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.     

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.     

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.     

Dynamic transition of α-helix to β-sheet structure in linear surfactin correlating to critical micelle concentration

Summary Linear heptapeptide surfactin was prepared by alkaline cleavage of the lactone ring of cyclic surfactin. The structure of linear surfactin was characterised and confirmed by FAB-mass-spectroscopy, FT-IR and HPLC analysis. It was found that linear surfactin easily forms micelles in aqueous solutions by coordinating -sheet formation from -helical monomolecules, and the cmc value found to be 1.28×10^–5 M. The CD spectra indicates conformational change of linear surfactin from -helical below cmc to -sheet above cmc.     

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.     

Temperature-induced formation of a non-native intermediate state of the All β-sheet protein CD2

Domain 1 of the cell adhesion protein CD2 (CD2-1) has an all β-structure typical of proteins belonging to the immunoglobin superfamily. It has a remarkable, ability to fold as a native monomer or a metastable intertwined dimer. To understand the origin of structural rearrangements of CD2-1, we have studied equilibrium unfolding of the protein using various biophysical spectroscopic techniques. At temperatures above approx 68°C, a partially folded state of CD2-1 (H state) with a distinct secondary structure, involving largely exposed aromatic and hydrophobic residues and a substantially perturbed tertiary structure, is observed. In contrast, an unfolded state (D state) of CD2-1 with random-coil-like secondary and tertiary structures is observed in 6 M GuHCl. This partially folded high-temperature state has increased negative molar ellipticity at 222 nm in far-ultraviolet CD spectra, implying formation of a non-native helical conformation. The existence of this non-native high-temperature intermediate is consistent with relatively high intrinsic helical propensities in the primary sequence of CD2-1. This conformation flexibility may be important in the observed domain swapping of CD2-1.     

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.     

Fractionation of a commercial cellulase preparation from Penicillium funiculosum and its use in the purification of a water soluble α-glucan from milled barley

A commercial enzyme preparation from a selected strain of Penicillium funiculosum has been partially purified using a single stage chromatofocusing fractionation to produce an amylase-free mixture of hydrolytic enzymes. This mixture has been used to remove the non-starch polysaccharides from aqueous extracts of laboratory milled barley. The structure of the resulting purified α-glucan fraction has been examined by gel filtration before and after enzymic debranching and by iodine staining. The mild damage caused to the starch within the barley kernel releases a low molecular weight amylopectin molecule with no detectable amylose in the product. In this respect the product is different from that produced after severe, direct grinding of the purified barley starch where some amylose appears in the water soluble extract. Although the small amount of α-glucan is not of any quantitative industrial significance in itself, it does reflect the extent and type of physical damage which is taking place in the starch granule. The model proposed to explain these results - a starch granule with a solid amylose/amylopectin core but with a number of patches of protruding amylopectin clusters - may have important implications in an industrial context.     

Characterization of a novel β-helix antifreeze protein from the desert beetle Anatolica polita

Many ectotherms organisms produce antifreeze proteins (AFPs) which inhibit the growth of ice by binding to the surface of ice crystals. In this study, a novel antifreeze protein gene from the desert beetle Anatolica polita (named as Apafp752) was expressed in a high level in Escherichia coli strain BL21 (DE3). An approximately 30 kDa fusion protein thioredoxin (Trx)-ApAFP752 was purified through Ni–NTA affinity chromatography and gel filtration chromatography. The activity of the purified fusion protein Trx-ApAFP752 was analyzed by thermal hysteresis activity (THA) and cryoprotection assay. The results suggested that Trx-ApAFP752 conferred freeze resistance on bacterium in a concentration- and time-dependent manner and the cryoprotective effect increased under alkaline conditions. Circular Dichroism (CD) spectrum analysis showed that the recombinant protein of ApAFP752 possessing β-sheet as the main structure was stable under a wide range of pH from 2.0 to 11.0 and thermal stability below 50 °C. The predicted 3D structure showed that Trx-ApAFP752 could form a β-helix structure on the antifreeze protein part, which placed most of the Thr in a regular array on one side of the protein to form a putative ice-binding surface.     

Grafting a short chameleon sequence from αB crystallin into a β-sheet scaffold protein

Many protein and peptide sequences are self-assembled into β-sheet-rich fibrous structures called amyloids. Their atomic details provide insights into fundamental knowledge related to amyloid diseases. To study the detailed structure of the amyloid, we have developed a model system that mimics the self-assembling process of the amyloid within a water-soluble protein, termed peptide self-assembly mimic (PSAM). PSAM enables capturing of a peptide sequence within a water-soluble protein, thus making structural and energetics-related studies possible. In this work, we extend our PSAM approach to a naturally occurring chameleon sequence from αB crystallin. We chose “Val–Leu–Gly–Asp–Val (VLGDV)”, a five amino-acid sequence, which forms a β-turn in the native structure and a β-barrel in the amyloid oligomer cylindrin, as a grafting sequence to the PSAM scaffold. The crystal structure revealed that the sequence grafting induced β-sheet bending at the grafted site. We further investigated the role of the central glycine residue and found that its role in the β-sheet bending is dependent on the neighboring residues. The ability of PSAM to observe the structural alterations induced by the grafted sequence provides an opportunity to evaluate the structural impact of a sequence from the peptide self-assembly.     

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.     

Membrane-bound structure and alignment of the antimicrobial β-sheet peptide gramicidin S derived from angular and distance constraints by solid state 19F-NMR

The antimicrobial properties of the cyclic -sheet peptide gramicidin S are attributed to its destabilizing effect on lipid membranes. Here we present the membrane-bound structure and alignment of a derivative of this peptide, based on angular and distance constraints. Solid-state ¹⁹F-NMR was used to study a ¹⁹F-labelled gramicidin S analogue in dimyristoylphosphatidylcholine bilayers at a lipid:peptide ratio of 80:1 and above. Two equivalent leucine side chains were replaced by the non-natural amino acid 4F-phenylglycine, which serves as a highly sensitive reporter on the structure and dynamics of the peptide backbone. Using a modified CPMG multipulse sequence, the distance between the two ¹⁹F-labels was measured from their homonuclear dipolar coupling as 6 Å, in good agreement with the known backbone structure of natural gramicidin S in solution. By analyzing the anisotropic chemical shift of the ¹⁹F-labels in macroscopically oriented membrane samples, we determined the alignment of the peptide in the bilayer and described its temperature-dependent mobility. In the gel phase, the ¹⁹F-labelled gramicidin S is aligned symmetrically with respect to the membrane normal, i.e., with its cyclic -sheet backbone lying flat in the plane of the bilayer, which is fully consistent with its amphiphilic character. Upon raising the temperature to the liquid crystalline state, a considerable narrowing of the ¹⁹F-NMR chemical shift dispersion is observed, which is attributed the onset of global rotation of the peptide and further wobbling motions. This study demonstrates the potential of the ¹⁹F nucleus to describe suitably labelled polypeptides in membranes, requiring only little material and short NMR acquisition times.     

Purification and partial characterization of β-glucosidase from papaya fruit

β-Glucosidase (I) was isolated from Carica papaya fruit pulp and purified ca 1000-fold to electrophoretic homogeneity. The procedure used ammonium sulphate fractionation followed by chromatography on Phenyl-Sepharose CL-4B and Sephacryl S-200 to separate α-mannosidase (II) and, in part, β-galactosidase (III) from (I). Final separation of (III) from (I) was achieved by preparative isoelectric focusing (PIEF). The glycosidases had pI of 5.2 (I), 4.9 (II) and 6.9 (III). M,s of 54 000 (I), 260 000 (II) and 67 000 (III) were determined by gel filtration. The M, of (I) estimated by SDS-PAGE was 27 000 suggesting that (I) consisted of two subunits. The optimum pH and optimum temperature of (I) were 5.0 and 50°, respectively, and the enzyme followed typical Michaelis kinetics with K_m and V_max of 1.1 × 10⁻⁴ M and 1.8 × 10⁻⁶ mol/hr, respectively, for p-nitrophenyl-β-d-glucoside (40°).     

On the mechanism of direct conversion of cellulose to ethanol by Fusarium oxysporum: effect of cellulase and β-glucosidase

Summary The effects of the three main enzymes involved in cellulose saccharification, namely cellobiohydrolase, carboxymethylcellulase and -glucosidase, on the direct conversion of cellulose to ethanol by Fusarium oxysporum F3 were investigated. Ethanol production was not affected when the activity of the former two enzymes was varied within a wide range. By contrast, -glucosidase markedly affected ethanol production showing an optimum level of 0.7–0.8 unit/ml growth medium. A significant decrease of cellulose bioconversion time to ethanol was obtained when -glucosidase activity was adjusted to this optimal level at the beginning of the fermentation process. Offprint requests to: B. J. Macris     

Purification and partial biochemical characterization of a membrane-bound type II-like α-glucosidase from the yeast morphotype of Sporothrix schenckii

The early steps of glycoprotein biosynthesis involve processing of the N-glycan core by endoplasmic reticulum α-glucosidases I and II which sequentially trim the outermost α1,2-linked and the two more internal α1,3-linked glucose units, respectively. We have demonstrated the presence of some components of the enzymic machinery required for glycoprotein synthesis in Sporothrix schenckii, the etiological agent of human and animal sporotrichosis. However, information on this process is still very limited. Here, a distribution analysis of α-glucosidase revealed that 38 and 50% of total enzyme activity were present in a soluble and in a mixed membrane fraction, respectively. From the latter, the enzyme was solubilized, purified to apparent homogeneity and biochemically characterized. Analysis of the enzyme by denaturing electrophoresis and size exclusion chromatography revealed molecular masses of 75.4 and 152.7 kDa, respectively, suggesting a homodimeric structure. Purified α-glucosidase cleaved the fluorogenic substrate 4-methylumbelliferyl-α-d-glucopyranoside with high affinity as judged from K_m and V_max values of 0.3 μM and 250 nmol of MU/min/mg protein, respectively. Analysis of linkage specificity using a number of glucose α-disaccharides as substrates demonstrated a clear preference of the enzyme for nigerose, an α1,3-linked disaccharide, over other substrates such as kojibiose (α1,2), trehalose (α1,1) and isomaltose (α1,6). Use of selective inhibitors of processing α-glucosidases such as 1-deoxynojirimycin, castanospermine and australine provided further evidence of the possible type of α-glucosidase. Accordingly, 1-deoxynojirimycin, a more specific inhibitor of α-glucosidase II than I, was a stronger inhibitor of hydrolysis of 4-methylumbelliferyl-α-d-glucopyranoside and nigerose than castanospermine, a preferential inhibitor of α-glucosidase I. Inhibition of hydrolysis of kojibiose and maltose by 1-deoxynojirimycin and castanoespermine was significantly lower than that of nigerose. Taken together, these properties are consistent with a type II-like α-glucosidase probably involved in N-glycan processing. To our knowledge, this is the first report of such an activity in a truly dimorphic fungus.     

Self-assembly of amphipathic β-sheet peptides: insights and applications

Amphipathic peptides composed of alternating polar and nonpolar residues have a strong tendency to self-assemble into one-dimensional, amyloid-like fibril structures. Fibrils derived from peptides of general (XZXZ)(n) sequence in which X is hydrophobic and Z is hydrophilic adopt a putative β-sheet bilayer. The bilayer configuration allows burial of the hydrophobic X side chain groups in the core of the fibril and leaves the polar Z side chains exposed to solvent. This architectural arrangement provides fibrils that maintain high solubility in water and has facilitated the recent exploitation of self-assembled amphipathic peptide fibrils as functional biomaterials. This article is a critical review of the development and application of self-assembling amphipathic peptides with a focus on the fundamental insight these types of peptides provide into peptide self-assembly phenomena.