Reversible transition between α-helix and β-sheet conformation of a transmembrane domain

Despite the important functions of protein transmembrane domains, their structure and dynamics are often scarcely known. The SNARE proteins VAMP/synaptobrevin and syntaxin 1 are implicated in membrane fusion. Using different spectroscopic approaches we observed a marked sensitivity of their transmembrane domain structure in regard to the lipid/peptide ratio. In the dilute condition, peptides corresponding to the complete transmembrane domain fold into an α-helix inserted at ∼ 35° to the normal of the membranes, an observation in line with molecular simulations. Upon an increase in the peptide/lipid ratio, the peptides readily exhibited transition to β-sheet structure. Moreover, the insertion angle of these β-sheets increased to 54° and was accompanied by a derangement of lipid acyl chains. For both proteins the transition from α-helix to β-sheet was reversible under certain conditions by increasing the peptide/lipid ratio. This phenomenon was observed in different model systems including multibilayers and small unilamellar vesicles. In addition, differences in peptide structure and transitions were observed when using distinct lipids (DMPC, DPPC or DOPC) thus indicating parameters influencing transmembrane domain structure and conversion from helices to sheets. The putative functional consequences of this unprecedented dynamic behavior of a transmembrane domain are discussed.     

Denaturation of β-sheet structure

A two-dimensional Ising model is used to study the thermal denaturation of parallel β-sheet structures in biomolecules.The fraction of intact hydrogen bonds and the excess heat capacity are evaluated as a function of the temperature.     

β-sheet to α-helix conversion and thermal stability of β-Galactosidase encapsulated in a nanoporous silica gel

The effect on protein conformation and thermal stability was studied for β-Galactosidase (β-Gal) encapsulated in the nanopores of a silicate matrix (E_β-Gal). Circular dichroism spectra showed that, compared with the enzyme in buffer (S_β-Gal), E_β-Gal exhibited a higher content of α-helix structure. Heating E_β-Gal up to 75?°C caused a decrease in the content of β-sheet structure and additional augments on E_β-Gal components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with S_β-Gal. Steady state fluorescence spectroscopy analysis evidenced an E_β-Gal structure less compact and more accessible to solvent and also less stable against temperature increase. While for S_β-Gal the denaturation midpoint (Tm) was 59?°C, for E_β-Galit was 48?°C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65?°C and E_β-Gal did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changes on the tertiary/cuaternary structure of E_β-Gal leading to the loss of thermal stability and enzymatic activity.     

Unfolding and partial refolding of a cellulase from the SDS-denatured state: From β-sheet to α-helix and back

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C₁₂E₈). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C₁₂E₈ leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C₁₂E₈ were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3–4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C₁₂E₈ recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C₁₂E₈ was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.     

Studies on the rules of β-strand alignment in a protein β-sheet structure

To further disclose the underlying mechanisms of protein β-sheet formation, studies were made on the rules of β-strands alignment forming β-sheet structure using statistical and machine learning approaches. Firstly, statistical analysis was performed on the sum of β-strands between each β-strand pairs in protein sequences. The results showed a propensity of near-neighbor pairing (or called “first come first pair”) in the β-strand pairs. Secondly, based on the same dataset, the pairwise cross-combinations of real β-strand pairs and four pseudo-β-strand contained pairs were classified by support vector machine (SVM). A novel feature extracting approach was designed for classification using the average amino acid pairing encoding matrix (APEM). Analytical results of the classification indicated that a segment of β-strand had the ability to distinguish β-strands from segments of α-helix and coil. However, the result also showed that a β-strand was not strongly conserved to choose its real partner from all the alternative β-strand partners, which was corresponding with the ordination results of the statistical analysis each other. Thus, the rules of “first come first pair” propensity and the non-conservative ability to choose real partner, were possible important factors affecting the β-strands alignment forming β-sheet structures.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

Minimal model systems for β-sheet secondary structure in proteins

Use of model systems to explore the forces that control β sheet formation was stymied for many years by the perception that small increments of β sheet necessarily aggregate. Recently, however, a number of short peptides (9–16 residues in length) that fold into two-stranded antiparallel β sheets (‘β hairpins’) have been reported; several short peptides (20–24 residues in length) that fold into three-stranded antiparallel β sheets have also been described. These model systems are beginning to provide fundamental insights into the origins of β sheet conformational stability.     

Pressure-dependent changes in the structure of the melittin α-helix determined by NMR

A novel method is described, which uses changes in NMR chemical shifts to characterise the structural change in a protein with pressure. Melittin in methanol is a small -helical protein, and its chemical shifts change linearly and reversibly with pressure between 1 and 2000 bar. An improved relationship between structure and HN shift has been calculated, and used to drive a molecular dynamics-based calculation of the change in structure. With pressure, the helix is compressed, with the H—O distance of the NH—O=C hydrogen bonds decreased by 0.021 ± 0.039 Å, leading to an overall compression along the entire helix of about 0.4 Å, corresponding to a static compressibility of 6 ×10^–6 bar^–1. The backbone dihedral angles and are altered by no more than ± 3° for most residues with a negative correlation coefficient of –0.85 between _i and _i–1, indicating that the local conformation alters to maintain hydrogen bonds in good geometries. The method is shown to be capable of calculating structural change with high precision, and the results agree with structural changes determined using other methodologies.     

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.     

The paradox of MHC-DRB exon/intron evolution: α-helix and β-sheet encoding regions diverge while hypervariable intronic simple repeats coevolve with β-sheet codons

Twenty-one different caprine and 13 ovine MHC-DRB exon 2 sequences were determined including part of the adjacent introns containing simple repetitive (gt)_n(ga)_m elements. The positions for highly polymorphic DRB amino acids vary slightly among ungulates and other mammals. From man and mouse to ungulates the basic (gt)_n(ga)_m structure is fixed in evolution for 7 × 10⁷ years whereas ample variations exist in the tandem (gt)_n and (ga)_m dinucleotides and especially their degenerated derivatives. Phylogenetic trees for the -helices and -pleated sheets of the ungulate DRB sequences suggest different evolutionary histories. In hoofed animals as well as in humans DRB -sheet encoding sequences and adjacent intronic repeats can be assembled into virtually identical groups suggesting coevolution of noncoding as well as coding DNA. In contrast a-helices and C-terminal parts of the first DRB domain evolve distinctly. In the absence of a defined mechanism causing specific, site-directed mutations, double-recombination or gene-conversion-like events would readily explain this fact. The role of the intronic simple (gt)_n(ga)_m repeat is discussed with respect to these genetic exchange mechanisms during evolution.     

Aromatic cluster mutations produce focal modulations of β-sheet structure

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain “clusters” at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design.     

Application of protein knockdown strategy targeting β-sheet structure to multiple disease-associated polyglutamine proteins

Polyglutamine diseases are a class of neurodegenerative diseases associated with the accumulation of aggregated mutant proteins. We previously developed a class of degradation-inducing agents targeting the β-sheet-rich structure typical of such aggregates, and we showed that these agents dose-, time-, and proteasome-dependently decrease the intracellular level of mutant huntingtin with an extended polyglutamine tract, which correlates well with the severity of Huntington’s disease. Here, we demonstrate that the same agents also deplete other polyglutamine disease-related proteins: mutant ataxin-3 and ataxin-7 in cells from spino-cerebellar ataxia patients, and mutant atrophin-1 in cells from dentatorubral-pallidoluysian atrophy patients. Targeting cross-β-sheet structure could be an effective design strategy to develop therapeutic agents for multiple neurodegenerative diseases.     

Toxic prefibrillar α-synuclein amyloid oligomers adopt a distinctive antiparallel β-sheet structure

Parkinson's disease is an age-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-amino-acid protein AS (α-synuclein). AS fibrillation follows a nucleation polymerization pathway involving diverse transient prefibrillar species varying in size and morphology. Similar to other neurodegenerative diseases, cytotoxicity is currently attributed to these prefibrillar species rather than to the insoluble aggregates. Nevertheless, the underlying molecular mechanisms responsible for cytotoxicity remain elusive and structural studies may contribute to the understanding of both the amyloid aggregation mechanism and oligomer-induced toxicity. It is already recognized that soluble oligomeric AS species adopt β-sheet structures that differ from those characterizing the fibrillar structure. In the present study we used ATR (attenuated total reflection)-FTIR (Fourier-transform infrared) spectroscopy, a technique especially sensitive to β-sheet structure, to get a deeper insight into the β-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel β-sheet structure, whereas fibrils adopt a parallel arrangement. The results are discussed in terms of regions of the protein involved in the early β-sheet interactions and the implications of such conformational arrangement for the pathogenicity associated with AS oligomers.     

Simulation of the β- to α-sheet transition results in a twisted sheet for antiparallel and an α-nanotube for parallel strands: implications for amyloid formation

α-sheet has been proposed to be the main constituent of the toxic amyloid intermediate. Molecular dynamics simulations on proteins known to be involved in amyloid diseases have demonstrated that β-sheet can, under certain conditions, spontaneously convert to α-sheet via ββ→α(R)α(L) peptide-plane flipping. Using torsion-angle driving to simulate this flip the transition has been investigated for parallel and antiparallel sheets. Concerted and sequential flipping processes were simulated, the former allowing direct calculation of helical parameters. For antiparallel sheet, the strands tend to splay apart during the transition. This can be understood by consideration of the geometry of repeating dipeptide conformations. At the end of the transition antiparallel α-sheet is slightly twisted, comprising gently curving strands. In parallel sheet, the strands maintain identical conformations and stay hydrogen bonded during the transition as they curl up to suggest a hitherto unseen structure, the multi-helix α-nanotube. Intriguingly, the α-nanotube has some of the characteristics of the parallel β-helix, a single-helix structure also implicated in amyloid. Unlike the β-helix, α-nanotube formation could involve identical strands aligning with each other in register as in most amyloids.     

Effect of alkyl alcohols on partially unfolded state of Proteinase K: Differential stability of α-helix and β-sheet rich regions of the enzyme

Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to α/β class of proteins and maintains structural integrity in the range of pH 7.0–3.0. Enzyme acquires partially unfolded conformation (U_P) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from α/β to β-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U_P state occurred in two steps with the formation of β state at low alcohol concentration followed by stabilization of β state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.     

Hydrophobic organization of α-helix membrane bundle in bacteriorhodopsin

The hydrophobic organization of the intramembraneα-helical bundle in bacteriorhodopsin (BRh) was assessed based on a new approach to characterization of spatial hydrophobic properties of transmembrane (TM)α-helical peptides. The method employs two independent techniques: Monte Carlo simulations of nonpolar solvent around TM peptides and analysis of molecular hydrophobicity potential on their surfaces. The results obtained by the two methods agree with each other and permit precise hydrophobicity mapping of TM peptides. Superimposition of such data on the experimentally derived spatial model of the membrane moiety together with 2D maps of hydrophobic hydrophilic contacts provide considerable insight into the hydrophobic organization of BRh. The helix bundle is stabilized to a large extent by hydrophobic interactions between helices—neighbors in the sequence of BRh, by long-range interactions in helix pairs C-E, C-F, and C-G, and by nonpolar contracts between retinal and helices C, D, E, F. Unlike globular proteins, no polar contacts between residues distantly separated in the sequence of BRh were found in the bundle. One of the most striking results of this study is the finding that the hydrophobic organization of BRh is significantly different from those in bacterial photoreaction centers. Thus, TMα-helices in BRh expose their most nonpolar sides to the bilayer as well as to the neighboring helices and to the interior of the bundle. Some of them contact lipids with their relatively hydrophilic surfaces. No correlation was found between disposition of the most hydrophobic and the most variable sides of the TM helices.     

Characterization and sequence prediction of structural variations in α-helix

Background

Centralised resources such as GenBank and UniProt are perfect examples of the major international efforts that have been made to integrate and share biological information. However, additional data that adds value to these resources needs a simple and rapid route to public access. The Distributed Annotation System (DAS) provides an adequate environment to integrate genomic and proteomic information from multiple sources, making this information accessible to the community. DAS offers a way to distribute and access information but it does not provide domain experts with the mechanisms to participate in the curation process of the available biological entities and their annotations.

Results

We designed and developed a Collaborative Annotation System for proteins called DAS Writeback. DAS writeback is a protocol extension of DAS to provide the functionalities of adding, editing and deleting annotations. We implemented this new specification as extensions of both a DAS server and a DAS client. The architecture was designed with the involvement of the DAS community and it was improved after performing usability experiments emulating a real annotation task.

Conclusions

We demonstrate that DAS Writeback is effective, usable and will provide the appropriate environment for the creation and evolution of community protein annotation.