Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of Human Carbonic Anhydrase-II

In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII.     

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.     

Structure and alignment of the membrane-associated antimicrobial peptide arenicin by oriented solid-state NMR spectroscopy

The antimicrobial arenicin peptides are cationic amphipathic sequences that strongly interact with membranes. Through a cystine ring closure a cyclic β-sheet structure is formed in aqueous solution, which persists when interacting with model membranes. In order to investigate the conformation, interactions, dynamics, and topology of their bilayer-associated states, arenicin 1 and 2 were prepared by chemical solid-phase peptide synthesis or by bacterial overexpression, labeled selectively or uniformly with (15)N, reconstituted into oriented membranes, and investigated by proton-decoupled (31)P and (15)N solid-state NMR spectroscopy. Whereas the (31)P NMR spectra indicate that the peptide induces orientational disorder at the level of the phospholipid head groups, the (15)N chemical shift spectra agree well with a regular β-sheet conformation such as the one observed in micellar environments. In contrast, the data do not fit the twisted β-sheet structure found in aqueous buffer. Furthermore, the chemical shift distribution is indicative of considerable conformational and/or topological heterogeneity when at the same time the (15)N NMR spectra exclude alignments of the peptide where the β-sheet lies side ways on the membrane surface. The ensemble of experimental constraints, the amphipathic character of the peptide, and in particular the distribution of the six arginine residues are in agreement with a boatlike dimer structure, similar or related to the one observed in micellar solution, that floats on the membrane surface with the possibility to oligomerize into higher order structures and/or to insert in a transmembrane fashion.     

The helix-to-sheet transition of an HIV-1 fusion peptide derivative changes the mechanical properties of lipid bilayer membranes

A short sequence on the gp41 envelope protein of HIV-1 is integral to infection by the virus. Without this sequence, termed the fusion peptide (FP), the virus is far less effective at fusing with the cellular membrane. One of the interesting features of the isolated FP is that it transitions between an α-helical conformation and a β-sheet conformation in lipid bilayer membranes as a function of lipid composition and concentration, and the transition correlates with fusion. To better understand how the conformations of the FP impact lipid bilayer membranes, a variant of the FP that does not strongly promote fusion, termed gp41rk, was studied. Circular dichroism spectroscopy, dynamic light scattering, small-angle neutron scattering (SANS) and neutron spin echo spectroscopy (NSE) were used to relate the conformation of gp41rk to the structure and mechanical properties of lipid bilayer membrane vesicles composed of a 7:3 molar ratio mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol). At a peptide-to-lipid ratio (P/L) of 1/200, it adopts an α-helical conformation, while gp41rk is a β-sheet at a P/L of 1/50 in the unilamellar vesicles. SANS reveals that the lipid bilayer membrane becomes thicker when gp41rk adopts a β-sheet conformation, which indicates that the high-concentration state of the peptide increases the order of the lipid acyl chains. At the same time, NSE demonstrates that the bilayer becomes more rigid, demonstrating that the β-sheet conformation, which correlates with fusion for the native FP sequence, stiffens the bilayer. The results have implications for the function of the FP.     

Isotope-edited vibrational circular dichroism study reveals a flexible N-terminal structure of islet amyloid peptide (NFGAIL) in amyloid fibril form: A site-specific local structural analysis

The short peptide fragment NFGAIL (IAPf) is a well-known amyloidogenic peptide (22–27), derived from human islet amyloid polypeptide(hIAPP), whose fibrillar structure is often used to better understand the wild-type hIAPP amyloid fibrils, associated with type II diabetes. Despite an extensive study, the fibrillar structure of IAPf at the amino acid residue level is still unclear. Herein, the vibrational circular dichroism(VCD) spectroscopic technique coupled with isotope labelling strategy has been used to study the site-specific local structure of IAPf amyloid fibrils. Two ¹³C labeled IAPfs were designed and used along with unlabelled IAPf to achieve this. The ¹³C labelled (on -C=O) glycine(IAPf-G) and phenylalanine (IAPf-F) residues were introduced into the IAPf sequence separately by replacing natural glycine (residue 24) and phenylalanine (residue 23), respectively. VCD spectral analysis on IAPf-G suggests that IAPf fibrils adopt parallel β-sheet conformation with glycine residues are part of β-sheet and in-register. Unlike IAPf-G, VCD analysis on IAPf-F reveals that phenylalanine residues exist in the turn/hairpin conformation rather than β-sheet region. Both VCD results thus suggest that IAPf amyloid fibril consists of a mixture of β-sheet as a major conformation involving GAIL and turn/hairpin as a minor conformation involving NF rather than an idealized β-sheet involving all the amino acids. While previous studies speculated that the full NFGAIL sequence could participate in the β-sheet formation, the present site-specific structural analysis of IAPf amyloid fibrils at residue level using isotope-edited VCD has gained significant attention. Such residue level information has important implications for understanding the role of NFGAIL sequence in the amyloid fibrillation of hIAPP.     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.     

天花粉蛋白小结构域N端同源片段的溶液构象：T18肽的圆二色性和荧光研究

应用圆二色性,内源荧光和疏水荧光探针法进一步研究Ｔｌ８肽的溶液构象及其相互转化。发现Ｔ１８肽在水溶液中为β折叠结构,且在高浓度（＞１ｍｇ／ｍｌ）时形成疏水聚合物;Ｌｙｓ１５和Ｉｌｅ３－Ｉｌｅ４是形成β折叠疏水簇的关键因素。并讨论了蛋白质肽链和溶剂环境对肽段二级结构的调制作用。     

High-resolution structure of a self-assembly-competent form of a hydrophobic peptide captured in a soluble beta-sheet scaffold

β-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide self-assemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer β-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that a penta-Ile peptide (Ile₅), which is insoluble and forms β-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of self-assembly. The 1.1-Å crystal structure revealed that the Ile₅ stretch forms a highly regular β-strand within this flat β-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile₅ peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat β-strand of the Ile₅ stretch primed for self-assembly is a low-energy conformation of the Ile₅ stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to “solubilize” an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides.     

Structural and biochemical characterization of Staphylococcus aureus clumping factor B/ligand interactions

Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg α-chain-derived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel β-sheet complementation as observed for the clumping factor A·γ-chain peptide complex. The β-sheet complementation is shorter in the ClfB·Fg α-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.     

Self-replicating protein conformations and information transfer: The adaptive β-sheet model

The deposition of proteins with a highly ordered β-sheet conformation is characteristic of several amyloid diseases. However, recent data indicate that amyloid formation is not always pathological, but may also be physiological. Based on the new insights in understanding the structural arrangements and the self-propagating properties of the β-conformational proteins in combination with the progress in elucidating the mechanisms of prion genetics and chaperone-controlled protein folding, a model of an adaptive β-sheet information system was developed. The idea is advanced that exogenous information can be specifically stored in the protease-resistant β-sheet rich protein aggregates in a prion-like mode creating a cytoplasmic molecular memory. The conformationally-bound information can then be transmitted to next cell generations by way of the self-propagating potential of the amyloidogenic structure. Controlled by a network of input-sensitive molecular chaperones, the β-conformation based information system could constitute a form of soft inheritance characterized by adaptability and plasticity. It is suggested that the β-system represents an evolutionary conserved primordial inheritance mechanism based on protein conformation.     

Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP^C) into a disease associated form (PrP^Sc). Recombinant PrP can be refolded into either an α-helical rich conformation (α-PrP) resembling PrP^C or a β-sheet rich, protease resistant form similar to PrP^Sc. Here, we generated tetracysteine tagged recombinant PrP, folded this into α- or β-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished β-PrP from α-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the α-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the β-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP^Sc from PrP^C. This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer’s, Huntington’s and Parkinson’s diseases.     

Role of β-Hairpin Formation in Aggregation: The Self-Assembly of the Amyloid-β(25-35) Peptide

The amyloid-β(25-35) peptide plays a key role in the etiology of Alzheimer's disease due to its extreme toxicity even in the absence of aging. Because of its high tendency to aggregate and its low solubility in water, the structure of this peptide is still unknown. In this work, we sought to understand the early stages of aggregation of the amyloid-β(25-35) peptide by conducting simulations of oligomers ranging from monomers to tetramers. Our simulations show that although the monomer preferentially adopts a β-hairpin conformation, larger aggregates have extended structures, and a clear transition from compact β-hairpin conformations to extended β-strand structures occurs between dimers and trimers. Even though β-hairpins are not present in the final architecture of the fibril, our simulations indicate that they play a critical role in fibril growth. Our simulations also show that β-sheet structures are stabilized when a β-hairpin is present at the edge of the sheet. The binding of the hairpin to the sheet leads to a subsequent destabilization of the hairpin, with part of the hairpin backbone dangling in solution. This free section of the peptide can then recruit an extra monomer from solution, leading to further sheet extension. Our simulations indicate that the peptide must possess sufficient conformational flexibility to switch between a hairpin and an extended conformation in order for β-sheet extension to occur, and offer a rationalization for the experimental observation that overstabilizing a hairpin conformation in the monomeric state (for example, through chemical cross-linking) significantly hampers the fibrillization process.     

Characterization of amyloidogenic intermediate states through a combined use of CD and NMR spectroscopy

Characterization of amyloidogenic intermediate states is of central importance in understanding the molecular mechanism of amyloid formation. In this study, we utilized CD and NMR spectroscopy to investigate secondary structure of the monomeric amyloidogenic intermediate of a β-structured SH3 domain, which was induced by trifluoroethanol (TFE). The combined biophysical studies showed that the native state SH3 domain is gradually converted to the amyloidogenic intermediate state at TFE concentrations of 20-26% (v/v) and the aggregation-prone state contains substantial amount of the β-sheet conformation (∼ 30%) with disordered (54%) and some helical characters (16%). Under weaker amyloidogenic conditions of higher TFE concentrations (> 40%), the β-sheet structures were gradually changed to helical conformations and the relative content of the helical and β-sheet conformations was highly correlated with the aggregation propensity of the SH3 domain. This indicates that the β-sheet characters of the amyloidogenic states may be critical to the effective amyloid formation.     

Membrane interactions of a self-assembling model peptide that mimics the self-association,structure and toxicity of Aβ(1–40)

β-amyloid peptide (Aβ) is a primary protein component of senile plaques in Alzheimer's disease (AD) and plays an important, but not fully understood role in neurotoxicity. Model peptides with the demonstrated ability to mimic the structural and toxicity behavior of Aβ could provide a means to evaluate the contributions to toxicity that are common to self-associating peptides from many disease states. In this work, we have studied the peptide–membrane interactions of a model β-sheet peptide, P_11-2 (CH₃CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH₂), by fluorescence, infrared spectroscopy, and hydrogen–deuterium exchange. Like Aβ(1–40), the peptide is toxic, and conditions which produce intermediate oligomers show higher toxicity against cells than either monomeric forms or higher aggregates of the peptide. Further, P_11-2 also binds to both zwitterionic (POPC) and negatively charged (POPC:POPG) liposomes, acquires a partial β-sheet conformation in presence of lipid, and is protected against deuterium exchange in the presence of lipids. The results show that a simple rationally designed model β-sheet peptide recapitulates many important features of Aβ peptide structure and function, reinforcing the idea that toxicity arises, at least in part, from a common mode of action on membranes that is independent of specific aspects of the amino acid sequence. Further studies of such well-behaved model peptide systems will facilitate the investigation of the general principles that govern the molecular interactions of aggregation-prone disease-associated peptides with cell and/or membrane surfaces.     

The surface of β-sheet proteins contains amphiphilic regions which may provide clues about protein folding

A major bottleneck in the field of biochemistry is our limited understanding of the processes by which a protein folds into its native conformation. Much of the work on this issue has focused on the conserved core of the folded protein. However, one might imagine that a ubiquitous motif for unaided folding or for the recognition of chaperones may involve regions on the surface of the native structure. We explore this possibility by an analysis of the spatial distribution of regions with amphiphilic α-helical potential on the surface of β-sheet proteins. All proteins, Including β-sheet proteins, contain regions with amphiphilic α-helical potential. That is, any α-helix formed by that region would be amphiphilic, having both hydrophobic and hydrophilic surfaces. In the three-dimensional structure of all β-sheet proteins analyzed, we have found a distinct pattern in the spatial distribution of sequences with amphiphilic α-helical potential. The amphiphilic regions occur in ring shaped clusters approximately 20 to 30 Å in diameter on the surface of the protein. In addition, these regions have a strong preference for positively charged amino acids and a lower preference for residues not favorable to α-helix formation. Although the purpose of these amphiphilic regions which are not associated with naturally occurring α-helix is unknown, they may play a critical role in highly conserved processes such as protein folding. © 1996 Wiley-Liss, Inc.     

Synthesis and β-sheet propensity of constrained N-amino peptides

The stabilization of β-sheet secondary structure through peptide backbone modification represents an attractive approach to protein mimicry. Here, we present strategies toward stable β-hairpin folds based on peptide strand N-amination. Novel pyrazolidinone and tetrahydropyridazinone dipeptide constraints were introduced via on-resin Mitsunobu cyclization between α-hydrazino acid residues and a serine or homoserine side chain. Acyclic and cyclic N-amino peptide building blocks were then evaluated for their effect on β-hairpin stability in water using a GB1-derived model system. Our results demonstrate the strong β-sheet stabilizing effect of the peptide N-amino substituent, and provide useful insights into the impact of covalent dipeptide constraint on β-sheet folding.     

Direct observations of shifts in the β-sheet register of a protein-peptide complex using explicit solvent simulations

Using explicit solvent molecular dynamics simulations, we were able to obtain direct observations of shifts in the hydrogen-bonding register of an intermolecular β-sheet protein-peptide complex. The β-sheet is formed between the FHA domain of cancer marker protein Ki67 (Ki67FHA) and a peptide fragment of the hNIFK signaling protein. Potential encounter complexes of the Ki67FHA receptor and hNIFK peptide are misregistered states of the β-sheet. Rearrangements of one of these misregistered states to the native state were captured in three independent simulations. All three rearrangements occurred by a common mechanism: an aromatic residue of the peptide (F263) anchors into a transient hydrophobic pocket of the receptor to facilitate the formation of native hydrogen bonds. To our knowledge, these simulations provide the first atomically detailed visualizations of a mechanism by which nature might correct for errors in the alignment of intermolecular β-sheets.     

Redesign of a WW domain peptide for selective recognition of single-stranded DNA

A β-sheet miniprotein based on the FBP11 WW1 domain sequence has been redesigned for the molecular recognition of ssDNA. A previous report showed that a β-hairpin peptide dimer, (WKWK)(2), binds ssDNA with low micromolar affinity but with little selectivity over duplex DNA. This report extends those studies to a three-stranded β-sheet miniprotein designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows about 10-fold selectivity for ssDNA over duplex DNA. The redesigned peptide no longer binds its native ligand, the polyproline helix, confirming that the peptide has been redesigned for the function of binding ssDNA. Structural studies provide evidence that this peptide consists of a well-structured β-hairpin made of strands 2 and 3 with a less structured first strand that provides affinity for ssDNA but does not improve the stability of the full peptide. These studies provide insight into protein-DNA interactions as well as a novel example of protein redesign.     

A new approach to secondary structure evaluation: Secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments

A new approach for evaluating the secondary structure of proteins by CD spectroscopy of overlapping peptide segments is applied to porcine adenylate kinase (AK1) and yeast guanylate kinase (GK3). One hundred seventy-six peptide segments of a length of 15 residues, overlapping by 13 residues and covering the complete sequences of AK1 and GK3, were synthesized in order to evaluate their secondary structure composition by CD spectroscopy. The peptides were prepared by solid phase multiple peptide synthesis method using the 9-fluorenylmethoxycarbonyl/tert-butyl strategy. The individual peptide secondary structures were studied with CD spectroscopy in a mixture of 30% trifluoroethanol in phosphate buffer (pH 7) and subsequently compared with x-ray data of AK1 and GK3. Peptide segments that cover α-helical regions of the AK1 or GK3 sequence mainly showed CD spectra with increasing and decreasing Cotton effects that were typical for appearing and disappearing α-helical structures. For segments with dominating β-sheet conformation, however, the application of this method is limited due to the stability and clustering of β-sheet segments in solution and due to the difficult interpretation of random-coiled superimposed β-sheet CD signals. Nevertheless, the results of this method especially for α-helical segments are very impressive. All α-helical and 71% of the β-sheet containing regions of the AK1 and GK3 could be identified. Moreover, it was shown that CD spectra of consecutive peptide content reveal the appearance and disappearance of α-helical secondary structure elements and help localizing them on the sequence string. © 1997 John Wiley & Sons, Inc. Biopoly 41: 213–231, 1997     

Conformational structure of bombesin as studied by vibrational and circular dichroism spectroscopy

Raman and Fourier transform infrared (FTIR) spectroscopies and circular dichroism (CD) have been applied to investigate the secondary structure of bombesin in the solid state and in phosphate buffer solution (pH 3.8). At concentrations around 10⁻⁵ M, circular dichroism reveals that bombesin exists as an irregular or disordered conformation. However, the secondary structure of the peptide appears to be a mixture of disordered structure and intermolecular β-sheets in 0.01 M sodium phosphate buffer when the peptide concentrations are higher than around 6.5 mM. The tendency of bombesin to form aggregated β-sheet species seems to be originated mainly in the sequence of the residues 7–14, as supported by the Raman spectra and β-sheet propensities (P_β) of the amino-acid residues. It is the hydrophobic force of this amino-acid sequence, and not a salt bridge effect, that is the factor responsible for the formation of peptide aggregates.