A de novo designed helix-turn-helix peptide forms nontoxic amyloid fibrils.

We report here that a monomeric de novo designed alpha-helix-turn-alpha-helix peptide, alpha t alpha, when incubated at 37 degrees C in an aqueous buffer at neutral pH, forms nonbranching, protease resistant fibrils that are 6-10 nm in diameter. These fibrils are rich in beta-sheet and bind the amyloidophilic dye Congo red. alpha t alpha fibrils thus display the morphologic, structural, and tinctorial properties of authentic amyloid fibrils. Surprisingly, unlike fibrils formed by peptides such as the amyloid beta-protein or the islet amyloid polypeptide, alpha t alpha fibrils were not toxic to cultured rat primary cortical neurons or PC12 cells. These results suggest that the potential to form fibrils under physiologic conditions is not limited to those proteins associated with amyloidoses and that fibril formation alone is not predictive of cytotoxic activity.

Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies.

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.)

Dissection of the de novo designed peptide alpha t alpha: stability and properties of the intact molecule and its constituent helices.

Alpha t alpha is a de novo designed 38-residue peptide [Fezoui et al. (1995) Protein Sci. 4, 286-295] that adopts a helical hairpin conformation in solution [Fezoui et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3675-3679; Fezoui et al. (1997) Protein Sci. 6, 1869-1877]. Since alpha t alpha was developed as a model system for protein folding at the stage where secondary structures interact and become mutually stabilizing, it is of interest to investigate the increase in stability that occurs with helix association. alpha t alpha was dissected into its component helices and the relative stabilities of the individual helices and the parent molecule were assessed. The Delta G0 of unfolding of alpha t alpha measured by guanidine hydrochloride denaturation was determined to be 3.4 kcal/mol. The equilibrium constant for folding of alpha t alpha was estimated from the Delta G0 as 338 and from hydrogen exchange measurements as 259. The stability of the helices in intact alpha t alpha over the individual helices increased by a factor of at least 37 based on amide proton exchange measurements. Sedimentation equilibrium studies showed very little association of the peptides to form either homo- or heterodimers, suggesting that helix association is stabilized by the high effective concentration of the helices caused by the presence of the connecting turn. The effects of salt and pH on the helicity of the component peptides are largely reflected in the intact molecule, implying that short-range interactions still make important contributions to the conformation of the intact molecule even though significant stabilization is caused by helix association.

Structure, thermostability, and conformational flexibility of hen egg-white lysozyme dissolved in glycerol.

Hen egg-white lysozyme dissolved in glycerol containing 1% water was studied by using CD and amide proton exchange monitored by two-dimensional 1H NMR. The far- and near-UV CD spectra of the protein showed that the secondary and tertiary structures of lysozyme in glycerol were similar to those in water. Thermal melting of lysozyme in glycerol followed by CD spectral changes indicated unfolding of the tertiary structure with a Tm of 76.0 +/- 0.2 degreesC and no appreciable loss of the secondary structure up to 85 degreesC. This is in contrast to the coincident denaturation of both tertiary and secondary structures with Tm values of 74.8 +/- 0.4 degreesC and 74.3 +/- 0.7 degreesC, respectively, under analogous conditions in water. Quenched amide proton exchange experiments revealed a greater structural protection of amide protons in glycerol than in water for a majority of the slowly exchanging protons. The results point to a highly ordered, native-like structure of lysozyme in glycerol, with the stability exceeding that in water.

Sequence requirements for stabilization of a peptide reverse turn in water solution--proline is not essential for stability.

To probe the sequence requirements for stabilization of a reverse turn conformation in a short peptide in water solution, the behavior of two series of peptides was investigated by nuclear magnetic resonance (NMR) spectroscopy. The peptides have the general sequences XPGDV and AXGDV, where X is a representative subset of all 20 naturally occurring amino acids. The residues chosen at positions 3 and 4, Gly and Asp, respectively, were shown to give the greatest population of reverse turns in a previous study [Dyson, H. J., Rance, M., Houghten, R. A., Lerner, R. A. & Wright, P. E. (1988) J. Mol. Biol. 201, 161-200]. Within this framework, the identity of the first residue of the turn (X in XPGDV) does not greatly influence the turn population, although a small but significant increase is observed for residues such as Ala which have a preference for backbone conformations in the alpha region of (phi,psi) space. The series AXGDV was initially studied for completeness only, since it was expected that the turn would not be stabilized in such a small linear peptide in the absence of proline. In contrast, it appears that a significant population of type II turn conformations is to be found in peptides in the series AXGDV, although proline remains one of the most favorable residues at position 2. These results indicate that while residues at all positions within the turn can influence the turn population, the presence of Gly-Asp as the third and fourth members of the sequence gives a strong bias towards type II turn formation regardless of the residues at positions 1 and 2. Our results give a final prediction that the sequence with the highest intrinsic propensity for turn formation is APGD.

Solution structure of alpha t alpha, a helical hairpin peptide of de novo design.

alpha t alpha is a 38-residue peptide designed to adopt a helical hairpin conformation in solution (Fezoui Y, Weaver DL Osterhout JJ, 1995, Protein Sci 4:286-295). A previous study of the carboxylate form of alpha t alpha by CD and two-dimensional NMR indicated that the peptide was highly helical and that the helices associated in approximately the intended orientation (Fezoui Y, Weaver DL, Osterhout JJ, 1994, Proc Natl Acad Sci USA 91:3675-3679). Here, the solution structure of alpha t alpha as determined by two-dimensional NMR is reported. A total of 266 experimentally derived distance restraints and 20 dihedral angle restraints derived from J-couplings were used. One-hundred initial structures were generated by distance geometry and refined by dynamical simulated annealing. Twenty-three of the lowest-energy structures consistent with the experimental restraints were analyzed. The results presented here show that alpha t alpha is comprised of two associating helices connected by a turn region.

Strategies and rationales for the de novo design of a helical hairpin peptide.

The de novo design of alpha t alpha, a helical hairpin peptide, is described, alpha t alpha (alpha-helix/turn/alpha-helix) was developed to provide a model system for protein folding at the level of secondary structure association and stabilization. According to the prevailing models of protein folding, the second step in the folding process is the association and stabilization of secondary structural elements or microdomains. A brief description of the design, along with CD and NMR evidence confirming the conformation of the peptide in solution, has been published (Fezoui Y, Weaver DL, Osterhout JJ, 1994, Proc Natl Acad Sci USA 91:3675-3679). The present work includes a full description of the design process, including the trade-offs that were made during the development of the peptide, a discussion of recent experimental results that were not available at the time of the original design, indications of areas where, in retrospect, the design might have been done differently, and a discussion of how the present work fits into the field of de novo protein design.

De novo design and structural characterization of an alpha-helical hairpin peptide: a model system for the study of protein folding intermediates.

The de novo design and structural characterization of an alpha-helical hairpin peptide (alpha-helix/turn/alpha-helix, alpha t alpha) are reported. The peptide is intended to provide a model system for the study of the interactions of secondary structural elements during protein folding. Both the diffusion-collision and framework models of protein folding envision that the earliest intermediates in protein folding are transient secondary structures or microdomains which interact and become mutually stabilizing. Design principles for the alpha t alpha peptide were drawn from the large body of work on the structure of peptides in solution. Computer modeling was not used in the design process. Study of alpha t alpha by circular dichroism and two-dimensional nuclear magnetic resonance indicates that the designed peptide is monomeric, helical, and stable in aqueous solution at room temperature. Analysis of two-dimensional nuclear magnetic resonance experiments indicates that the two helices and the turn form in the intended positions and that the helices associate in the designed orientation. Development of alpha t alpha represents an advance in protein design in that both the secondary structural elements and designed tertiary interactions have been realized and can be detected in solution by nuclear magnetic resonance. The resulting model system resembles a protein folding intermediate and will allow the study of interacting helices in a context that approximates an early stage in protein folding.

1H NMR studies of the solution conformations of an analogue of the C-peptide of ribonuclease A.

Two-dimensional NMR experiments have been performed on a peptide, succinyl-AE-TAAAKFLRAHA-NH2, related to the amino-terminal sequence of ribonuclease A. This peptide contains 50-60% helix in 0.1 M NaCl solution, pH 5.2, 3 degrees C, as measured by circular dichroism. NOESY spectra of the peptide in aqueous solution at low temperatures show a number of NOE connectivities that are used to determine the highly populated conformations of the peptide in solution. Short-range dNN(i, i + 1) and d alpha N(i, i + 1) connectivities and medium-range d alpha beta(i, i + 3) and d alpha N(i, i + 3) connectivities are detected. The pattern of NOE connectivities unambiguously establishes the presence of helix in this peptide. The magnitudes of the 3JHN alpha coupling constants and the intensities of the dNN(i, i + 1) and d alpha N(i,i + 1) NOEs allow the evaluation of the position of the helix along the peptide backbone. These data indicate that the amino terminus of the peptide is less helical than the remainder of the peptide. The observation of several long-range NOEs that are atypical of helices indicates the presence of a high population of peptide molecules in which the first three residues are distorted out of the helical conformation. The absence of these NOEs in a related peptide, RN-31, in which Arg 10 has been changed to Ala, suggests that this distortion at the amino-terminal end of the peptide arises from the formation of a salt bridge between Glu 2 and Arg 10.(ABSTRACT TRUNCATED AT 250 WORDS)

pH dependence of folding of iso-2-cytochrome c.

Starting from a standard unfolded state (3.0 M guanidine hydrochloride, pH 7.2), the kinetics of refolding of iso-2-cytochrome c have been investigated as a function of final pH between pH 3 and pH 10. Absorbance in the ultraviolet and visible spectral regions and tryptophan fluorescence are used to monitor folding. Over most of the pH range, fast and slow folding phases are detected by both fluorescence and absorbance probes. Near neutral pH, the rate of fast folding appears to be the same when monitored by absorbance and fluorescence probes. At higher and lower pH, there are two fast folding reactions, with absorbance-detected fast folding occurring in a slightly faster time range than fluorescence-detected fast folding. The rates of both fast folding reactions pass through broad minima near neutral pH, indicating involvement of ionizable groups in rate-limiting steps. The rates of slow folding also depend on the final pH. At acid pH, there appears to be a single slow folding phase for both fluorescence and absorbance probes. At neutral pH, the absorbance-detected and fluorescence-detected slow folding phases separate into distinct kinetic processes which differ in rate and relative amplitude. At high pH, absorbance-detected slow folding is no longer observed, while fluorescence-detected slow folding is decreased in amplitude. In contrast, the equilibrium and kinetic properties of proline imide bond isomerization, believed to be involved in the slow folding reactions, are largely independent of pH. The results suggest that the pH dependence of slow folding involves coupling of pH-sensitive structure to proline imide bond isomerization.(ABSTRACT TRUNCATED AT 250 WORDS)

Slow refolding kinetics in yeast iso-2 cytochrome c.

In refolding of iso-2 cytochrome c from Saccharomyces cerevisiae, there are two slow folding reactions, tau 1a and tau 1b. The slower of the slow reactions, tau 1a = 100-200 s, is observed only by absorbance changes, while tau 1b (10-20-fold faster) is detected by fluorescence changes. The temperature dependence of the rates of these reactions has been measured: for kinetic experiments ending below the folding-unfolding transition zone (pH 7.2, 0.3 M guanidine hydrochloride, 5-30 degrees C), the activation enthalpies are delta H++ = 27 kcal/mol for tau 1a and 21 kcal/mol for tau 1b. Double-jump (unfolding, then refolding) experiments demonstrate that the two sets of species responsible for the slow folding reactions are generated slowly but at different rates under unfolding conditions (3 M guanidine hydrochloride, pH 7.2, 20 degrees C). Finally, as a test for changes in the population of the slow refolding species under different unfolding conditions, the amplitudes for slow refolding have been measured as a function of the initial unfolding conditions with the final refolding conditions held constant. Over the range accessible to measurement in the absence of interference from other reactions, the amplitudes for fluorescence-detected (alpha 1b) and absorbance-detected (alpha 1a) slow folding are independent of guanidine hydrochloride concentration and pH in the initial conditions. Although a full description requires a more complex explanation, many of the properties of the slow folding species are those expected for proline imide bond isomerization.

pH-induced conformation changes and equilibrium unfolding in yeast iso-2 cytochrome c.

The relationship between pH-induced conformational changes in iso-2 cytochrome c from Saccharomyces cerevisiae and the guanidine hydrochloride induced unfolding transition has been investigated. Comparison of equilibrium unfolding transitions at acid, neutral, and alkaline pH shows that stability toward guanidine hydrochloride denaturation is decreased at low pH but increased at high pH. In the acid range the decrease in stability of the folded protein is correlated with changes in the visible spectrum, which indicate conversion to a high-spin heme state--probably involving the loss of heme ligands. The increase in stability at high pH is correlated with a pH-induced conformational change with an apparent pK near 8. As in the case of homologous cytochromes c, this transition involves the loss of the 695-nm absorbance band with only minor changes in other optical parameters. For the unfolded protein, optical spectroscopy and 1H NMR spectroscopy are consistent with a random coil unfolded state in which amino acid side chains serve as (low-spin) heme ligands at both neutral and alkaline pH. However, the paramagnetic region of the proton NMR spectrum of unfolded iso-2 cytochrome c indicates a change in the (low-spin) heme-ligand complex at high pH. Apparently, the folded and unfolded states of the (inactive) alkaline form differ from the corresponding states of the less stable native protein.

Factors from wheat germ that enhance the activity of eukaryotic initiation factor eIF-2. Isolation and characterization of Co-eIF-2 alpha.

Two factors have been isolated from wheat germ that enhance the ability of initiation factor 2(eIF-2) to form a ternary complex with GTP and Met-tRNAf. One of these factors, Co-eIF-2 beta, is a monomeric protein with a molecular weight of approximately 83,000 (Lax, S. R., Osterhout, J.J., and Ravel, J.M. (1982) J. Biol. Chem. 257, 8233-8237). The purification and properties of Co-eIF-2 alpha are described in this report. The most highly purified preparations of Co-eIF-2 alpha contain two polypeptides with molecular weights of 21,000 and 19,000. Both Co-eIF-2 alpha and Co-eIF-2 beta are heat-stable factors that stimulate ternary complex formation in the presence and absence of Mg2+ and overcome the inhibitory effect of aurintricarboxylic acid. Co-eIF-2 alpha differs from Co-eIF-2 beta in that Co-eIF-2 beta stimulates the formation of a binary complex between eIF-2 and GDP and Co-eIF-2 alpha does not. Also. The stimulatory effects of Co-eIF-2 alpha and Co-eIF-2 beta on the ternary complex formation are close to additive, strongly suggesting that the two factors function independently. Neither Co-eIF-2 alpha nor Co-eIF-2 beta enhances the rate of exchange between GDP bound to eIF-2 and free GDP, indicating that neither factor functions as a guanine nucleotide exchange factor.

Factors from wheat germ that enhance the activity of eukaryotic initiation factor eIF-2. Isolation and characterization of Co-eIF-2 beta.

A factor has been isolated from wheat germ that enhances the ability of initiation factor 2 (eIF-2) to form a ternary complex with GTP and Met-tRNAf and enhances the binding of Met-tRNAf to 40 s ribosomal subunits. This factor, designated Co-eIF2 beta, is a monomeric protein with a molecular weight of approximately 83,000. Wheat germ eIF-2 forms a stable binary complex with GDP but not with GTP. Co-eIF-2 beta enhances the formation of an eIF-2 . GDP complex, but does not enable eIF-2 to form a stable complex with GTP.