Α-helical Propensity Research Articles

Characterization of the disordered‐to‐α‐helical transition of IA3 by SDSL‐EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA₃ is used as a model system demonstrating SDSL-EPR characterization of conformational changes in small IDP systems. IA₃ has 68 amino acids, is unstructured in solution, and becomes α-helical upon addition of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE). Two single cysteine substitutions, one in the N-terminus (S14C) and one in the C-terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h((+1))/h(₀), the local tumbling volume (V(L)), and the percent change of the h(₋₁) intensity were utilized to quantitatively monitor TFE-induced conformational changes. The values of h((+1)/)h(₀) as a function of TFE titration varied in a sigmoidal manner and were fit to a two-state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA₃. The other parameters provide site-specific information and show that S14C-SL undergoes a conformational change resulting in more restricted motion than N58C-SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α-helical propensity of the N-terminal segment of IA₃. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured-to-helical conformations in IDPs by SDSL.

Exploring the trigger sequence of the GCN4 coiled‐coil: Biased molecular dynamics resolves apparent inconsistencies in NMR measurements

Trigger sequences are indispensable elements for coiled-coil formation. The monomeric helical trigger sequence of the yeast transcriptional activator GCN4 has been investigated recently using several solution NMR observables including nuclear Overhauser enhancement (NOE) intensities and 3J(HN, HCα)-coupling constants, and a set of 20 model structures was proposed. Constrained to satisfy the NOE-derived distance bounds, the NMR model structures do not appear to reproduce all the measured 3J(HN-HCα)-coupling constant values, indicating that the α-helical propensity is not uniform along the GCN4 trigger sequence. A recent methodological study of unrestrained and restrained molecular dynamics (MD) simulations of the GCN4 trigger sequence in solution showed that only MD simulations incorporating time-averaged NOE distance restraints and instantaneous or local-elevation 3J-coupling restraints could satisfy the entire set of the experimental data. In this report, we assess by means of cluster analyses the model structures characteristic of the two simulations that are compatible with the measured data and compare them with the proposed 20 NMR model structures. Striking characteristics of the MD model structures are the variability of the simulated configurations and the indication of entropic stability mediated by the aromatic N-terminal residues 17Tyr and 18His, which are absent in the set of NMR model structures.

α-Helical nascent polypeptide chains visualized within distinct regions of the ribosomal exit tunnel

As translation proceeds, the nascent polypeptide chain passes through a tunnel in the large ribosomal subunit. Although this ribosomal exit tunnel was once thought only to be a passive conduit for the growing nascent chain, accumulating evidence suggests that it may in fact play a more active role in regulating translation and initial protein folding events. Here we have determined single-particle cryo-electron microscopy reconstructions of eukaryotic 80S ribosomes containing nascent chains with high alpha-helical propensity located within the exit tunnel. The maps enable direct visualization of density for helices as well as allowing the sites of interaction with the tunnel wall components to be elucidated. In particular regions of the tunnel, the nascent chain adopts distinct conformations and establishes specific contacts with tunnel components, both ribosomal RNA and proteins, that have been previously implicated in nascent chain-ribosome interaction.

Designed low amphipathic peptides with α-helical propensity exhibiting antimicrobial activity via a lipid domain formation mechanism

Although several low amphipathic peptides have been known to exhibit antimicrobial activity, their mode of action has not been completely elucidated. In this study, using designed low amphipathic peptides that retain different α-helical content and hydrophobicity, we attempted to investigate the mechanism of these properties. Calorimetric and thermodynamic analyses demonstrated that the peptides induce formation of two lipid domains in an anionic liposome at a high peptide-to-lipid ratio. On the other hand, even at a low peptide-to-lipid ratio, they caused minimal membrane damage, such as flip-flop of membrane lipids or leakage of calcein molecules from liposomes, and never translocated across membranes. Interaction energies between the peptides and anionic liposomes showed good correlation with antimicrobial activity for both Escherichia coli and Bacillus subtilis. We thus propose that the domain formation mechanism in which antimicrobial peptides exhibit activity solely by forming lipid domains without membrane damage is a major determinant of the antimicrobial activity of low amphipathic peptides. These peptides appear to stiffen the membrane such that it is deprived of the fluidity necessary for biological functions. We also showed that to construct the lipid domains, peptides need not form stable and cooperative structures. Rather, it is essential for peptides to only interact tightly with the membrane interface via strong electrostatic interactions, and slight differences in binding strength are invoked by differences in hydrophobicity. The peptides thus designed might pave the way for “clean” antimicrobial reagents that never cause release of membrane elements and efflux of their inner components.

The First N-terminal Amino Acids of α-Synuclein Are Essential for α-Helical Structure Formation In Vitro and Membrane Binding in Yeast

α-Synuclein (α-syn), a protein implicated in Parkinson's disease, is structurally diverse. In addition to its random-coil state, α-syn can adopt an α-helical structure upon lipid membrane binding or a β-sheet structure upon aggregation. We used yeast biology and in vitro biochemistry to detect how sequence changes alter the structural propensity of α-syn. The N-terminus of the protein, which adopts an α-helical conformation upon lipid binding, is essential for membrane binding in yeast, and variants that are more prone to forming an α-helical structure in vitro are generally more toxic to yeast. β-Sheet structure and inclusion formation, on the other hand, appear to be protective, possibly by sequestering the protein from the membrane. Surprisingly, sequential deletion of residues 2 through 11 caused a dramatic drop in α-helical propensity, vesicle binding in vitro, and membrane binding and toxicity in yeast, part of which could be mimicked by mutating aspartic acid at position 2 to alanine. Variants with distinct structural preferences, identified here by a reductionist approach, provide valuable tools for elucidating the nature of toxic forms of α-syn in neurons.

Distinctions between Hydrophobic Helices in Globular Proteins and Transmembrane Segments as Factors in Protein Sorting

Transmembrane (TM) segments in proteins can be distinguished in amino acid sequences as continuous stretches of hydrophobic residues. However, examination of a data base of helical water-soluble (globular) proteins revealed that nearly one-third contained helices of sufficient length to span a bilayer (> or =19 residues) that had mean hydrophobicity > or =actual TM segments. We now report that synthetic peptides corresponding to these globular protein sequences, which we termed "delta-helices," behave like native TM sequences and readily insert into membrane mimetic environments in helical conformations. As well, certain delta-helix sequences can integrate into the membrane bilayer when placed into a membrane-targeted chimeric protein. We establish that delta-helices can be distinguished computationally from bona fide TM segments by the decreased frequency of occurrence of Ile/Val residues and by their relatively decreased solvent accessibilities (versus other globular helices) within tertiary structure. The further observations that (i) delta-helices generally contain three or more charged residues and (ii) delta-helices display relatively even distribution of these charged residues along their lengths, rather than concentration near their N and C termini as observed for TM segments, may constitute key recognition factors in diverting delta-helices from the membrane in vivo. Although a discrete biological role for delta-helices remains to be pinpointed, our overall results suggest that such segments may be required for globular protein folding and identify additional factors that may be important in the correct selection of TM segments by the cellular machinery.

The trigger sequence in the GCN4 leucine zipper: α-helical propensity and multistate dynamics of folding and dimerization

We investigate the importance of the trigger sequence in the folding and dimerization of the GCN4 leucine zipper. We examine the role of the enhanced propensity of the amino acids in the trigger sequence to form an alpha-helix. Using computer simulations, we calculate heat capacities, free energy profiles, and the probability for successful dimerization as a function of the strength of the alpha-helical propensity of the trigger sequence. Our results elucidate the experimentally observed importance of the trigger sequence for dimerization and why it is not necessary for the trigger to have a specific "consensus" sequence of amino acids. We also find that a stronger trigger sequence not only increases the probability for dimerization but also changes the dimerization dynamics by introducing multiple intermediate states.

Search for α-helical propensity in the receptor-bound conformation of glucagon-like peptide-1

To elucidate the receptor-bound conformation of glucagon-like peptide-1 (GLP-1), a series of conformationally constrained GLP-1 analogues were synthesized by introducing lactam bridges between Lys i and Glu i +4 to form α-helices at various positions. The activity and affinity of these analogues to GLP-1 receptors suggested that the receptor-bound conformation comprises two α-helical segments between residues 11-21 and 23-34. It is notable that the N-terminal α-helix is extended to Thr 11, and that Gly 22 plays a pivotal role in arranging the two α-helices. Based on these findings, a highly potent bicyclic GLP-1 analogue was synthesized which is the most conformationally constrained GLP-1 analogue reported to date.

Structural Characterization and Oligomerization of PB1-F2, a Proapoptotic Influenza A Virus Protein

Recently, a novel 87-amino acid influenza A virus protein with proapoptotic properties, PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most known human influenza A virus isolates. Here we characterize the molecular structure of a biologically active synthetic version of the protein (sPB1-F2). Western blot analysis, chemical cross-linking, and NMR spectroscopy afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization mediated by two distinct domains located in the N and C termini, respectively. CD and (1)H NMR spectroscopic analyses indicate that the stability of structured regions in the molecule clearly depends upon the hydrophobicity of the solvent. In aqueous solutions, the behavior of sPB1-F2 is typical of a largely random coil peptide that, however, adopts alpha-helical structure upon the addition of membrane mimetics. (1)H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong alpha-helical propensity comprising amino acid residues Ile(55)-Lys(85) connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp(9) and Lys(20). The C-terminal helix is not a true amphipathic helix and is more compact than previously predicted. It corresponds to a positively charged region previously shown to include the mitochondrial targeting sequence of PB1-F2. The consequences of the strong oligomerization and helical propensities of the molecule are discussed and used to formulate a hypothetical model of its interaction with the mitochondrial membrane.

Semirational design of Jun-Fos coiled coils with increased affinity: Universal implications for leucine zipper prediction and design

Activator protein-1 (AP-1) is a crucial transcription factor implicated in numerous cancers. For this reason, nine homologues of the AP-1 leucine zipper region have been characterized: Fos (c-Fos, FosB, Fra1, and Fra2), Jun (c-Jun, JunB, and JunD), and semirational library-designed winning peptides FosW and JunW. The latter two were designed to specifically target c-Fos or c-Jun. They have been identified by using protein-fragment complementation assays combined with growth competition. This assay removes nonspecific, unstable, and protease susceptible library members from the pool, leaving winners with excellent drug potential. Thermal melts of all 45 possible dimeric interactions have been surveyed, with the FosW-c-Jun complex displaying a melting temperature (T(m)) of 63 degrees C, compared to only 16 degrees C for wild-type c-Fos-c-Jun interaction. This impressive 70,000-fold K(D) decrease is largely due to optimized core packing, alpha-helical propensity, and electrostatics. Contrastingly, due to a poor c-Fos core, c-Fos-JunW dimerizes with lower affinity. However the T(m) far exceeds wild-type c-Fos-c-Jun and averaged JunW and c-Fos, indicating a preference over either homodimer. Finally, and with wider implications, we have compiled a method for predicting interaction of parallel, dimeric coiled coils, using our T(m) data as a training set, and applying it to 59 bZIP proteins previously reported. Our algorithm, unlike others to date, accounts for helix propensity, which is found to be integral in coiled coil stability. Indeed, in applying the algorithm to these 59(2) bZIP interactions, we were able to correctly identify 92% of all strong interactions and 92% of all noninteracting pairs.

Advances in antimicrobial peptide immunobiology

Antimicrobial peptides are ancient components of the innate immune system and have been isolated from organisms spanning the phylogenetic spectrum. Over an evolutionary time span, these peptides have retained potency, in the face of highly mutable target microorganisms. This fact suggests important coevolutionary influences in the host-pathogen relationship. Despite their diverse origins, the majority of antimicrobial peptides have common biophysical parameters that are likely essential for activity, including small size, cationicity, and amphipathicity. Although more than 900 different antimicrobial peptides have been characterized, most can be grouped as belonging to one of three structural classes: (1) linear, often of alpha-helical propensity; (2) cysteine stabilized, most commonly conforming to beta-sheet structure; and (3) those with one or more predominant amino acid residues, but variable in structure. Interestingly, these biophysical and structural features are retained in ribosomally as well as nonribosomally synthesized peptides. Therefore, it appears that a relatively limited set of physicochemical features is required for antimicrobial peptide efficacy against a broad spectrum of microbial pathogens. During the past several years, a number of themes have emerged within the field of antimicrobial peptide immunobiology. One developing area expands upon known microbicidal mechanisms of antimicrobial peptides to include targets beyond the plasma membrane. Examples include antimicrobial peptide activity involving structures such as extracellular polysaccharide and cell wall components, as well as the identification of an increasing number of intracellular targets. Additional areas of interest include an expanding recognition of antimicrobial peptide multifunctionality, and the identification of large antimicrobial proteins, and antimicrobial peptide or protein fragments derived thereof. The following discussion highlights such recent developments in antimicrobial peptide immunobiology, with an emphasis on the biophysical aspects of host-defense polypeptide action and mechanisms of microbial resistance.

A novel dileucine lysosomal-sorting-signal mediates intracellular EGF-receptor retention independently of protein ubiquitylation

One of the main goals of this study was to understand the relationship between an epidermal growth factor (EGF) receptor dileucine (LL)-motif (679-LL) required for lysosomal sorting and the protein ubiquitin ligase CBL. We show that receptors containing 679-AA (di-alanine) substitutions that are defective for ligand-induced degradation nevertheless bind CBL and undergo reversible protein ubiquitylation similar to wild-type receptors. We also demonstrate that 679-LL but not CBL is required for EGF receptor downregulation by an endosomal membrane protein encoded by human adenoviruses that uncouples internalization from post-endocytic sorting to lysosomes. 679-LL is necessary for endosomal retention as well as degradation by the adenovirus protein, and is also transferable to reporter molecules. Using NMR spectroscopy, we show that peptides with wild-type 679-LL or mutant 679-AA sequences both exhibit alpha-helical structural propensities but that this structure is not stable in water. A similar analysis carried out in hydrophobic media showed that the alpha-helical structure of the wild-type peptide is stabilized by specific interactions mediated by side-chains in both leucine residues. This structure distinguishes 679-LL from other dileucine-based sorting-signals with obligatory amino-terminal acidic residues that are recognized in the form of an extended beta or beta-like conformation. Taken together, these data show that 679-LL is an alpha-helical stabilizing motif that regulates a predominant step during lysosomal sorting, involving intracellular retention under both sub-saturating and saturating conditions.

The α-Helical Propensity of the Cytoplasmic Domain of Phospholamban: A Molecular Dynamics Simulation of the Effect of Phosphorylation and Mutation

We have used molecular dynamics simulations to investigate the effect of phosphorylation and mutation on the cytoplasmic domain of phospholamban (PLB), a 52-residue protein that regulates the calcium pump in cardiac muscle. Simulations were carried out in explicit water systems at 300 K for three peptides spanning the first 25 residues of PLB: wild-type (PLB(1-25)), PLB(1-25) phosphorylated at Ser16 and PLB(1-25) with the R9C mutation, which is known to cause human heart disease. The unphosphorylated peptide maintains a helical conformation from 3 to 15 throughout a 26-ns simulation, in agreement with spectroscopic data. Comparison with simulations of a fourth peptide truncated at Pro21 showed the importance of the region from 17 to 21 in preventing local unfolding of the helix. The results suggest that residues 11-16 are more likely to unfold when specific capping motifs are not present. It is proposed that protein kinase A exploits the intrinsic flexibility of the 11-21 region when binding PLB. In agreement with available CD and NMR data, the simulations show a decrease in the helical content upon phosphorylation. The phosphorylated peptide is characterized by helix spanning residues 3-11, followed by a turn that optimizes the salt-bridge interaction between the side chains of the phosphorylated Ser-16 and Arg-13. Replacing Arg-9 with Cys results in unfolding of the helix from C9 and an overall decrease of the helical conformation. The simulations show that initiation of unfolding is due to increased solvent accessibility of the backbone atoms near the smaller Cys. It is proposed that the loss of inhibitory potency upon Ser-16 phosphorylation or R9C mutation of PLB is due to a similar mechanism, in which the partial unfolding of the cytoplasmic helix of PLB results in a conformation that interacts with the cytoplasmic domain of the calcium pump to relieve its inhibition.

Detection and characterization of an acid-induced folding intermediate of endostatin

Endostatin, an important angiogenesis inhibitor, is very acid resistant. We are particularly interested in knowing that whether or not endostatin can form a folding intermediate during acid titration. 1H-NMR, CD spectrum, and ANS binding assay show that endostatin at pH 2.0 contains little tertiary structure, but retains substantial secondary structure with strong ANS binding, and Na 2SO 4 or TFE is found to strongly stabilize endostatin at pH 2.0. All these observations are consistent with the formation of a folding intermediate at pH 2.0. Kinetics studies show that sulfate anions significantly slow down the process for endostatin to reach its equilibrium state at pH 2.0. A regular increase in the amount of α-helix content of the intermediate of endostatin at pH 2.0 is found when the concentration of TFE is increased in the range of 0–40%, suggesting that endostatin has an intrinsic α-helical propensity.

The C-terminal domain of measles virus nucleoprotein belongs to the class of intrinsically disordered proteins that fold upon binding to their physiological partner

The nucleoprotein of measles virus consists of an N-terminal domain, N CORE (aa 1–400), resistant to proteolysis, and a C-terminal domain, N TAIL (aa 401–525), hypersensitive to proteolysis and not visible by electron microscopy. Using two complementary computational approaches, we predict that N TAIL belongs to the class of natively unfolded proteins. Using different biochemical and biophysical approaches, we show that N TAIL is indeed unstructured in solution. In particular, the spectroscopic and hydrodynamic properties of N TAIL indicate that this protein domain belongs to the premolten globule subfamily within the class of intrinsically disordered proteins. The isolated N TAIL domain was shown to be able to bind to its physiological partner, the phosphoprotein (P), and to undergo an induced folding upon binding to the C-terminal moiety of P [J. Biol. Chem. 278 (2003) 18638]. Using a computational analysis, we have identified within N TAIL a putative α-helical molecular recognition element (α-MoRE, aa 488–499), which could be involved in binding to P via induced folding. We report the bacterial expression and purification of a truncated form of N TAIL (N TAIL2, aa 401–488) devoid of the α-MoRE. We show that N TAIL2 has lost the ability to bind to P, thus supporting the hypothesis that the α-MoRE may play a role in binding to P. We have further analyzed the α-helical propensities of N TAIL2 and N TAIL using circular dichroism in the presence of 2,2,2-trifluoroethanol. We show that N TAIL2 has a lower α-helical potential compared to N TAIL, thus suggesting that the α-MoRE may be indeed involved in the induced folding of N TAIL.

Conformational Polymorphism of the Amyloidogenic Peptide Homologous to Residues 113–127 of the Prion Protein

Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of “conformation switching” facilitated by structured “seeds” consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113–127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113–127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113–127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the α-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113–127), CD studies on another synthetic peptide, PrP (113–120) were carried out. PrP(113–120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for β-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113–127) has been investigated. At higher concentration, PrP (113–127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113–127) takes high α-helical propensity. The environment-dependent conformational polymorphism of PrP (113–127) and its marked tendency to form stable β-sheet structure at acidic pH could account for its conformation switching behavior from α-helix to β-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113–127).

Are trigger sequences essential in the folding of two-stranded α-helical coiled-coils?

The amino acid residues comprising the interface between strands of the coiled-coil motif are usually hydrophobic and make a major contribution to coiled-coil folding and stability. However, in some cases the presence of excellent hydrophobic residues at the coiled-coil interface is insufficient for folding. It has been proposed that a “consensus trigger sequence” exists that is necessary within the coiled-coil domains of various proteins to trigger folding. Therefore, in this study we designed a 31-residue hybrid sequence based on sequences from the two-stranded parallel coiled-coil domains of the yeast transcriptional activator GCN4 and the actin-bundling protein Dictyostelium discoideum cortexillin I. The hybrid and its analogs were studied by CD spectroscopy and analytical ultracentrifugation. The hybrid had stable residues in the core “a” and “d” positions in the 3–4 hydrophobic repeat, denoted (abcdefg)n, but did not have a consensus trigger sequence and did not possess appreciable secondary structure as determined by CD spectroscopy. The substitutions in the parent peptide were introduced at positions other than “a” and “d”, altering a variety of interactions including α-helical propensity, interchain and intrachain electrostatics, and hydrophobicity. Although the substitutions did not bring the overall sequence in closer agreement to the consensus trigger sequence, they increased coiled-coil folding and stability. Therefore, our results suggest that the combination of stabilizing effects along a protein sequence is a more general indicator of protein folding in coiled-coils than the identification of a specific trigger sequence. We propose that surpassing a critical threshold stability value using any type or combination of stabilizing effects will allow coiled-coils to fold, in the absence of a specific trigger sequence per se.

Structures of thermolabile mutants of human glutathione transferase P1-1

An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix α6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the α-helical propensity of the isolated α6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37°C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.

The Helix-Destabilizing Propensity Scale ofd-Amino Acids: The Influence of Side Chain Steric Effects

Although d-amino acids are found in various naturally occurring peptides and frequently used for structure−activity studies, not much is known about their impact on the helical secondary structures formed by l-amino acids. Although several previous accounts reported on the α-helical propensity of l-amino acids, the present contribution addresses this subject for the first time for the corresponding d-enantiomers of all of the proteinogenic amino acids. Thus, the helix-destabilizing abilities of the 19 d-amino acids in the host sequence acetyl-KLALKLALxxLKLALKLA-amide (x9,10-KLA) were evaluated by means of circular dichroism (CD) spectroscopy, nuclear magnetic resonance, and reversed-phase HPLC. CD and HPLC data enabled calculation of differences in the free energy of helix formation for d-amino acid x relative to glycine (ΔΔGt) or to the corresponding l-amino acid (ΔΔGd-l). The data show that the helix-destabilizing propensity is highly dependent on the amino acid side chain and not related to the structu...

The p3 Peptide, a Naturally Occurring Fragment of the Amyloid-β Peptide (Aβ)Found in Alzheimer's Disease, Has a Greater Aggregation Propensity In Vitro Than Full-Length Aβ, But Does Not Bind Cu2+.

Cerebellar preamyloid from both Down’s syndrome and Alzheimer’s disease contains the p3 fragment (Aβ 17–40/42) as a major amyloid-β peptide (Aβ) component. The p3 peptide was previously shown to form amyloid in vitro, but less readily than full-length Aβ. Here we show that the p3 peptide has a greater β-sheet-forming propensity than full-length Aβ. Using circular dichroism spectroscopy we determined that in aqueous solutions the p3 peptide forms β-sheet structure more readily than full-length Aβ. The p3 peptide also has a lower α-helical propensity than full-length Aβ in the structure-forming solvent trifluoroethanol. These results indicate that the lower amyloidogenicity of the p3 peptide is not related to an inability to form β-sheet structure. In this study we also show that, unlike full-length Aβ, the p3 peptide does not bind Cu2+ ions. This inability to bind copper ions may explain why the p3 peptide appears to play a lesser role in Down’s syndrome and Alzheimer’s disease related neurodegeneration than does full-length Aβ.