gave the UVRR spectrum of the R structure, but in the presence of inositol-hexakis-phosphate (IHP) for which the Fe-His bond of the alpha heme is broken, UVRR bands of Trp residues behaved half-T-like while Tyr bands remained R-like. The half-ligated nitrosylHb, alpha(NO)beta(deoxy), in the presence of IHP at pH 5.6, gave T-like UVRR spectra for both Tyr and Trp, but binding of CO to its beta heme (alpha(NO)beta(CO)) changed the UVRR spectrum to half-T-like. Binding of NO to its beta heme (NOHb) changed the UVRR spectrum to 70% T-type for Trp but almost R-type for Tyr. When the pH was raised to 8.2 in the presence of IHP, the UVRR spectrum of NOHb was the same as that of COHb. EPR spectra of these Hbs indicated that the Fe-His bond of alpha(NO) heme is partially cleaved. On the other hand, the UVRR spectra of alpha(NO)beta(deoxy) in the absence of IHP at pH 8.8 showed the T-like UVRR spectrum, but the EPR spectrum indicated that 40-50% of the Fe-His bond of alpha hemes was intact. Therefore, it became evident that there is a qualitative correlation between the cleavage of the Fe-His bond of alpha heme and T-like contact of Trp-beta 37. We note that the behaviors of Tyr and Trp residues at the alpha 1-beta 2 interface are not synchronous. It is likely that the behaviors of Tyr residues are controlled by the ligation of beta heme through His-beta 92(F8)-->Val-beta 98(FG5)-->Asp-beta 99(G1 )-->Tyr-alpha 42(C7) or Tyr-beta 145(HC2).     

Raman optical activity and circular dichroism reveal dramatic differences in the influence of divalent copper and manganese ions on prion protein folding

The binding of divalent copper ions to the full-length recombinant murine prion protein PrP23-231 at neutral pH was studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UV CD). The effect of the Cu2+ ions on PrP structure depends on whether they are added after refolding of the protein in water or are present during the refolding process. In the first case ROA reveals that the hydrated alpha-helix is lost, with UV CD revealing a drop from approximately 25% to approximately 18% in the total alpha-helix content. The lost alpha-helix could be that comprising residues 145-156, located within the region associated with scrapie PrP formation. In the second case, ROA reveals the protein's structure to be almost completely disordered/irregular, with UV CD revealing a drop in total alpha-helix content to approximately 5%. Hence, although Cu2+ binding takes place exclusively within the unfolded/disordered N-terminal region, it can profoundly affect the structure of the folded/alpha-helical C-terminal region. This is supported by the finding that refolding in the presence of Cu2+ of a mutant in which the first six histidines associated with copper binding to the N-terminal region are replaced by alanine has a similar alpha-helix content to the metal-free protein. In contrast, when the protein is refolded in the presence of divalent manganese ions, ROA indicates the alpha-helix is reinforced, with UV CD revealing an increase in total alpha-helix content to approximately 30%. The very different influence of Cu2+ and Mn2+ ions on prion protein structure may originate in the different stability constants and geometries of their complexes.     

Circular dichroism and UV-resonance Raman investigation of the temperature dependence of the conformations of linear and cyclic elastin

We used electronic circular dichroism (CD) and UV resonance Raman (UVRR) spectroscopy at 204 nm excitation to examine the temperature dependence of conformational changes in cyclic and linear elastin peptides. We utilize CD spectroscopy to study global conformation changes in elastin peptides, while UVRR is utilized to probe the local conformation and hydrogen bonding of Val and Pro peptide bonds. Our results indicate that at 20 degrees C cyclic elastin predominantly populates distorted beta-strand, beta-type II and beta-type III turn conformations. At 60 degrees C, the beta-type II turn population increases, while the distorted beta-strand population decreases. Linear elastin predominantly adopts distorted beta-strand and beta-type III turn conformations with some beta-type II turn population at 20 degrees C. Increasing temperature to 60 degrees C results in a small increase in the turn population.     

Conformational modifications of alpha gliadin and globulin proteins upon complex coacervates formation with gum Arabic as studied by Raman microspectroscopy

As a molecular model of gelatin-free coacervates, complexes of pea globulin and alpha gliadin proteins with gum arabic prepared at different acidic pH values are studied using Raman microspectrometry. Raman spectra confirm higher content of beta-sheets and random coils in pea globulin and dominating alpha-helical structures in alpha gliadin. For protein-gum arabic complexes, Raman data support the existence of specific pH conditions for optimal complex coacervation (pH 2.75 for globulin and pH 3.0 for gliadin(1)), when (i) pH-induced conformational perturbations of free protein structure are the strongest and (ii) compensation of these perturbations by gum arabic is the most pronounced. Conformations implied in the protein-gum complexes are mainly beta-sheets in pea globulin and alpha-helix in alpha gliadin. The role of electrostatic and non-Coulombic interactions (intermolecular hydrogen bonds) in stabilizing of protein-polysaccharide complexes is discussed in relation with the overall structure and the charge density profile of these two proteins.     

Conformational intermediates in the folding of a coiled-coil model peptide of the N-terminus of tropomyosin and alpha alpha-tropomyosin.

Circular dichroism was used to study the folding of alpha alpha-tropomyosin and AcTM43, a 43-residue peptide designed to serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (< 25 degrees C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled-coil alpha-helix of native alpha alpha-tropomyosin. The second curve resembled the spectrum of single-stranded short alpha-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the alpha-helical content increased before most of the coiled coil was formed. When the CD spectrum of striated muscle alpha alpha-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 degrees C, there was a decrease in the coiled coil and an increase in the conventional alpha-helix type spectrum without formation of random coil. The major transition, occurring at 40 degrees C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.     

Intermediacy of poly(L-proline) II and beta-strand conformations in poly(L-lysine) beta-sheet formation probed by temperature-jump/UV resonance Raman spectroscopy

Ultraviolet resonance Raman spectroscopy (UVRR) in combination with a nanosecond temperature jump (T-jump) was used to investigate early steps in the temperature-induced alpha-helix to beta-sheet conformational transition of poly(L-lysine) [poly(K)]. Excitation at 197 nm from a tunable frequency-quadrupled Ti:sapphire laser provided high-quality UVRR spectra, containing multiple conformation-sensitive amide bands. Although un-ionized poly(K) (pH 11.6) is mainly alpha-helical below 30 degrees C, there is a detectable fraction (approximately 15%) of unfolded polypeptide, which is mainly in the poly(L-proline) II (PPII) conformation. However, deviations from the expected amide I and II signals indicate an additional conformation, suggested to be beta-strand. Above 30 degrees C un-ionized poly(K) forms a beta-sheet at a rate (minutes) which increases with increasing temperature. A 22-44 degrees C T-jump is accompanied by prompt amide I and II difference signals suggested to arise from a rapid shift in the PPII/beta-strand equilibrium. These signals are superimposed on a subsequently evolving difference spectrum which is characteristic of PPII, although the extent of conversion is low, approximately 2% at the 3 micros time limit of the experiment. The rise time of the PPII signals is approximately 250 ns, consistent with melting of short alpha-helical segments. A model is proposed in which the melted PPII segments interconvert with beta-strand conformation, whose association through interstrand H-bonding nucleates the formation of beta-sheet. The intrinsic propensity for beta-strand formation could be a determinant of beta-sheet induction time, with implications for the onset of amyloid diseases.     

Substrate polarization by residues in delta 5-3-ketosteroid isomerase probed by site-directed mutagenesis and UV resonance Raman spectroscopy.

delta 5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of delta 5-3-ketosteroids to delta 4-3-ketosteroids by the stereospecific transfer of the steroid 4 beta-proton to the 6 beta-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y14(0) mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y14(0) mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y14(0) enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm-1, respectively, on binding to Y14(0) KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue. UVRR spectra of the Y14(0) enzyme obtained using 200 nm excitation show enhancement of the amide II and S Raman bands. The secondary structure of KSI was estimated from the amide II and S intensities and was found to be low in alpha-helical structure. The alpha-helix content was estimated to be in the range of 0-25% (i.e., 10 +/- 15%).     

Structural characterization of the filamentous bacteriophage PH75 from Thermus thermophilus by Raman and UV-resonance Raman spectroscopy

The filamentous bacteriophage PH75, which infects the thermophile T. thermophilus, assembles in vivo at 70 degrees C and is stable to at least 90 degrees C. Although a high-resolution structure of PH75 is not available, the virion is known to comprise a closed single-stranded (ss) DNA circle of 6500 nucleotides sheathed by a capsid comprising 2700 copies of a 46-residue subunit (pVIII). Here, we employ Raman and UV-resonance Raman (UVRR) spectroscopy to identify structural details of the pVIII and DNA constituents of PH75 that may be related to the high thermostability of the native virion assembly. Analysis of the Raman amide I and amide III signatures reveals that the capsid subunit secondary structure is predominantly (87%) alpha-helical but contains a significant number of residues (6 +/- 1 or 13 +/- 3%) differing from the canonical alpha-helix. This minor structural component is not apparent in capsid subunits of the mesophilic filamentous phages, fd, Pf1, and Pf3, previously examined at similar spectral resolution. The Raman signature of PH75 also differs from those of fd, Pf1, and Pf3 by virtue of an unusual alanine marker (898 cm(-)(1) band), which is attributed to C(alpha)-H hydrogen-bond donation by subunit Ala residues. Because alanines of the PH75 subunit occur primarily within sXXXs motifs (where s is a small side chain, e.g. Gly, Ala, Ser), and because the occurrence of such motifs in alpha-helices is believed to thermostabilize interhelix associations via C(alpha)-H...O interactions [G. Kleiger et al. (2002) Biochemistry 41, 5990-5997], we propose that such hydrogen bonding may explain both the alanyl and amide I/III markers of PH75 capsid subunits and that C(alpha)-H...O interactions may serve as a significant source of virion thermostabilization. Raman and UVRR signatures of PH75 are also distinguished from those of fd, Pf1, and Pf3 by several marker bands that are indicative of hydrophilic Trp and Tyr environments, including hydrogen bonding interactions of aromatic ring substituents. These interactions are likewise proposed as contributors to the high thermostability of PH75 vis-a-vis fd, Pf1, and Pf3. Finally, PH75 is the only filamentous phage exhibiting UVRR markers diagnostic of a highly base-stacked ssDNA genome incorporating the low energy C2'-endo/anti deoxynucleoside conformation. The present results suggest that both intersubunit interactions and genome organization contribute to the enhanced thermostability of PH75 relative to mesophilic filamentous bacteriophages.     

Spectroscopic detection of β ‐sheet structure in nascent Aβ oligomers

Deep‐UV resonance Raman (UVRR) spectroscopy and circular dichroism (CD) were employed to study the secondary structure of Aβ(1–42) in fresh samples with increasing fractions of oligomeric peptide. A feature with a minimum at ～217 nm appeared in CD spectra of samples containing oligomeric Aβ(1–42). UVRR spectra more closely resembled those of disordered proteins. The primary difference between UVRR spectra was the ratio of the 1236 cm^–1 to 1260 cm^–1 amide III peak intensities, which shifted in favor of the 1236 cm^–1 band as the fraction of oligomeric peptide increased. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Helix bundles and coiled coils in alpha-spectrin and tropomyosin: a theoretical CD study

The dipole interaction model is used to investigate the effects of interactions between helices and supertwisting of helices by determining whether the predicted UV absorption and CD spectra for the three-helix bundle and coiled coil are significantly different from spectra for the single straight alpha-helix. Crystallographic data by Yan et al. for alpha-spectrin are used to construct a three-helix bundle of poly(L-alanine) modeling the protein. Backbone torsion angles represented by Fourier series are used to generate supertwisted helices and coiled coil models of poly(L-alanine) that have pitch, radius, and residue repeat similar to experimental crystallographic data on tropomyosin. Calculated CD spectra are compared with available experimental data. Theoretical spectra for the three-helix bundle and the supertwisted structures are quite similar to predictions for the straight alpha-helix of the same length with similar torsion angles, suggesting that CD is primarily dependent on the average backbone conformation and would not be a sensitive tool for distinguishing between single straight helices and closely packed or twisted alpha-helices.     

Folding of a peptide corresponding to the alpha-helix in bovine pancreatic trypsin inhibitor

A short peptide corresponding to the alpha-helical region of BPTI shows partial folding in aqueous solution (pH 7) as judged by circular dichroism (CD). Folding is temperature and denaturant sensitive, and the peptide is monomeric. The difference CD spectrum, obtained from spectra at two temperatures, indicates that the peptide folds as an alpha-helix. Difference CD spectroscopy provides a sensitive assay for helix formation in peptides exhibiting small amounts of structure. Helix stability in this peptide shows a marked pH dependence which is consistent with stabilizing charged side-chain interactions with the helix dipole and/or salt bridge formation.     

Factors governing 3(10)-helix vs alpha-helix formation in peptides: percentage of C(alpha)-tetrasubstituted alpha-amino acid residues and sequence dependence

As an additional step toward the dissection of the factors responsible for the onset of 3(10)-helix vs alpha-helix in peptides, in this paper we describe the results of a three-dimensional (3D) structural analysis by x-ray diffraction of the N(alpha)-acylated heptapeptide alkylamide mBrBz-L-Iva-L-(alphaMe)Val-L-Abu-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe characterized by a single (L-Abu3) C(alpha)-trisubstituted and six C(alpha)-tetrasubstituted alpha-amino acids. We find that in the crystal state this peptide is folded in a mixed helical structure with short elements of 3(10)-helix at either terminus and a central region of alpha-helix. This finding, taken together with the published NMR and x-ray diffraction data on the all C(alpha)-methylated parent sequence and its L-Val2 analog (also the latter heptapeptide has a single C(alpha)-trisubstituted alpha-amino acid) strongly supports the view that one C(alpha)-trisubstituted alpha-amino acid inserted near the N-terminus of an N(alpha)-acylated heptapeptide alkylamide sequence may be enough to switch a regular 3(10)-helix into an essentially alpha-helical conformation. As a corollary of this work, the x-ray diffraction structure of the N(alpha)-protected, C-terminal tetrapeptide alkylamide Z-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe, also reported here, is clearly indicative of the preference of this fully C(alpha)-methylated, short peptide for the 3(10)-helix. As the same terminally blocked sequence is mixed 3(10)/alpha-helical in the L-Abu3 heptapeptide amide but regular 3(10)-helical in the tetrapeptide amide and in the parent heptapeptide amide, these results point to an evident plasticity even of a fully C(alpha)-methylated short peptide.     

Heme structure of hemoglobin M Iwate [alpha 87(F8)His-->Tyr]: a UV and visible resonance Raman study

Heme structures of a natural mutant hemoglobin (Hb), Hb M Iwate [alpha87(F8)His-->Tyr], and protonation of its F8-Tyr were examined with the 244-nm excited UV resonance Raman (UVRR) and the 406.7- and 441.6-nm excited visible resonance Raman (RR) spectroscopy. It was clarified from the UVRR bands at 1605 and 1166 cm(-)(1) characteristic of tyrosinate that the tyrosine (F8) of the abnormal subunit in Hb M Iwate adopts a deprotonated form. UV Raman bands of other Tyr residues indicated that the protein takes the T-quaternary structure even in the met form. Although both hemes of alpha and beta subunits in metHb A take a six-coordinate (6c) high-spin structure, the 406.7-nm excited RR spectrum of metHb M Iwate indicated that the abnormal alpha subunit adopts a 5c high-spin structure. The present results and our previous observation of the nu(Fe)(-)(O(tyrosine)) Raman band [Nagai et al. (1989) Biochemistry 28, 2418-2422] have proved that F8-tyrosinate is covalently bound to Fe(III) heme in the alpha subunit of Hb M Iwate. As a result, peripheral groups of porphyrin ring, especially the vinyl and the propionate side chains, were so strongly influenced that the RR spectrum in the low-frequency region excited at 406.7 nm is distinctly changed from the normal pattern. When Hb M Iwate was fully reduced, the characteristic UVRR bands of tyrosinate disappeared and the Raman bands of tyrosine at 1620 (Y8a), 1207 (Y7a), and 1177 cm(-)(1) (Y9a) increased in intensity. Coordination of distal His(E7) to the Fe(II) heme in the reduced alpha subunit of Hb M Iwate was proved by the observation of the nu(Fe)(-)(His) RR band in the 441.6-nm excited RR spectrum at the same frequency as that of its isolated alpha chain. The effects of the distal-His coordination on the heme appeared as a distortion of the peripheral groups of heme. A possible mechanism for the formation of a Fe(III)-tyrosinate bond in Hb M Iwate is discussed.     

Tipping the Scale from Disorder to Alpha-helix: Folding of Amphiphilic Peptides in the Presence of Macroscopic and Molecular Interfaces

Secondary amphiphilicity is inherent to the secondary structural elements of proteins. By forming energetically favorable contacts with each other these amphiphilic building blocks give rise to the formation of a tertiary structure. Small proteins and peptides, on the other hand, are usually too short to form multiple structural elements and cannot stabilize them internally. Therefore, these molecules are often found to be structurally ambiguous up to the point of a large degree of intrinsic disorder in solution. Consequently, their conformational preference is particularly susceptible to environmental conditions such as pH, salts, or presence of interfaces. In this study we use molecular dynamics simulations to analyze the conformational behavior of two synthetic peptides, LKKLLKLLKKLLKL (LK) and EAALAEALAEALAE (EALA), with built-in secondary amphiphilicity upon forming an alpha-helix. We use these model peptides to systematically study their aggregation and the influence of macroscopic and molecular interfaces on their conformational preferences. We show that the peptides are neither random coils in bulk water nor fully formed alpha helices, but adopt multiple conformations and secondary structure elements with short lifetimes. These provide a basis for conformation-selection and population-shift upon environmental changes. Differences in these peptides’ response to macroscopic and molecular interfaces (presented by an aggregation partner) can be linked to their inherent alpha-helical tendencies in bulk water. We find that the peptides’ aggregation behavior is also strongly affected by presence or absence of an interface, and rather subtly depends on their surface charge and hydrophobicity.     

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.     

Helix-turn-helix peptides that form alpha-helical fibrils: turn sequences drive fibril structure

Models of apolipoprotein A-I (apo A-I), the main protein of high-density lipoprotein, predict that it contains 10 amphiphilic, alpha-helical segments connected by turns. We synthesized four peptides with two identical 18-residue, amphiphilic, alpha-helical segments (Anantharamaiah, G. M., et al. (1985) J. Biol. Chem. 260, 10248-10255) connected by putative turn sequences from apo A-I: (1) Ac-DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF-NH2, (2) Ac-DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF-NH2, (3) Ac-DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF-NH2, and (4) Ac-DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF-NH2. Surprisingly, peptides 1-3 formed fibrils after incubation (37 degrees C, 10 mM sodium phosphate, pH 7.60), but in contrast to beta-sheet amyloid fibrils, these did not bind thioflavin T and they induced a blue shift in the spectrum of Congo red. CD (peptides 1-3) and FTIR (peptides 1 and 2) of the fibrils showed significant alpha-helical character. Synchrotron X-ray fiber diffraction on a magnetically aligned sample of 1 confirmed the alpha-helical character in the fibrils and indicated that the helical axes are oriented perpendicular to the fibril axis. In contrast, peptide 4, containing two Gly residues but no Pro in the turn, formed only a small amount of nonfibrillar precipitate after prolonged incubation. Peptide 4P (peptide 4 with a Pro in place of the central Ala) and peptide 5, containing a PEG block in lieu of the central turn, were similar to peptide 4 in not forming fibrils, possibly because the region linking the helices was unstructured. These studies indicate that varying turn sequences between longer amphiphilic alpha-helical segments can drive the structure of fibrils.