The use of deuterium exchange for the study of the distortion of α-helices in proteins

The geometrical distortion of the α-helical structure of the globular proteins sperm-whale myoglobin, bacteriophage T4 lysozyme and hen egg-white lysozyme have been studied by means of deuterium exchange in solution. It was examined with the use of infrared spectroscopy in the region of the amide A band. The parameters of this band are known to be dependent on the length and geometry of the peptide hydrogen bond. In this way an estimation of the structural heterogeneity of the polypeptide backbone of the protein molecule has been achieved by studying the half-width of the amide A band during successive deuteration of the protein in heavy water solution. For all the proteins studied the peptide groups with broad amide A bands were exchanged at the first stage. These groups have been assigned as belonging to the unordered form of the molecule. The α-helical fragments were assigned to have smaller values of half-widths of the amide A band, and these were exchanged at the second stage. From these data α-helical fragments were shown to be characterized by a set of geometrical distortions. The results obtained also disclose a correlation between the degree of geometrical distortion of α-helical structure in the protein molecule and the dynamic accessibility of their peptide groups to a water molecule.     

Cysteine synthesis in plants: protein-protein interactions of serine acetyltransferase from Arabidopsis thaliana

The biosynthesis of cysteine represents the final step of sulfate assimilation in bacteria and plants. It is catalyzed by the sequential action of serine acetyltransferase (SAT) and O -acetylserine (thiol) lyase (OAS-TL) which form a cysteine synthase (CS) complex in vitro . SAT and OAS-TL from Arabidopsis thaliana have previously been cloned, and now the first evidence is presented for the CS complex and SAT self-interaction in vivo employing the yeast two-hybrid system. Application of this method proved to be an efficient tool for the analysis of protein-protein interactions within a plant metabolic protein complex. Mapping of SAT domain structure revealed two new, independent domains with specific functions in protein-protein interaction. Analysis using truncated proteins proved the C-terminus of SAT to be sufficient for association with OAS-TL and to correlate with the putative transferase activity domain. SAT/SAT interaction was localized in the central region of the protein and occured also between SAT isoforms. Both protein interaction domains coincided with distinct α-helical and β-sheet clusters and together correlated with the minimal protein structure required for SAT catalysis as shown by functional complementation of an Escherichia coli mutant. The homo- and hetero-oligomerization properties are discussed with respect to the assumed function of the CS complex in metabolic channeling and activation of SAT by interaction with OAS-TL.     

Antimicrobial 14-helical beta-peptides: potent bilayer disrupting agents

The interactions of two amphiphilic and cationic, nine-residue beta-peptides with liposomal membranes were studied. These beta-peptides are shown to form 14-helices in the presence of bilayers. Membrane binding and membrane permeabilization occur preferentially in the presence of anionic lipids. The beta-peptides have the ability to cause tranbilayer diffusion of phospholipids, form pores, and promote lipid mixing between liposomes. These beta-peptides have previously been shown to display antimicrobial activity comparable to that of a longer beta-peptide, beta-17, which adopts a different type of helical conformation (12-helix), and to the 23 amino acid (Ala(8,13,18))-magainin-II-amide, which adopts an alpha-helical conformation. In addition, these 14-helical beta-peptides show relatively low hemolytic activity. The biological potency and microbial specificity of the 14-helical beta-peptides, despite their relatively short length, suggests that 14-helices can be particularly disruptive to microbial membranes.     

The structure of the MAPK scaffold, MP1, bound to its partner, p14. A complex with a critical role in endosomal map kinase signaling

Scaffold proteins of the mitogen-activated protein kinase (MAPK) pathway have been proposed to form an active signaling module and enhance the specificity of the transduced signal. Here, we report a 2-A resolution structure of the MAPK scaffold protein MP1 in a complex with its partner protein, p14, that localizes the complex to late endosomes. The structures of these two proteins are remarkably similar, with a five-stranded beta-sheet flanked on either side by a total of three helices. The proteins form a heterodimer in solution and interact mainly through the edge beta-strand in each protein to generate a 10-stranded beta-sheet core. Both proteins also share structural similarity with the amino-terminal regulatory domains of the membrane trafficking proteins, sec22b and Ykt6p, as well as with sedlin (a component of a Golgi-associated membrane-trafficking complex) and the sigma2 and amino-terminal portion of the mu2 subunits of the clathrin adaptor complex AP2. Because neither MP1 nor p14 have been implicated in membrane traffic, we propose that the similar protein folds allow these relatively small proteins to be involved in multiple and simultaneous protein-protein interactions. Mapping of highly conserved, surface-exposed residues on MP1 and p14 provided insight into the potential sites of binding of the signaling kinases MEK1 and ERK1 to this complex, as well as the areas potentially involved in other protein-protein interactions.     

Right-handed 14-helix in beta 3-peptides from L-aspartic acid monomers

beta-Peptides made from L-aspartic acid monomers form a new class of beta 3-peptides. Here we report the first three-dimensional NMR solution structure of a beta 3-hexapeptide (1) from L-aspartic acid monomers in 2,2,2-trifluoroethanol (TFE). We show that 1 forms a right-handed 14-helical structure in TFE. alpha-peptides from naturally occurring L-amino acids adopt a right-handed alpha-helix whereas beta 3-peptides formed from beta 3-amino acids derived from naturally occurring L-amino acids form left-handed 14-helices. The right-handed 14-helical conformation of 1 is a better mimic of alpha-peptide conformations. Using the NMR structure of 1 in TFE, we further study the conformation of 1 in water, as well as two similar beta 3-peptides (2 and 3) in water and TFE by molecular dynamics (MD) simulations. NMR and MD results suggest loss of secondary structure of 1 in water and show that it forms a fully extended structure. 2 and 3 contain residues with oppositely charged side chains that engage in salt-bridge interactions and dramatically stabilize the 14-helical conformation in aqueous media.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: A consequence of the formation of “native-like conformation”

The influence of n-propanol on the overall α-helical conformation of β-globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of α-helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of α-helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of α-globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The α-helical conformation induced into α- and β-globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the α-helical conformation of both α- and β-chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced “α-helical conformation” of globins and the “α-helical conformation” of α- and β-chains. A cross-correlation of the n-propanol induced increase in the fluorescence of β-globin with the corresponding increase in the α-helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the α-helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a “native-like structure.” The induction of α-helical conformation into these proteins in the presence of n-propanol and the consequent generation of “native-like conformation” is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an α-helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume α-helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high α-helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of α-helical proteins. Consistent with this concept, the induction of α-helical conformation into shorter polypeptide fragments of 30 residues, (e.g., α_1-30, which exists in an α-helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

Conformational studies of the α-helical proteins from wool keratin by c.d.

The conformation and conformational change of wool keratin $\text{S-}\text{carboxymethylated}$ low-sulphur proteins (SCMKA), which are α-helical fibrous proteins, have been investigated in aqueous solution by means of c.d. Comparisons of various methods proposed for c.d. analysis of protein secondary structure are made using least-squares curve-fitting of the observed c.d. spectra of SCMKA with a linear combination of the corresponding reference spectra of secondary structures. It has been found that (i) the most satisfactory results are obtained with the method¹³ which takes into account the β-turn contribution: (ii) SCMKA is 52–54% α-helical in water and has little β-form, (iii) the addition of n-propanol produces, even at higher concentrations of n-propanol, little chnage in spectra with respect to helical character in water; (iv) SCMKA undergoes a thermally-induced conformational transition from α-helix to random coil around 50 C; and (v) $\text{S-}\text{aminoethylated}$ low-sulphur proteins with positively charged protecting groups are /_~50% α-helical in water, which is similar to SCMKA, showing that the protecting groups introduced in the low-sulphur proteins are little effect upon their conformation in water     

14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如:新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide.     

Mechanistic insights into protein precipitation by alcohol

Ethanol is used to precipitate proteins during various processes, including purification and crystallization. To elucidate the mechanism of protein precipitation by alcohol, we have investigated the solubility and structural changes of protein over a wide range of alcohol concentrations. Conformation of hen egg-white lysozyme was changed from native to α-helical rich structure in the presence of ethanol at concentrations above 60%. The solubility of lysozyme was reduced with increasing ethanol concentration, although gel formation at ethanol concentrations between 60% and 75% prevented accurate solubility measurements. SH-modified lysozyme showed largely unfolded structure in water and α-helical structure in the presence of ethanol. More importantly, solubility of the chemically modified lysozyme molecules decreased with increasing ethanol concentration. There is no indication of increased solubility upon unfolding of the lysozyme molecules by ethanol, indicating that any favorable interaction of ethanol with the hydrophobic side chains, if indeed occuring, is offset by the unfavorable interaction of ethanol with the hydrophilic side chains and peptide bonds.     

Structural principles of the globular organization of protein chains. A stereochemical theory of globular protein secondary structure

The paper reveals the types of amino acid sequences of polypeptide chain regions of globular protein which form a regular (α or β) or irregular conformation in the native globule. The study was made taking into account general “architectural” principles of packing of polypeptide chains in globular proteins and considering the interactions of proteins with water molecules. An a priori theory is developed which permits the identification, in good agreement with experiment, of α-helical and β-structural regions in globular proteins from their primary structure.     

Helical repeat structure of apoptosis inhibitor 5 reveals protein-protein interaction modules

Apoptosis inhibitor 5 (API5) is an anti-apoptotic protein that is up-regulated in various cancer cells. Here, we present the crystal structure of human API5. API5 exhibits an elongated all α-helical structure. The N-terminal half of API5 is similar to the HEAT repeat and the C-terminal half is similar to the ARM (Armadillo-like) repeat. HEAT and ARM repeats have been implicated in protein-protein interactions, suggesting that the cellular roles of API5 may be to mediate protein-protein interactions. Various components of multiprotein complexes have been identified as API5-interacting protein partners, suggesting that API5 may act as a scaffold for multiprotein complexes. API5 exists as a monomer, and the functionally important heptad leucine repeat does not exhibit the predicted a dimeric leucine zipper. Additionally, Lys-251, which can be acetylated in cells, plays important roles in the inhibition of apoptosis under serum deprivation conditions. The acetylation of this lysine also affects the stability of API5 in cells.     

Crystal structure of N-glycosylated human glypican-1 core protein: structure of two loops evolutionarily conserved in vertebrate glypican-1

Glypicans are a family of cell-surface proteoglycans that regulate Wnt, hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling. Loss-of-function mutations in glypican core proteins and in glycosaminoglycan-synthesizing enzymes have revealed that glypican core proteins and their glycosaminoglycan chains are important in shaping animal development. Glypican core proteins consist of a stable α-helical domain containing 14 conserved Cys residues followed by a glycosaminoglycan attachment domain that becomes exclusively substituted with heparan sulfate (HS) and presumably adopts a random coil conformation. Removal of the α-helical domain results in almost exclusive addition of the glycosaminoglycan chondroitin sulfate, suggesting that factors in the α-helical domain promote assembly of HS. Glypican-1 is involved in brain development and is one of six members of the vertebrate family of glypicans. We expressed and crystallized N-glycosylated human glypican-1 lacking HS and N-glycosylated glypican-1 lacking the HS attachment domain. The crystal structure of glypican-1 was solved using crystals of selenomethionine-labeled glypican-1 core protein lacking the HS domain. No additional electron density was observed for crystals of glypican-1 containing the HS attachment domain, and CD spectra of the two protein species were highly similar. The crystal structure of N-glycosylated human glypican-1 core protein at 2.5 Å, the first crystal structure of a vertebrate glypican, reveals the complete disulfide bond arrangement of the conserved Cys residues, and it also extends the structural knowledge of glypicans for one α-helix and two long loops. Importantly, the loops are evolutionarily conserved in vertebrate glypican-1, and one of them is involved in glycosaminoglycan class determination.     

Mimicry of bioactive peptides via non-natural,sequence-specific peptidomimetic oligomers

Non-natural, sequence-specific peptidomimetic oligomers are being designed to mimic bioactive peptides, with potential therapeutic application. Cationic, facially amphipathic helical beta-peptide oligomers have been developed as magainin mimetics. Non-natural mimics of HIV-Tat protein, lung surfactant proteins, collagen, and somatostatin are also being developed. Pseudo-tertiary structure in beta-peptides and peptoids may herald the creation of entirely artificial proteins.     

Aging of Dry Desiccation-Tolerant Pollen Does Not Affect Protein Secondary Structure

Protein secondary structure and membrane phase behavior in aging Typha latifolia pollen were studied by means of Fourier transform infrared microspectroscopy (FTIR). Membranes isolated from fresh pollen occurred mainly in the liquid crystalline phase at room temperature, whereas the membrane fluidity of aged pollen was drastically decreased. This decrease did not result in large-scale irreversible protein aggregation, as was concluded from in situ FTIR assessment of the amide-1 bands. Curve-fitting on the infrared absorbance spectra enabled estimation of the proportion of different classes of protein secondary structure. Membrane proteins had a relatively large amount of [alpha]-helical structure (48%; band at 1658 cm-1), and turn-like structures (at 1637 and 1680 cm-1) were also detected. The secondary protein structure of isolated cytoplasmic proteins resembled that of proteins in whole pollen and was conserved upon drying in the absence of sucrose. The isolated cytoplasmic proteins had a large amount of [alpha]-helical structure (43%), and also [beta]-sheet (at 1637 and 1692 cm-1) and turn structures were detected. Heat-denaturing experiments with intact hydrated pollen showed low (1627 cm-1) and high (1692 cm-1) wave number bands indicating irreversible protein aggregates. The results presented in this paper show that FTIR is an extremely suitable technique to study protein secondary structure in intact plant cells of different hydration levels and developmental stages.     

Computational identification of self-inhibitory peptides from envelope proteins

Fusion process is known to be the initial step of viral infection and hence targeting the entry process is a promising strategy to design antiviral therapy. The self-inhibitory peptides derived from the enveloped (E) proteins function to inhibit the protein-protein interactions in the membrane fusion step mediated by the viral E protein. Thus, they have the potential to be developed into effective antiviral therapy. Herein, we have developed a Monte Carlo-based computational method with the aim to identify and optimize potential peptide hits from the E proteins. The stability of the peptides, which indicates their potential to bind in situ to the E proteins, was evaluated by two different scoring functions, dipolar distance-scaled, finite, ideal-gas reference state and residue-specific all-atom probability discriminatory function. The method was applied to α-helical Class I HIV-1 gp41, β-sheet Class II Dengue virus (DENV) type 2 E proteins, as well as Class III Herpes Simplex virus-1 (HSV-1) glycoprotein, a E protein with a mixture of α-helix and β-sheet structural fold. The peptide hits identified are in line with the druggable regions where the self-inhibitory peptide inhibitors for the three classes of viral fusion proteins were derived. Several novel peptides were identified from either the hydrophobic regions or the functionally important regions on Class II DENV-2 E protein and Class III HSV-1 gB. They have potential to disrupt the protein-protein interaction in the fusion process and may serve as starting points for the development of novel inhibitors for viral E proteins.     

Amphiphilic ��-Helical Potential: A Putative Folding Motif Adding Few Constraints to Protein Evolution

Evidence from a number of studies indicates that protein folding is dictated not only by factors stabilizing the native state, but also by potentially independent factors that create folding pathways. How natural selection might cope simultaneously with two independent factors was addressed in this study within the framework of the "Lim-model" of protein folding, which postulates that the early stages of folding of all globular proteins, regardless of their native structure, are directed at least in part by potential to form amphiphilic α-helices. For this purpose, the amphiphilic α-helical potential in randomly ordered amino acid sequences and the conservation in phylogeny of amphiphilic α-helical potential within various proteins were assessed. These analyses revealed that amphiphilic α-helical potential is a common occurrence in random sequences, and that the presence of amphiphilic α-helical potential is present but not conserved in phylogeny within a given protein. The results suggest that the rapid formation of molten globules and the variable behavior of those globules depending on the protein may be a fundamental property of polymers of naturally occurring amino acids more so than a trait that must be derived or maintained by natural selection. Further, the results point toward the utility of randomly occurring process in protein function and evolution, and suggest that the formation of efficient pathways that determine early processes in protein folding, unlike the formation of stable, native protein structure, does not present a substantial hurdle during the evolution of amino acid sequences.     

How many protein-protein interactions types exist in nature?

"Protein quaternary structure universe" refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions.     

Towards assignment of secondary structures by anti-peptide antibodies. Specificity of the immune response to a beta-turn.

In an attempt to assign secondary structure elements to protein primary structures with antibodies, we synthesized a model peptide (beta-peptide: TVTVTDPGQTVTY) with a putative beta-turn structure and analysed the anti-peptide antibodies for their specificity towards the turn sequence. At least 50% of the peptide fraction adopts the intended conformation of a beta-turn (DPGQ) inserted between the two segments of an antiparallel beta-sheet structure. The specific anti-beta-peptide antibodies of the hyperimmune response bind the beta-turn containing epitope of the immunogenic beta-peptide with a three orders of magnitude higher affinity than the synthetic control peptide (Gly-peptide: GGGGGDPGQGGGG). The affinity of the antibodies with specificity for the beta-turn region increases from the primary to the hyperimmune response. Therefore, probing of secondary structure elements, i.e., of individual beta-turn regions, by anti-peptide antibodies now seems feasible for proteins of known sequence and may result in sequence assignments of secondary structures.