Normal mode spectrum of the parallel-chain β-sheet

Normal mode calculations have been carried out for parallel-chain β-sheet structures. These include the parallel-chain pleated sheet of poly(L -alanine) and the parallel-chain rippled sheet of polyglycine. Dipole derivative coupling has been included for amide I and II modes, and the effects of parallel-sheet and antiparallel-sheet arrangements of varying separation have been examined for the poly(L -alanine) case. Some amide and nonamide modes are distinctly different from their antiparallel-chain counterparts, thus providing a basis for distinguishing between such structures from their ir and Raman spectra. As in our previous studies, these results emphasize the need for both kinds of spectral data in order to draw definitive conclusions about conformation.     

Folding mechanism of three structurally similar β-sheet proteins

The folding mechanism of cellular retinoic acid binding protein I (CRABP I), cellular retinol binding protein II (CRBP II), and intestinal fatty acid binding protein (IFABP) were investigated to determine if proteins with similar native structures have similar folding mechanisms. These mostly β-sheet proteins have very similar structures, despite having as little as 33% sequence similarity. The reversible urea denaturation of these proteins was characterized at equilibrium by circular dichroism and fluorescence. The data were best fit by a two-state model for each of these proteins, suggesting that no significant population of folding intermediates were present at equilibrium. The native states were of similar stability with free energies (linearly extrapolated to 0 M urea, ΔG) of 6.5, 8.3, and 5.5 kcal/mole for CRABP I, CRBP II, and IFABP, respectively. The kinetics of the folding and unfolding processes for these proteins was monitored by stopped-flow CD and fluorescence. Intermediates were observed during both the folding and unfolding of all of these proteins. However, the overall rates of folding and unfolding differed by nearly three orders of magnitude. Further, the spectroscopic properties of the intermediate states were different for each protein, suggesting that different amounts of secondary and/or tertiary structure were associated with each intermediate state for each protein. These data show that the folding path for proteins in the same structural family can be quite different, and provide evidence for different folding landscapes for these sequences. Proteins 33:107–118, 1998. © 1998 Wiley-Liss, Inc.     

Unusually stable β-sheet formation in an ionic self-complementary oligopeptide

A 16-residue amphiphilic oligopeptide (EAK16) with every other residue alanine and also containing glutamic acid and lysine (Ac-NH-AEAEAKAKAEAEAKAK-CONH₂) is able to form an unusually stable β-sheet structure. The β-sheet structure is stable at very low concentrations in water and at high temperatures. Various pH changes at 1.5, 3, 7, and 11 had little effect on the stability of the β-sheet structure. The β-sheet structure was not altered significantly even in the presence of 0.1% SDS, 7 molar guanidine hydrochloride, or 8 molar urea. One of the structural characteristics of the EAK16 is its ionic self-complementarity in that ionic bonds and hydrogen bonds between Glu and Lys can form readily between two oligopeptide β-sheet structures. This structural feature is probably one of the factors that promotes its extreme stability. This is the first example of such an extended ionic self-complementarity in a protein structure. EAK16 and its related peptides may have applications as useful biomaterials. It also offers a good model for studying the mechanism of β-sheet formation. Because the oligopeptide can self-assemble to form a membranous structure, it may have relevance to origin of life research. © 1994 John Wiley & Sons, Inc.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Five-stranded β-sheet sandwiched with two α-helices: A structural link between restriction endonucleases EcoRI and EcoRV

Examination of crystal structures of restriction endonucleases EcoRI and EcoRV complexes with their cognate DNA revealed a common structural element, which forms the core of both proteins. This element consists of a five-stranded β-sheet and two α-helices packed against it and could be described as α–β sandwich in which helices and β-strands lie in two stacked layers. While the spatial structure of this α–β sandwich is conserved in both enzymes, there are no detectable similarities between amino acid sequences except of a few residues involved in active site formation. Probably, other restriction endonucleases which have similar organization of the active site might possess similar structural element regardless of DNA sequence recognized and recognition elements in the enzyme used. © 1994 Wiley-Liss, Inc.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Protein structure and the sequential structure of mRNA: α-Helix and β-sheet signals at the nucleotide level

A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed. We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting protein. The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain. A complete search for GenBank nucleotide sequences coding for structural entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment. By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets. These signals do not originate from the clustering of rare codons, but from the similarity of codons coding for very abundant amino acid residues at the N- and C-termini of helices and sheets. No correlation between the positioning of rare codons and the location of structural units was found. The mRNA signals were also compared with conserved nucleotide features of 16S-like ribosomal RNA sequences and related to mechanisms for maintaining the correct reading frame by the ribosome. © 1996 Wiley-Liss, Inc.     

NMR study of the reconstitution of the β-sheet of thioredoxin by fragment complementation

The study of complementary protein fragments is thought to be generally useful to identify early folding intermediates. A prerequisite for these studies is the reconstitution of the native-like structure by fragment complementation. Structural analysis of the complementation of the domain-sized proteolytic fragments of E. coli thioredoxin, using a combination of H-exchange and 2D NMR experiments as a fingerprint technique, provide evidence for the extensive reconstitution of a native β-sheet, with local conformational adjustments near the cleavage site. Remarkably, the antiparallel β-strand between the fragments shows a native-like protection of the amide protons to solvent exchange. Our results indicate that these fragments can be useful to study the early events in the still little understood formation of β-sheets. © 1995 Wiley-Liss, Inc.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

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

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

Syntheses and anti‐tubercular activity of β‐substituted and α,β‐disubstituted peptidyl β‐aminoboronates and boronic acids

Tuberculosis is still affecting millions of people worldwide, and new resistant strains of Mycobacterium tuberculosis are being found. It is therefore necessary to find new compounds for treatment. In this paper, we report the synthesis and in vitro testing of peptidyl β‐aminoboronic acids and β‐aminoboronates with anti‐tubercular activity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Plasma gelsolin facilitates interaction between β2 glycoprotein I and α5β1 integrin

Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL) that directly recognizes plasma β₂‐glycoprotein I (β₂GPI). Tissue factor (TF), the major initiator of the extrinsic coagulation system, is induced on monocytes by aPL in vitro, explaining in part the pathophysiology in APS. We previously reported that the mitogen‐activated protein kinase (MAPK) pathway plays an important role in aPL‐induced TF expression on monocytes. In this study, we identified plasma gelsolin as a protein associated with β₂GPI by using immunoaffinity chromatography and mass spectrometric analysis. An in vivo binding assay showed that endogenous β₂GPI interacts with plasma gelsolin, which binds to integrin a₅β₁ through fibronectin. The tethering of β₂GPI to monoclonal anti‐β₂GPI autoantibody on the cell surface was enhanced in the presence of plasma gelsolin. Immunoblot analysis demonstrated that p38 MAPK protein was phosphorylated by monoclonal anti‐β₂GPI antibody treatment, and its phosphorylation was attenuated in the presence of anti‐integrin a₅β₁ antibody. Furthermore, focal adhesion kinase, a downstream molecule of the fibronectin‐integrin signalling pathway, was phosphorylated by anti‐β₂GPI antibody treatment. These results indicate that molecules including gelsolin and integrin are involved in the anti‐β₂GPI antibody‐induced MAPK pathway on monocytes and that integrin is a possible therapeutic target to modify a prothrombotic state in patients with APS.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Ecosystem stability in space: α, β and γ variability

The past two decades have seen great progress in understanding the mechanisms of ecosystem stability in local ecological systems. There is, however, an urgent need to extend existing knowledge to larger spatial scales to match the scale of management and conservation. Here, we develop a general theoretical framework to study the stability and variability of ecosystems at multiple scales. Analogously to the partitioning of biodiversity, we propose the concepts of alpha, beta and gamma variability. Gamma variability at regional (metacommunity) scale can be partitioned into local alpha variability and spatial beta variability, either multiplicatively or additively. On average, variability decreases from local to regional scales, which creates a negative variability–area relationship. Our partitioning framework suggests that mechanisms of regional ecosystem stability can be understood by investigating the influence of ecological factors on alpha and beta variability. Diversity can provide insurance effects at the various levels of variability, thus generating alpha, beta and gamma diversity–stability relationships. As a consequence, the loss of biodiversity and habitat impairs ecosystem stability at the regional scale. Overall, our framework enables a synthetic understanding of ecosystem stability at multiple scales and has practical implications for landscape management.     

The Muscle-Specific Laminin Receptor α7β1 Integrin Negatively Regulates α5β1 Fibronectin Receptor Function

α7β1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the α7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with α7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the α7β1. α7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of α7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the α5β1 fibronectin receptor. Although cell surface expression of α5β1 was reduced by a factor of 20–25% in α7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of¹²⁵I-fibronectin for its surface receptor was decreased by 50% in α7 transfectants, indicating that the α5β1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn²⁺, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn²⁺restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in α7 transfectants. These data indicate that α7 expression leads to the functional down regulation of α5β1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of anegative cooperativitybetween α7 and α5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.