Identification and analysis of residues contained on β → α loops of the dual‐substrate (βα)8 phosphoribosyl isomerase A specific for its phosphoribosyl anthranilate isomerase activity

A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient‐like dual‐substrate (βα)₈ phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis–Menten enzyme kinetics for both isomerase activities in wild‐type PriA from S. coelicolor and in selected single‐residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important β → α loop 5, namely, Arg¹³⁹, which was postulated on structural grounds to be important for the dual‐substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser⁸¹Thr and PriA_Arg¹³⁹Asn showed that these residues, which are contained on β → α loops and in close proximity to the N‐terminal phosphate‐binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X‐ray crystallographic structure of PriA_Arg¹³⁹Asn elucidated at 1.95 Å herein strongly implicates the occurrence of conformational changes in this β → α loop as a major structural feature related to the evolution of the dual‐substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates—within a bifunctional and thus highly constrained active site—without compromising its structural stability.     

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     

Re‐engineering a β‐lactamase using prototype peptides from a library of local structural motifs

B. licheniformis exo‐small β‐lactamase (ESBL) has a complex architecture with twelve α helices and a five‐stranded beta sheet. We replaced, separately or simultaneously, three of the ESBL α helices with prototype amphiphatic helices from a catalog of secondary structure elements. Although the substitutes bear no sequence similarity to the originals and pertain to unrelated protein families, all the engineered ESBL variants were found able to fold in native like structures with in vitro and in vivo enzymic activity. The triple substituted variant resembles a primitive protein, with folding defects such as a strong tendency to oligomerization and very low stability; however it mimics a non homologous recombinant abandoning the family sequence space while preserving fold. The results test protein folding and evolution theories.     

The expression of α2β1 integrin and α smooth muscle actin in fibroblasts grown on collagen

The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β₁ family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α₂β₁ integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α₂β₁ integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α₂β₁ integrin on their membranes. Using [³⁵S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α₂β₁ integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α₂β₁ integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd.     

Induction of mouse β integrin expression following transfection with human α4 chain

We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy‐sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ‐like proteins, plants also encode a hybrid βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in‐depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast‐containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre‐CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.     

Gimmicks of gamma‐aminobutyric acid (GABA) in pancreatic β‐cell regeneration through transdifferentiation of pancreatic α‐ to β‐cells

In vivo regeneration of lost or dysfunctional islet β cells can fulfill the promise of improved therapy for diabetic patients. To achieve this, many mitogenic factors have been attempted, including gamma‐aminobutyric acid (GABA). GABA remarkably affects pancreatic islet cells’ (α cells and β cells) function through paracrine and/or autocrine binding to its membrane receptors on these cells. GABA has also been studied for promoting the transformation of α cells to β cells. Nonetheless, the gimmickry of GABA‐induced α‐cell transformation to β cells has two different perspectives. On the one hand, GABA was found to induce α‐cell transformation to β cells in vivo and insulin‐secreting β‐like cells in vitro. On the other hand, GABA treatment showed that it has no α‐ to β‐cell transformation response. Here, we will summarize the physiological effects of GABA on pancreatic islet β cells with an emphasis on its regenerative effects for transdifferentiation of islet α cells to β cells. We will also critically discuss the controversial results about GABA‐mediated transdifferentiation of α cells to β cells.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

The β‐cyanoalanine synthase pathway: beyond cyanide detoxification

Production of cyanide through biological and environmental processes requires the detoxification of this metabolic poison. In the 1960s, discovery of the β ‐cyanoalanine synthase ( β ‐CAS) pathway in cyanogenic plants provided the first insight on cyanide detoxification in nature. Fifty years of investigations firmly established the protective role of the β ‐CAS pathway in cyanogenic plants and its role in the removal of cyanide produced from ethylene synthesis in plants, but also revealed the importance of this pathway for plant growth and development and the integration of nitrogen and sulfur metabolism. This review describes the β ‐CAS pathway, its distribution across and within higher plants, and the diverse biological functions of the pathway in cyanide assimilation, plant growth and development, stress tolerance, regulation of cyanide and sulfide signalling, and nitrogen and sulfur metabolism. The collective roles of the β ‐CAS pathway highlight its potential evolutionary and ecological importance in plants.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Human embryonic stem cell differentiation into insulin secreting β‐cells for diabetes

hESC (human embryonic stem cells), when differentiated into pancreatic β ILC (islet‐like clusters), have enormous potential for the cell transplantation therapy for Type 1 diabetes. We have developed a five‐step protocol in which the EBs (embryoid bodies) were first differentiated into definitive endoderm and subsequently into pancreatic lineage followed by formation of functional endocrine β islets, which were finally matured efficiently under 3D conditions. The conventional cytokines activin A and RA (retinoic acid) were used initially to obtain definitive endoderm. In the last step, ILC were further matured under 3D conditions using amino acid rich media (CMRL media) supplemented with anti‐hyperglycaemic hormone‐Glp1 (glucagon‐like peptide 1) analogue Liraglutide with prolonged t_½ and Exendin 4. The differentiated islet‐like 3D clusters expressed bonafide mature and functional β‐cell markers‐PDX1 (pancreatic and duodenal homoeobox‐1), C‐peptide, insulin and MafA. Insulin synthesis de novo was confirmed by C‐peptide ELISA of culture supernatant in response to varying concentrations of glucose as well as agonist and antagonist of functional 3D β islet cells in vitro. Our results indicate the presence of almost 65% of insulin producing cells in 3D clusters. The cells were also found to ameliorate hyperglycaemia in STZ (streptozotocin) induced diabetic NOD/SCID (non‐obese diabetic/severe combined immunodeficiency) mouse up to 96 days of transplantation. This protocol provides a basis for 3D in vitro generation of long‐term in vivo functionally viable islets from hESC.     

Expression of γ‐tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal‐induced oxidative stresses by reducing reactive oxygen species

The γ‐tocopherol methyltransferase (γ‐TMT) is an important enzyme regulating synthesis of four tocopherols (α, γ, β and δ). In this report, we investigated the role of γ‐TMT in regulating abiotic stress within chloroplasts. The At γ‐tmt overexpressed via the tobacco chloroplast genome accumulated up to 7.7% of the total leaf protein, resulting in massive proliferation of the inner envelope membrane (IEM, up to eight layers). Such high‐level expression of γ‐TMT converted most of γ‐tocopherol to α‐tocopherol in transplastomic seeds (~10‐fold higher) in the absence of abiotic stress. When grown in 400 mm NaCl, α‐tocopherol content in transplastomic TMT leaves increased up to 8.2‐fold and 2.4‐fold higher than wild‐type leaves. Likewise, under heavy metal stress, α‐tocopherol content in the TMT leaves increased up to 7.5‐fold, twice higher than in the wild type. Under extreme salt stress, the wild type accumulated higher starch and total soluble sugars, but TMT plants were able to regulate sugar transport. Hydrogen peroxide and superoxide content in wild type increased up to 3‐fold within 48 h of NaCl stress when compared to TMT plants. The ion leakage from TMT leaves was significantly less than wild‐type plants under abiotic stress and with less malondialdehyde, indicating lower lipid peroxidation. Taken together, these studies show that α‐tocopherol plays a crucial role in the alleviation of salt and heavy metal stresses by decreasing ROS, lipid peroxidation and ion leakage, in addition to enhancing vitamin E conversion. Increased proliferation of the IEM should facilitate studies on retrograde signalling from chloroplast to the nucleus.     

Occurrence of Phomopsis longicolla β Conidia in Naturally Infected Soybean

Although Phomopsis longicolla is primarily known as a seedborne pathogen, it can be isolated from all parts of the plant. The disease lesions observed on the basal parts of soybean stems were slightly sunken with irregular shapes and sizes, bordered by a thin black margin. Within the lesions themselves, large and diffusely distributed pycnidia with α and β conidia, typical of the genus Phomopsis, were observed. The percentages of the two types of conidia varied considerably, but β conidia were predominant in most of the pycnidia. The presence of these reproductive organs indicated that the symptoms could have been caused by Phomopsis sojae. However, after isolation on a nutritive medium, all cultural and morphological characteristics clearly indicated that the isolated fungus was P. longicolla, whose identification was subsequently confirmed by sequencing three genomic regions. Monosporic isolates, with different ratios of α and β conidia, exhibited a high level of pathogenicity on soybean, after artificial inoculation. Both types of conidia were observed on the stems of the inoculated soybean plants. Beta conidia also formed quickly on medium made of soybean seeds and mature stems after exposure to low temperatures (?10°C). This study suggests that P. longicolla is capable of a massive production of β conidia, not only in old fungal cultures as it had until now been believed, but also in infected soybean plants in the field.     

Selection and structural analysis of de novo proteins from an α3β3 genetic library

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.     

Molten globule state of equine β-Lactoglobulin

The acid-unfolded state of equine β-lactoglobulin was characterized by means of circular dichroism, nuclear magnetic resonance, analytical gel-filtration chromatography, and analytical centrifugation. The acid-unfolded state of equine β-lactoglobulin has a substantial secondary structure as shown by the far-ultraviolet circular dichroism spectrum but lacks persistent tertiary packing of the side chains as indicated by the near-ultraviolet circular dichroism and nuclear magnetic resonance spectra. It is nearly as compact as the native conformation as shown by the gel filtration and sedimentation experiments, and it has the exposed hydrophobic surface as indicated by its tendency to aggregate. All of these characteristics indicate that the acid-unfolded state of equine β-lactoglobulin is a molten globule state. The α helix content in the acid-unfolded state, which has been estimated from the circular dichroism spectrum, is larger than that in the native state, suggesting the presence of nonnative α helices in the molten globule state. This result suggests the generality of the intermediate with nonnative α helices during the folding of proteins having the β-clam fold. © 1997 Wiley-Liss Inc.     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Aromatic interactions with naphthylalanine in a β‐hairpin peptide

Stable peptides have been explored as epitope mimics for protein–protein and protein–nucleic acid interactions; however, presentation of a regular structure is critical. Aromatic interactions are ubiquitous and are competent at stabilizing a β‐hairpin fold. The greatest stabilization has been reported from pairs of tryptophan side chains. Naphthylalanine residues are often used as tryptophan replacements, but it is not clear if 1‐naphthylalanine or 2‐naphthylalanine is adequate at replicating the geometry and stability observed with tryptophan aromatic interactions. Herein, a 12‐residue peptide has been constructed with laterally disposed aromatic amino acids. A direct comparison is made between tryptophan and other bicyclic, unnatural amino acids. Significant stabilization is gained from all bicyclic amino acids; however, geometric analysis shows that only 1‐naphthylalanine adopts a similar edge to face geometry as tryptophan, whereas the 2‐naphthylalanine appears most similar to a substituted phenylalanine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of external electrostatic field on the stability of β sheet structures

To explore the effect of an external electrostatic field (EEF) on the stability of protein conformations, the molecular dynamic modeling approach was applied to evaluate the effect of an EEF along the x or y direction on a water cluster containing a parallel or antiparallel β sheet structure. The β sheet structure contained two strands with a (Gly)₃ sequence separated by a distance d along the x direction. The mean forces between the two strands along the x direction were computed from the trajectories of molecular dynamics simulations. In the absence of the EEF, the forces between the two strands in vacuum were repulsive and attractive in the parallel and antiparallel β sheet structures, respectively. In contrast, the mean forces between the two strands in water were attractive in both the parallel and antiparallel β sheet structures. This is because the electric interactions between the two strands were shielded by water, and the hydrophobic effect dominated the interaction between the two strands. When an EEF >50 MV/cm was applied to the water cluster, the attractive force between the two strands in the parallel and antiparallel β sheet structures decreased and increased, respectively. Further, the binding affinity between the two strands in the parallel and antiparallel β sheet structures also decreased and increased, respectively. This is because the large EEF leads to dielectric saturation, and consequently reduces the effects of the dielectric shielding and hydrophobic interactions. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 861–870, 2014.