Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes

Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic α-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% α-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% α-helical in the dry state. However, the dry protein contained 15% β-sheets. FTIR spectroscopy revealed the β-sheets to be largely due to aggregation. β-Sheet content was reduced and α-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates.     

Prediction of the structure of the replication initiator protein DnaA

The secondary structure of DnaA protein and its interaction with DNA and ribonucleotides has been predicted using biochemical, biophysical techniques, and prediction methods based on multiple-sequence alignment and neural networks. The core of all proteins from the DnaA family consists of an “open twisted α/β structure,” containing five α-helices alternating with five β-strands. In our proposed structural model the interior of the core is formed by a parallel β-sheet, whereas the α-helices are arranged on the surface of the core. The ATP-binding motif is located within the core, in a loop region following the first β-strand. The N-terminal domain (80 aa) is composed of two α-helices, the first of which contains a potential leucine zipper motif for mediating protein-protein interaction, followed by a β-strand and an additional α-helix. The N-terminal domain and the α/β core region of DnaA are connected by a variable loop (45–70 aa); major parts of the loop region can be deleted without loss of protein activity. The C-terminal DNA-binding domain (94 aa) is mostly α-helical and contains a potential helix-loop-helix motif. DnaA protein does not dimerize in solution; instead, the two longest C-terminal α-helices could interact with each other, forming an internal “coiled coil” and exposing highly basic residues of a small loop region on the surface, probably responsible for DNA backbone contacts. © 1997 Wiley-Liss Inc.     

Differences in the structural stability and cooperativity between monomeric variants of natural and de novo Cro proteins revealed by high-pressure Fourier transform infrared spectroscopy

It is widely accepted that pressure affects the structure and dynamics of proteins; however, the underlying mechanism remains unresolved. Our previous studies have investigated the effects of pressure on fundamental secondary structural elements using model peptides, because these peptides represent a basis for understanding the effects of pressure on more complex structures. This study targeted monomeric variants of naturally occurring bacteriophage λ Cro (natural Cro) and de novo designed λ Cro (SN4m), which are α + β proteins. The sequence of SN4m is 75% different from that of natural Cro, but the structures are almost identical. Consequently, a comparison of the folding properties of these proteins is of interest. Pressure- and temperature-variable Fourier transform infrared spectroscopic analyses revealed that the α-helices and β-sheets of natural Cro are cooperatively and reversibly unfolded by pressure and temperature, whereas those of SN4m are not cooperatively unfolded by pressure; i.e., the α-helices of SN4m unfold at significantly higher pressures than the β-sheets and irreversibly unfold with increases in temperature. The higher unfolding pressure for the α-helices of SN4m indicates the presence of an intermediate structure of SN4m that does not retain β-sheet structure but does preserve the α-helices. These results demonstrate that the α-helices of natural Cro are stabilized by global tertiary contacts among the α-helices and the β-sheets, whereas the α-helices of SN4m are stabilized by local tertiary contacts between the α-helices.     

Relationship between structure and amino acid sequence of strongly twisted and coiled β-hairpins in globular proteins

β-Hairpins are widespread in proteins, and it is possible to find them both within β-sheets and separately. In this work, a comparative analysis of amino acid sequences of β-strands within strongly twisted β-hairpins from different structural protein subclasses has been conducted. Strongly twisted and coiled β-hairpin generates in the space a right double helix out of β-strands that are connected by a loop region (connections). The frequencies of amino acid residues on the internal (concave) and external (convex) surfaces of strongly twisted β-hairpins have been determined (220 β-hairpins from nonhomologous proteins were studied). The concave surface of these β-hairpins is mainly generated by hydrophobic residues, while the convex surface by hydrophilic residues; accordingly, the alternation of hydrophobic internal and hydrophilic external residues is observed in their amino acid sequences. Amino acid residues of glycine and alanine (especially in places of the largest twisting of the strands) were anomalously frequently found in internal positions of strongly twisted and coiled β-hairpins. It was established that internal positions never contain the proline residues, while external positions in the twisting region contain them in a relatively large amount. It was demonstrated that at least one amino acid residue in α_L- or ε-conformation is required for generation of relatively short (up to 7 amino acid residues) connection. As a rule, these positions are occupied by glycines. Thus, not only the alternation of hydrophobic and hydrophilic amino acid residues, but also the presence of one or two glycine residues in the connection region and the excess of glycines and alanines in the places of the largest strand twisting on the concave surface, as well as the presence of prolines on the convex surface, are required to generate a strongly twisted and coiled β-hairpin.     

Basis for large differences in the cooperativity of the formation of antiparallel β-sheets and clusters of interacting α-helices in isolated chains

Configuration partition functions that describe the intramolecular formation of antiparallel β-sheets and clusters of antiparallel interacting α-helices are very nearly of the same form. They can be interconverted by a simple change in notation and the addition of one weighting factor for each cluster of interacting α-helices. This extra weighting factor is the Zimm–Bragg σ which must be less than one. When it is assigned a reasonable numerical value, it plays an important role in the determination of the nature of the transition from the disordered chain to the ordered structure. It causes the formation of clusters of interacting α-helices to be more cooperative than the formation of antiparallel β-sheets in isolated chains.     

Secondary structure of proteins associated in thin films

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L -lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy¹ and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all α-helical myoglobin, decreased the molar ellipticity of the α/β RNase A, and increased the molar ellipticity of all-β Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less α-helix and more β-sheet structure. The results suggest that the protein α-helices are less stable in films and that the secondary structure may rearrange into β-sheets at high temperature. Two heterooligomeric peptides and poly (L -lysine), all in solution at neutral pH with “random coil” conformation, formed films with variable degrees of their secondary structure in β-sheets or β-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a “random” structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable α-helix or β-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent. © 1993 John Wiley & Sons, Inc.     

Amyloid beta-protein monomer folding: free-energy surfaces reveal alloform-specific differences

Alloform-specific differences in structural dynamics between amyloid β-protein (Aβ) 40 and Aβ42 appear to underlie the pathogenesis of Alzheimer's disease. To elucidate these differences, we performed microsecond timescale replica-exchange molecular dynamics simulations to sample the conformational space of the Aβ monomer and constructed its free-energy surface. We find that neither peptide monomer is unstructured, but rather that each may be described as a unique statistical coil in which five relatively independent folding units exist, comprising residues 1-5, 10-13, 17-22, 28-37, and 39-42, which are connected by four turn structures. The free-energy surfaces of both peptides are characterized by two large basins, comprising conformers with either substantial α-helix or β-sheet content. Conformational transitions within and between these basins are rapid. The two additional hydrophobic residues at the Aβ42 C-terminus, Ile41 and Ala42, significantly increase contacts within the C-terminus, and between the C-terminus and the central hydrophobic cluster (Leu17-Ala21). As a result, the β-structure of Aβ42 is more stable than that of Aβ40, and the conformational equilibrium in Aβ42 shifts towards β-structure. These results suggest that drugs stabilizing α-helical Aβ conformers (or destabilizing the β-sheet state) would block formation of neurotoxic oligomers. The atomic-resolution conformer structures determined in our simulations may serve as useful targets for this purpose. The conformers also provide starting points for simulations of Aβ oligomerization—a process postulated to be the key pathogenetic event in Alzheimer's disease.     

A protein structural class prediction method based on novel features

In this study, a 12-dimensional feature vector is constructed to reflect the general contents and spatial arrangements of the secondary structural elements of a given protein sequence. Among the 12 features, 6 novel features are specially designed to improve the prediction accuracies for α/β and α + β classes based on the distributions of α-helices and β-strands and the characteristics of parallel β-sheets and anti-parallel β-sheets. To evaluate our method, the jackknife cross-validating test is employed on two widely-used datasets, 25PDB and 1189 datasets with sequence similarity lower than 40% and 25%, respectively. The performance of our method outperforms the recently reported methods in most cases, and the 6 newly-designed features have significant positive effect to the prediction accuracies, especially for α/β and α + β classes.     

Displaying inter-main chain hydrogen bond patterns in proteins

Two computer graphics techniques for displaying hydrogen bonds between the main chains of different proteins are described, and illustrated for two thiol proteases. (The X-ray crystallography was performed by Kamphuis et al. in 1984,¹ and by Baker and Dodson in 1980.²) One is a three-dimensional model that can be manipulated in space; the hydrogen bonds are represented with the smoothed α-carbon plot of the polypeptide chain. In the other type of display, hydrogen bonds are viewed in relation to the one-dimensional sequence. Both types of picture facilitate visualization of hydrogen bond patterns, such that loop motifs, as well as α-helices and β-sheets, can be examined easily. We suggest that such displays are useful as a general means of displaying whole proteins and whole domains because they reveal more information than do conventional simplified pictures of proteins, which focus exclusively on α-helices and β-sheets. These techniques can be implemented on a UNIX-based computer graphics workstation. (UNIX is a trademark of Bell Telephone laboratories.)     

Local hydrophobicity stabilizes secondary structures in proteins

The probability of occurrence of helix and β-sheet residues in 47 globular proteins was determined as a function of local hydrophobicity, which was defined by the sum of the Nozaki-Tanford transfer free energies at two nearest-neighbors on both sides of the amino acid sequence. In general, hydrophilic amino acids favor neither helix nor β-sheet formations when neighbor residues are also hydrophilic but favor helix formation at higher local hydrophobicity. On the other hand, some hydrophobic amino acids such as Met, Leu, and Ile favor helix formation when neighbor residues are hydrophilic. None of the hydrophobic amino acids favor β-sheet formation with hydrophilic neighbors, but most of them strongly favor β-sheet formation at high local hydrophobicity. When the average of 20 amino acids is taken, both helix and β-sheet residue probabilities are higher at higher local hydrophobicity, although the increase is steeper for β-sheets. Therefore, β-sheet formation is more influenced by local hydrophobicity than helix formation. Generally, helices are nearer the surface and tend to have hydrophilic and hydrophobic faces at opposite sides. The tendency of alternating regions of hydrophilic and hydrophobic residues in a helical sequence was revealed by calculating the correlation of the Nozaki-Tanford values. Such amphipathic helices may be important in protein–protein and protein–lipid interactions and in forming hydrophilic channels in the membrane. The choice of 30 nonhomologous proteins as the data set did not alter the above results.     

The role of local tight packing of hydrophobic groups in β-structure

An analysis of the tendency of hydrophobic groups to tight packing on the surface of β-sheets based on well-known parameters of β-sheets and hydrophobic groups was conducted. This analysis shows the existence of very limited numbers and clearly outlined architecture families of regular parts for the majority of β-structure-containing domains. Each family of architecture strongly depends on the number of β-strands in the pure β-domains and on the existence and number of additional α-helixes and on the mutual arrangements β-strands and α-helixes along the chain in mixed α/β-domains. This paper demonstrates that the tendency of hydrophobic groups to the local tight packing on the surface of β-sheets is probably the main reason for the twist of β-sheets. © 1993 Wiley-Liss, Inc.     

A novel structural tree for wrap-proteins,a subclass of (α+β)-proteins

In this paper, a novel structural subclass of (α+β)-proteins is presented. A characteristic feature of these proteins and domains is that they consist of strongly twisted and coiled β-sheets wrapped around one or two α-helices, so they are referred to here as wrap-proteins. It is shown that overall folds of the wrap-proteins can be obtained by stepwise addition of α-helices and/or β-strands to the strongly twisted and coiled β-hairpin taken as the starting structure in modeling. As a result of modeling, a structural tree for the wrap-proteins was constructed that includes 201 folds of which 49 occur in known nonhomologous proteins.     

Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.     

Protein design as a challenge for peptide chemists

All efforts to turn the ultimate goal in protein de novo design into reality–the construction of new macromolecules with predetermined three-dimensional structure and well-defined functionality–failed because the mechanism of folding has still to be unravelled. In the present review, various attempts to apply synthetic tools for inducing native-like structural features in peptides in order to bypass the folding problem are described. Besides well-established methods for the nucleation and stabilization of secondary structures, e.g. α-helices, β-sheets and β-turns, topological templates as ‘built-in’ folding devices have more recently become the key elements for the induction of protein-like folding units (template-assembled synthetic proteins, TASP). Progress in the synthetic strategy and structural characterization of this new type of macromolecules opens the way for the design of functional TASP molecules.     

Structural features of split and unsplit βαβ-units

In this study, 1064 nonhomologous “unsplit”, “one-strand split” and “two-strand split” right-handed βαβ-units having standard α-helices and loops up to seven residues in length have been analyzed. It was found that the α-helices in these kinds of βαβ-units have different distributions of the hydrophobic and hydrophilic amino acid residues along the chain. In the unsplit βαβ-units, most α-helices have hydrophobic residues in positions N4-N7-N8-N11 or N6-N7-N10, where N1 is the first N-terminal residue. In the one-strand split βαβ-units, most α-helices have hydrophobic residues in positions N4-N7-N8-N11 and those in two-strand split βαβ-units in positions N4-N5-N8-N12. On the other hand, in all kinds of βαβ-units, there are commonly occurring hydrophobic stripes of type C4-C7-C8 at the C-terminal parts of the α-helices. As a rule, the C- and N-terminal hydrophobic stripes overlap and the extent of their overlapping determine the length of α-helices.     

Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus

We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca²⁺-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 Å resolution. The lpAFP structure displayed a relatively planar surface, which encompasses two loop regions (Cys86-Lys89 and Asn91-Cys97) and a short β-strand (Trp109-Leu112) with three unstructured segments (Gly57-Ile58, Ala103-Ala104, and Pro113-His118). Electrostatic calculation of the protein surface showed that the relatively planar surface was divided roughly into a hydrophobic area (composed of the three unstructured segments lacking secondary structure) and a hydrophilic area (composed of the loops and β-strand). Site-directed mutation of Ile58 with Phe at the center of the hydrophobic area decreased activity significantly, whereas mutation of Leu112 with Phe at an intermediate area between the hydrophobic and hydrophilic areas retained complete activity. In the hydrophilic area, a peptide-swap mutant in the loops retained 60% activity despite simultaneous mutations of eight residues. We conclude that the epicenter of the ice-binding site of lpAFP is the hydrophobic region, which is centered by Ile58, in the relatively planar surface. We built an ice-binding model for lpAFP on the basis of a lattice match of ice and constrained water oxygen atoms surrounding the hydrophobic area in the lpAFP structure. The model in which lpAFP has been docked to a secondary prism (2-1-10) plane, which is different from the one determined for Ca²⁺-independent type II AFP from sea raven (11-21), appears to explain the results of the mutagenesis analysis.     

Monte Carlo studies on equilibrium globular protein folding. II. Beta-barrel globular protein models

In the context of dynamic Monte Carlo simulations on a model protein confined to a tetrahedral lattice, the interplay of protein size and tertiary structure, and the requirements for an all-or-none transition to a unique native state, are investigated. Small model proteins having a primary sequence consisting of a central bend neutral region flanked by two tails having an alternating hydrophobic/hydrophilic pattern of residues are seen to undergo a continuous transition to a beta-hairpin collapsed state. On increasing the length of the tails, the beta-hairpin structural motif is found to be in equilibrium with a four-member beta-barrel. Further increase of the tail length results in the shift of the structural equilibrium to the four-member beta-barrel. The random coil to beta-barrel transition is of an all-or-none character, but while the central turn is always the desired native bend, the location of the turns involving the two external strands is variable. That is, beta-barrels having the external stands that are two residues out of register are also observed in the transition region. Introduction into the primary sequence of two additional regions that are at the very least neutral toward turn formation produces an all-or-none transition to the unique, native, four-member beta-barrel. Various factors that can augment the stability of the native conformation are explored. Overall, these folding simulations strongly indicate that the general rules of globular protein folding are rather robust--namely, one requires a general pattern of hydrophobic/hydrophilic residues that allow the protein to have a well-defined interior and exterior and the presence of regions in the amino acid sequence that at the very least are locally indifferent to turn formation. Since no site-specific interactions between hydrophobic and hydrophilic residues are required to produce a unique four-member beta-barrel, these simulations strongly suggest that site specificity is involved in structural fine-tuning.     

Role of cholesterol in functional association between K(+)-Cl(-) cotransporter-3a and Na+,K(+)-ATPase

The conformation of amyloid-beta peptide (Aβ) determines if toxic aggregates are formed. The peptide structure by its turn depends on the environment and molecule-molecule interactions. We characterized the secondary structure of Aβ-(1-40) in surfactant solutions and interacting with monolayers. The peptide adopts β-sheet structure in solutions of ionic surfactants at sub-micelle concentrations and α-helix in the presence of ionic micelles. Uncharged micelles induce β-sheets. Aβ-(1-40) alters the critical micelle concentration value of the non-ionic surfactant, underlining hydrophobic interactions. At ionic monolayers the peptide forms β-sheets when its concentration at the surface is high enough. These results suggest that only electrostatic interactions of charged micelles that surround completely the peptide are able to induce non-aggregated α-helix structure.     

Temporary secondary structures in tau,an intrinsically disordered protein

The tau protein belongs to the category of intrinsically disordered proteins, which in their native state do not have an average stable structure and fluctuate between many conformations. In its physiological state, tau helps nucleating and stabilising the microtubules in the axons of the neurons. On the other hand, the same tau is involved in the development of Alzheimer disease, when it aggregates in paired helical filaments forming fibrils, which form insoluble tangles. The beginning of the pathological aggregation of tau has been attributed to a local transition of protein portions from random coil to a β-sheet. These structures would very likely be transient; therefore, we performed a molecular dynamics simulation of tau to gather information on the existence of segments of tau endowed with a secondary structure. We combined the results of our simulation with small-angle X-ray scattering experimental data to extract from the dynamics a set of most probable conformations of tau. The analysis of these conformations highlights the presence of transient secondary structures such as turns, β-bridges, β-sheets and α-helices. It also shows that a large segment of the N-terminal region is found near the repeats domain in a globular-like shape.