Secondary conformations and temperature effect on structural transformation of amyloid beta (1-28), (1-40) and (1-42) peptides

Secondary structure of three amyloid b-peptides [A beta(1-28), A beta(1-40) and A beta(1-42)] in the solid state was respectively determined by Fourier transform infrared (FT-IR) microspectroscopy. Their thermal-dependent structural transformation were also investigated by FT-IR microspectroscopy equipped with a thermal analyzer. The present result demonstrates that the solid-state A beta(1-28), A beta(1-40) and A beta(1-42) peptides showed a significant IR spectral difference in the amide I and II bands. The secondary conformation of A beta(1-28) peptide was the combination of major beta-sheet and minor alpha-helix with little random coil structures, but A beta(1-40) peptide showed the co-existence of major beta-sheet and minor random coil with little alpha-helix structures. A beta(1-42) peptide mainly consisted of the predominant b-sheet structure. Although the intact A beta(1-28), A beta(1-40) or A beta(1-42) peptide exhibits a different secondary structure, a similar beta-conformation may form after thermal treatment. A thermal-dependent transition was found for solid A beta(1-28) and A beta(1-40) peptides near 40 degrees C and 45 degrees C, respectively. There was no transition temperature for solid A beta(1-42) peptide, however, due to only a very little level of alpha-helix and random coil structure containing in the solid A beta(1-42) peptide. The thermal denaturation plays an important role in the structural transformation from alpha-helix/random coil to beta-sheet.     

NMR studies in aqueous solution fail to identify significant conformational differences between the monomeric forms of two Alzheimer peptides with widely different plaque-competence, A beta(1-40)(ox) and A beta(1-42)(ox).

NMR studies of amyloid beta-peptides (A beta) in aqueous solution provide a novel way in which to characterize the apparent Alzheimer's disease-related conformational polymorphism of A beta. In the aqueous medium, neither of the polypeptides A beta(1-40)(ox) or A beta(1-42)(ox) (both of which contain a methionine sulfoxide at position 35) is folded into a globular structure, but they both deviate from random coil behavior by local conformational preferences of several short segments along the amino-acid sequence. Differences between the solution structures of A beta(1-40)(ox) and A beta(1-42)(ox) are indicated only by decreased flexibility of the region from about residue 32 to the C-terminus in A beta(1-42)(ox) when compared to A beta(1-40)(ox). The lack of the observation of more extensive conformational differences between the two molecules is intriguing, considering that A beta(1-42)(ox) in aqueous solution has much higher plaque-competence than A beta(1-40)(ox).     

Pressure-induced transformation of alpha-helix to beta-sheet in the secondary structures of amyloid beta (1-40) peptide exacerbated by temperature

The effect of pressure on the conformational structure of amyloid beta (1-40) peptide (A beta(1-40)), exacerbated with or without temperature, was determined by Fourier transform infrared (FT-IR) microspectroscopy. The result indicates the shift of the maximum peak of amide I band of intact solid A beta(1-40) from 1655 cm(-1) (alpha-helix) to 1647-1643 cm(-1) (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm(-1) assigned to beta-antiparallel sheet structure was also evident. Furthermore, the peak at 1540 cm(- 1) also shifted to 1527 (1529) cm(-1) in amide II band. The former was assigned to the combination of alpha-helix and random coil structures, and the latter was due to beta-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed A beta(1-40) samples were obtained from 33% to 22% for alpha-helix/random coil structures and from 47% to 57% for beta-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from alpha-helix to random coil and to beta- sheet structure. The structural transformation of the compressed A beta(1-40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of beta-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the beta-sheet structure transformed. The thermal-dependent transition temperatures of solid A beta(1-40) prepared by different pressures were near 55-60 degrees C.     

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.     

Using chimeric immunity proteins to explore the energy landscape for alpha-helical protein folding

To address the role of sequence in the folding of homologous proteins, the folding and unfolding kinetics of the all-helical bacterial immunity proteins Im2 and Im9 were characterised, together with six chimeric derivatives of these proteins. We show that both Im2 and Im9 fold rapidly (k(UN)(H(2)O)) approximately 2000 s(-1) at pH 7.0, 25 degrees C) in apparent two-state transitions, through rate-limiting transition states that are highly compact (beta(TS)0.93 and 0.96, respectively). Whilst the folding and unfolding properties of three of the chimeras (Im2 (1-44)(Im9), Im2 (1-64)(Im9 )and Im2 (25-44)(Im9)) are similar to their parental counterparts, in other chimeric proteins the introduced sequence variation results in altered kinetic behaviour. At low urea concentrations, Im2 (1-29)(Im9) and Im2 (56-64)(Im9) fold in two-state transitions via transition states that are significantly less compact (beta(TS) approximately 0.7) than those characterised for the other immunity proteins presented here. At higher urea concentrations, however, the rate-limiting transition state for these two chimeras switches or moves to a more compact species (beta(TS) approximately 0.9). Surprisingly, Im2 (30-64)(Im9) populates a highly collapsed species (beta(I)=0.87) in the dead-time (2.5 ms) of stopped flow measurements. These data indicate that whilst topology may place significant constraints on the folding process, specific inter-residue interactions, revealed here through multiple sequence changes, can modulate the ruggedness of the folding energy landscape.     

Thermodynamics of the coil <==> beta-sheet transition in a membrane environment

Biologically important peptides such as the Alzheimer peptide Abeta(1-40) display a reversible random coil <==>beta-structure transition at anionic membrane surfaces. In contrast to the well-studied random coil left arrow over right arrow alpha-helix transition of amphipathic peptides, there is a dearth on information on the thermodynamic and kinetic parameters of the random coil left arrow over right arrow beta-structure transition. Here, we present a new method to quantitatively analyze the thermodynamic parameters of the membrane-induced beta-structure formation. We have used the model peptide (KIGAKI)(3) and eight analogues in which two adjacent amino acids were substituted by their d-enantiomers. The positions of the d,d pairs were shifted systematically along the three identical segments of the peptide chain. The beta-structure content of the peptides was measured in solution and when bound to anionic lipid membranes with circular dichroism spectroscopy. The thermodynamic binding parameters were determined with isothermal titration calorimetry and the binding isotherms were analysed by combining a surface partition equilibrium with the Gouy-Chapman theory. The thermodynamic parameters were found to be linearly correlated with the extent of beta-structure formation. beta-Structure formation at the membrane surface is characterized by an enthalpy change of DeltaH(beta)=-0.23 kcal/mol per residue, an entropy change of DeltaS(beta)=-0.24 cal/mol K residue and a free energy change of DeltaG(beta)=-0.15 kcal/mol residue. An increase in temperature induces an unfolding of beta-structure. The residual free energy of membrane-induced beta-structure formation is close to that of membrane-induced alpha-helix formation.     

Thermally induced coil-to-helix transition of sodium gellan gum with different molar masses in aqueous salt solutions

Using 5 samples of well-purified Na-gellans (Na-gellans G1-G5, weight-average molar mass M(w) = 120 x 10(3)-32 x 10(3) at 40 degrees C), the effects of molar mass on the coil-to-double-helix transition in aqueous solutions with 25 mM NaCl were studied by light scattering and circular dichroism (CD) measurements, viscometry, and differential scanning calorimetry (DSC). From the temperature dependence of M(w), molar ellipticity at 201 nm [theta]201, intrinsic viscosity [eta], and DSC exothermic curves, it was found that the coil-to-double-helix transitions for G1-G5 samples took place at almost the same temperature. The [eta] and M(w) obtained in the temperature range from 40 to 25 degrees C can be explained by a simple coil/double-helix equilibrium model using the double-helix contents determined from CD data. The van't Hoff's transition enthalpy deltaH(vH) of Na-gellans depended on M(w). It is concluded that the coil-to-double-helix transitions of Na-gellans are all-or-none type transitions, and are accelerated with increasing M(w).     

Investigations of the molecular mechanism of metal-induced Abeta (1-40) amyloidogenesis

Transition-metal ions (Cu2+ and Zn2+) play critical roles in the Abeta plaque formation. However, precise roles of the metal ions in the Abeta amyloidogenesis have been controversial. In this study, the molecular mechanism of the metal-induced Abeta oligomerization was investigated with extensive metal ion titration NMR experiments. Upon additions of the metal ions, the N-terminal region (1-16) of the Abeta (1-40) peptide was selectively perturbed. In particular, polar residues 4-8 and 13-15 were more strongly affected by the metal ions, suggesting that those regions may be the major binding sites of the metal ions. The NMR signal changes of the N-terminal region were dependent on the peptide concentrations (higher peptide concentrations resulted in stronger signal changes), suggesting that the metal ions facilitate the intermolecular contact between the Abeta peptides. The Abeta (1-40) peptides (>30 microM) were eventually oligomerized even at low temperature (3 degrees C), where the Abeta peptides are stable as monomeric forms without the metal ions. The real-time oligomerization process was monitored by 1H/15N HSQC NMR experiments, which provided the first residue-specific structural transition information. Hydrophobic residues 12-21 initially underwent conformational changes due to the intermolecular interactions. After the initial structural rearrangements, the C-terminal residues (32-40) readjusted their conformations presumably for effective oligomerization. Similar structural changes of the metal-free Abeta (1-40) peptides were also observed in the presence of the preformed oligomers, suggesting that the conformational transitions may be the general molecular mechanism of the Abeta (1-40) amyloidogenesis.     

Heterogeneous exchange behavior of Samia cynthia ricini silk fibroin during helix-coil transition studied with (13)C NMR

The structure and structural transition of the glycine residue adjacent to the N-terminal alanine residue of the poly(L-alanine), (Ala)(12-13), region in Samia cynthia ricini silk fibroin was studied using (13)C nuclear magnetic resonance (NMR). Most of the glycine carbonyl peaks in the (13)C solution NMR spectrum of [1-(13)C]glycine-silk fibroin could be assigned to the primary structure from the comparison of the (13)C chemical shifts of seven glycine-containing tripeptides. The slow exchange between helix and coil forms in the NMR time scale was observed with increasing temperature exclusively for the underlined glycine residue in the Gly-Gly-(Ala)(12-13) sequence during fast helix-coil transition of the (Ala)(12-13) region.     

Conformational transitions of the hydrophobic polyacids

The conformational transitions of the alternating maleic acid copolymers with styrene (MA-St)n, and alpha-methyl styrene (MA-MSt)(n) in aqueous solutions were studied by means of various methods. The following results were obtained: 1) The conformational transitions of (MA-St)n from the compact to extended coil form are observed in various salt solutions, as in aqueous NaCl, and the compact form is stabilized by Rb+ and Cs+, but destabilized by Li+. The coions, Br-, I-, ClO4- and SCN- affect scarcely the stability of the compact form. 2) The temperature coefficient of viscosity d In [eta]/dT of (MA-St)n in 0.09 M NaCl was positive for the compact form, but negative for the coil form, and it reflects the transition. 3) The difference between specific heats for the compact and coil forms of (MA-St)n in 0.03 M NaCl is determined to be about 15% of the corresponding heat of transfer of benzene to aqueous medium. 4) A remarkable dilution of the bound monomeric acridine orange to the compact form (MA-St)n is observed and the dimerization free energy of the dye in the compact form is about -2.1 kcal mole at 25 degrees C. 5) Potentiometric, dilatometric and viscometric titrations of (MA-MSt)n in aqueous NaCl at 25 degrees C show a similar conformational transition to that of (MA-St)n. Also, the difference in the molar extinction coefficient at 261 nm indicates the transition. The compact form of (MA-MSt)n is more unstable than that of (MA-St)n. From the results, the compact conformations and the transition mechanism of both the polyacids were discussed in comparison with the results for the maleic acid copolymers with n-alkyl vinyl ethers.     

Capillary electrophoresis is a sensitive monitor of the hairpin-random coil transition in DNA oligomers

Capillary electrophoresis (CE) has been used to characterize the hairpin-random coil transition of four octamers in the GCxxxxGC minihairpin family, where xxxx is GAAA, TTTC, TTTT, or AAAA. The transition can be monitored by CE because differences in the frictional coefficients of the hairpin and coil forms of each octamer lead to a difference of approximately 9% in the free solution mobilities of the two conformations. The GAAA octamer is unusually stable, with a melting temperature of 65 degrees C. The TTTT octamer forms a minihairpin with a melting temperature of 29 degrees C, the TTTC octamer has a melting temperature of 16 degrees C, and the AAAA octamer has a melting temperature below 0 degrees C. The thermal transitions of the TTTT, TTTC, and AAAA octamers are well fitted by a structure prediction algorithm; however, the GAAA minihairpin is considerably more stable than predicted. The melting temperature of the GAAA minihairpin is reduced to 47 degrees C in aqueous buffers containing 7.2M urea and to 33 degrees C in buffers containing 7.2M urea plus 40% (v/v) formamide. The combined results indicate that CE is a sensitive technique for monitoring conformational transitions in small DNA molecules.     

Secondary structure conversions of Alzheimer's Abeta(1-40) peptide induced by membrane-mimicking detergents

The amyloid beta peptide (Abeta) with 39-42 residues is the major component of amyloid plaques found in brains of Alzheimer's disease patients, and soluble oligomeric peptide aggregates mediate toxic effects on neurons. The Abeta aggregation involves a conformational change of the peptide structure to beta-sheet. In the present study, we report on the effect of detergents on the structure transitions of Abeta, to mimic the effects that biomembranes may have. In vitro, monomeric Abeta(1-40) in a dilute aqueous solution is weakly structured. By gradually adding small amounts of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate to a dilute aqueous solution, Abeta(1-40) is converted to beta-sheet, as observed by CD at 3 degrees C and 20 degrees C. The transition is mainly a two-state process, as revealed by approximately isodichroic points in the titrations. Abeta(1-40) loses almost all NMR signals at dodecyl sulfate concentrations giving rise to the optimal beta-sheet content (approximate detergent/peptide ratio = 20). Under these conditions, thioflavin T fluorescence measurements indicate a maximum of aggregated amyloid-like structures. The loss of NMR signals suggests that these are also involved in intermediate chemical exchange. Transverse relaxation optimized spectroscopy NMR spectra indicate that the C-terminal residues are more dynamic than the others. By further addition of SDS or lithium dodecyl sulfate reaching concentrations close to the critical micellar concentration, CD, NMR and FTIR spectra show that the peptide rearranges to form a micelle-bound structure with alpha-helical segments, similar to the secondary structures formed when a high concentration of detergent is added directly to the peptide solution.     

Conformation of the antifreeze glycoprotein of polar fish

High-field proton and 13C NMR spectroscopy has been used to test and refine the recent proposal, based on vacuum uv circular dichroism results, of a threefold left-handed helical conformation for antifreeze glycoprotein (AFGP). Partial assignment of the protons of the glycotripeptide repeating unit has been made by comparison with spectra of model compounds, by selective decoupling, and by measurements of nuclear Overhauser effect (nOe). At 40 degrees C, AFGP fraction 8 (Mr 2600) shows 2-Hz linewidths which broaden at lower temperature. Neither 1H nor 13C chemical shifts depend strongly on temperature, suggesting no abrupt conformational transition. The nOe between alanine alpha and beta protons vary with temperature and with field strength, from small positive enhancements at 50 degrees C and 80 MHz to large negative effects at 3 degrees C and 300 MHz, indicating a substantial change of rotational correlation time with temperature. The higher-molecular-weight fraction 1-4 shows negative nOe at all temperatures. The CD spectra of fraction 1-4 show bands characteristic of the polyproline II structure at both 3 and 50 degrees C, while those bands in fraction 8 are weaker at 50 than 3 degrees C. The 1H nOe, the 13C T1, and CD data are interpreted as indicating that AFGP fraction 8 is an extended "rod-like" conformation at low temperature which becomes a flexible coil at high temperature, while fraction 1-4 is a flexible rod with sufficient segmental mobility to eliminate any long-range order.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

Multivariate curve resolution: a powerful tool for the analysis of conformational transitions in nucleic acids

A successful application is reported of the multivariate curve resolution alternating least-squares method (MCR-ALS) for the analysis of nucleic acid melting and salt-induced transitions. Under conditions where several structures co-exist in a conformational equilibrium, MCR-ALS analysis of the UV and circular dichroism (CD) spectra at different temperatures, ionic strength and oligonucleotide concentration allows for the resolution of concentration profiles and pure spectra of the different species. The methodology is illustrated by the case of the cyclic oligonucleotide d<pTGCTCGCT>. The melting transition of this molecule at different oligonucleotide concentrations was studied at 0, 2 and 10 mM MgCl₂ by UV and CD spectroscopy. In addition, salt titration experiments were carried out at 21.0 and 54.0°C. The MCR-ALS analysis indicates that three different conformations of this molecule co-exist in solution. In agreement with previous NMR studies, these conformations were assigned to a monomeric dumbbell-like structure, a dimeric four-stranded conformation and a disordered (random coil) structure. The MCR-ALS methodology allows for a detailed analysis of how this equilibrium is affected by temperature, salt and oligonucleotide concentration.     

Temperature-induced conformational changes in amyloid beta(1-40) peptide investigated by simultaneous FT-IR microspectroscopy with thermal system

Temperature-dependent secondary structures of the amyloid beta(1-40) peptide in the solid state were studied by simultaneous Fourier transform infrared/differential scanning calorimetry (FT-IR/DSC) microspectroscopic system with the heating-cooling-reheating cycle. The result indicates that a thermal transition temperature at 45 degrees C was easily obtained from the three-dimensional plot of the transmission FT-IR spectra as a function of temperature. Furthermore, the thermal-dependent conformational transformations, due to denaturation and aggregation, of solid amyloid beta(1-40) were mainly evidenced by reducing the compositions from 37 to 20-24% for alpha-helical and random coil structures but increasing the components from 27 to 45% for intermolecular beta-sheet structures. Thermal-irreversible behavior and a poor thermal stability of solid amyloid beta(1-40) were also observed from the poor restoration of the secondary conformational changes in the heated sample.     

pH-dependent aggregate forms and conformation of Alzheimer amyloid beta-peptide (12-24)

The conformational transition to a beta-structure and the aggregation process of Alzheimer amyloid beta-peptide (12-24) [abbreviated as A beta(12-24)] were studied. The influence of sample dissolution methods for the aggregate structure was examined by electron microscopy (EM). The difference in the width of the aggregate of A beta(12-24) depended on the pH immediately after sample dissolution. Two types of sample dissolution methods, F and R, were employed. For dissolution method F, the peptide sample was immediately dissolved in water and then adjusted to pH 2.2 by adding buffer, while for dissolution method R, the peptide was directly dissolved in the buffer solution. In the latter case, the starting pH was 3.0. Slight fibrils (10-12 nm in diameter) were observed with method F, and wider ribbon-like aggregates (17-20 nm in diameter) with method R, despite the same pH range. A difference between methods F and R was also detected in the CD spectra, especially at pHs near 5.0. The CD intensity of the 214 nm band with method R changed with pH, with the highest value at pH 3.7, whereas that with method F was unchanged at pHs below 5.0. The temperature-dependent CD results showed that a thermostable aggregate of A beta(12-24) occurs at higher pHs than 3.0. NMR analysis showed that deprotonation of the C-terminal carboxylate group in A beta(12-24) triggered the aggregate formation, and the transition from a random coil to a beta-conformation in the C-terminal region of V18-V24 was detected on analysis of the (3)Ja(N) coupling constant in the pH range of 2.2 to 3.0.     

A comparison of the hairpin stability of the palindromic d(CGCG(A/T)4CGCG) oligonucleotides.

The palindromic deoxyribonucleotides 5'-CGCGA-TATCGCG-3' and 5'-CGCGTTAACGCG-3' have been characterized by 1H NMR spectroscopy. The NMR data identified both B-DNA duplex conformations and hairpin conformations, the latter with loop regions consisting of the four central nucleotides. The resonances of the various conformations were assigned by use of two-dimensional NMR methods. The relative stability of the various conformations was investigated as a function of temperature, ionic strength and nucleotide concentration. The duplexes were found to be stabilized at high ionic strength and at low temperature, while the hairpins were stabilized at low ionic strength and at medium temperature. The thermodynamics of the duplex-hairpin and the hairpin-random coil transitions were examined, and compared to the other two oligonucleotide in the palindromic d(CGCG(A/T)4CGCG) oligonucleotide family. The relative stabilities of the duplex conformations with respect to the random coil conformations are similar for the d(CGCGAATTCGCG), d(CGCGATATCGCG) and d(CGCGTATACGCG) oligonucleotides. The duplex conformation of d(CGCGTTAACGCG) is less stable. The hairpin of d(CGCGTTAACGCG) seems also to be less stable relative to the random coil conformation than in the case of the other oligonucleotides at an equal oligonucleotide concentration. A cruciform intermediate between the duplex and hairpin conformations is suggested to explain some discrepancies observed in this work in case of the d(CGCGTTAACGCG) oligonucleotide. This is similar to what has been reported for the d(CGCGTATACGCG) oligonucleotide.     

Characterizations of distinct amyloidogenic conformations of the Abeta (1-40) and (1-42) peptides

Major constituents of the amyloid plaques found in the brain of Alzheimer's patients are the 39-43 residue beta-amyloid (Abeta) peptides. Extensive in vitro as well as in vivo biochemical studies have shown that the 40- and 42-residue Abeta peptides play major roles in the neurodegenerative pathology of Alzheimer's disease. Although the two Abeta peptides share common aggregation properties, the 42-residue peptide is more amyloidogenic and more strongly associated with amyloid pathology. Thus, characterizations of the two Abeta peptides are of critical importance in understanding the molecular mechanism of Abeta amyloid formation. In this report, we present combined CD and NMR studies of the monomeric states of the two peptides under both non-amyloidogenic (<5 degrees C) and amyloid-forming conditions (>5 degrees C) at physiological pH. Our CD studies of the Abeta peptides showed that initially unfolded Abeta peptides at low temperature (<5 degrees C) gradually underwent conformational changes to more beta-sheet-like monomeric intermediate states at stronger amyloidogenic conditions (higher temperatures). Detailed residue-specific information on the structural transition was obtained by using NMR spectroscopy. Residues in the N-terminal (3-12) and 20-22 regions underwent conformational changes to more extended structures at the stronger amyloidogenic conditions. Almost identical structural transitions of those residues were observed in the two Abeta peptides, suggesting a similar amyloidogenic intermediate for the two peptides. The 42-residue Abeta (1-42) peptide was, however, more significantly structured at the C-terminal region (39-42), which may lead to the different aggregation propensity of the two peptides.     

Hints of nonhierarchical folding of acidic fibroblast growth factor

We have analyzed by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) the helical propensity of the all-beta protein acidic fibroblast growth factor (aFGF) and two peptides corresponding to beta-strand 8 (beta8 peptide, amino acids 95-107) and the beta-strand 8/turn/beta-strand 9 hairpin (beta8/9 peptide, amino acids 95-114), which has been involved in receptor binding. A secondary structure prediction of aFGF carried out by several procedures labels the 95-104 sequence as predominantly alpha-helical. A titration of aFGF with 2,2,2-trifluoroethanol (TFE) induces a change in the far-UV CD spectrum of the protein giving rise to a prominent alpha-helical shape (22% alpha-helix). The cooperativity of the transition and the moderate TFE concentrations used (midpoint at 24%) suggest that the effect of TFE is specific. Moreover, a titration performed at pH 2 yields a higher amount of alpha-helix (55%) at a smaller TFE concentration. Synthetic peptides containing the beta8 and beta8/9 sequences display a random coil conformation at pH 7 but acquire alpha-helical structure in the presence of TFE, methanol, and SDS micelles. At pH below 3.0 a significant amount (20-30%) of alpha-helical conformation is present in both the beta8 and beta8/9 peptides even in the absence of other solvent additives. The secondary structure of the peptides was determined by proton nuclear magnetic resonance (1H NMR). These results suggest that the 95-114 sequence of aFGF has helical propensity and that the protein may fold nonhierarchically in the early steps of folding, acquiring its final beta-structure by a later interaction with the rest of the polypeptide.