Important Role In Folding Research Articles

The structure of salt bridges between Arg(+) and Glu(-) in peptides investigated with 2D-IR spectroscopy: Evidence for two distinct hydrogen-bond geometries.

Salt bridges play an important role in protein folding and in supramolecular chemistry, but they are difficult to detect and characterize in solution. Here, we investigate salt bridges between glutamate (Glu(-)) and arginine (Arg(+)) using two-dimensional infrared (2D-IR) spectroscopy. The 2D-IR spectrum of a salt-bridged dimer shows cross peaks between the vibrational modes of Glu(-) and Arg(+), which provide a sensitive structural probe of Glu(-)⋯Arg(+) salt bridges. We use this probe to investigate a β-turn locked by a salt bridge, an α-helical peptide whose structure is stabilized by salt bridges, and a coiled coil that is stabilized by intra- and intermolecular salt bridges. We detect a bidentate salt bridge in the β-turn, a monodentate one in the α-helical peptide, and both salt-bridge geometries in the coiled coil. To our knowledge, this is the first time 2D-IR has been used to probe tertiary side chain interactions in peptides, and our results show that 2D-IR spectroscopy is a powerful method for investigating salt bridges in solution.

Novel numerical characterization of protein sequences based on individual amino acid and its application.

The hydrophobicity and hydrophilicity of amino acids play a very important role in protein folding and its interaction with the environment and other molecules, as well as its catalytic mechanism. Based on the two physicochemical indexes, a 2D graphical representation of protein sequences is introduced; meanwhile, a new numerical characteristic has been proposed to compute the distance of different sequences for analysis of sequence similarity/dissimilarity on the basis of this graphical representation. Furthermore, we apply the new distance in the similarities/dissimilarities of ND5 proteins of nine species and predict the four major classes based on the dataset containing 639 domains. The results show that the method is simple and effective.

DPP6 Domains Responsible for Its Localization and Function

Dipeptidyl peptidase-like protein 6 (DPP6) is an auxiliary subunit of the Kv4 family of voltage-gated K(+) channels known to enhance channel surface expression and potently accelerate their kinetics. DPP6 is a single transmembrane protein, which is structurally remarkable for its large extracellular domain. Included in this domain is a cysteine-rich motif, the function of which is unknown. Here we show that this cysteine-rich domain of DPP6 is required for its export from the ER and expression on the cell surface. Disulfide bridges formed at C349/C356 and C465/C468 of the cysteine-rich domain are necessary for the enhancement of Kv4.2 channel surface expression but not its interaction with Kv4.2 subunits. The short intracellular N-terminal and transmembrane domains of DPP6 associates with and accelerates the recovery from inactivation of Kv4.2, but the entire extracellular domain is necessary to enhance Kv4.2 surface expression and stabilization. Our findings show that the cysteine-rich domain of DPP6 plays an important role in protein folding of DPP6 that is required for transport of DPP6/Kv4.2 complexes out of the ER.

Abstract 1255: The Mediterranean herb rosemary modulates ER stress proteins to promote androgen receptor degradation in prostate cancer cells

Abstract Background: Prostate cancer is one of the most prevalent forms of cancer among men, especially in the United States. Epidemiological studies have shown that consumption of natural dietary phytochemicals helps in prevention of many types of cancers. One such dietary phytochemical, rosemary (Rosmarinus officinalis), widely used in Mediterranean cuisine is well known for its antitumor activities. However, not much is known about the effect of rosemary on pathogenesis of prostate cancer. Androgens play a critical role in progression of prostate cancer and hence androgen receptor has been a target for prostate cancer treatment since a long time. Herein we have examined the effect of rosemary on prostate cancer progression. Specifically we have examined how rosemary promotes endoplasmic reticulum (ER) stress and modulates the activity and expression of androgen receptor (AR). Materials and Methods: Prostate cancer cell lines, 22Rv1 and LNCaP cells, were used for all experiments. Cell proliferation and cell viability were studied using BrdU and MTT assay respectively. Western blotting, ELISA and siRNA were used to determine the association between ER stress and AR degradation. Results: Rosemary inhibited growth and decreased the cell viability of 22Rv1 and LNCaP cells in a dose-dependent manner. The anti-proliferative effect was associated with apoptosis as evidenced by a significant increase in levels of cleaved caspase-3 protein and TUNEL assay. Moreover, upon treatment with rosemary we saw a dose-dependent decrease in expression of AR in 22Rv1 and LNCaP cells along with decreased PSA levels in LNCaP cells. Rosemary induced apoptosis was associated with ER stress as evidenced by up-regulation of ER stress proteins PERK, IRE1, BiP, CHOP, XBP-1 and caspase- 4. ER stress proteins are known to play an important role in folding of misfolded proteins and degrading continuously misfolded proteins. We therefore explored the relationship between ER stress and AR degradation. Interestingly, we observed that AR degradation was dependent on ER stress proteins BiP and CHOP. Conclusion: Rosemary induces ER stress-mediated apoptotic cell death in prostate cancer cell lines, 22Rv1and LNCaP cells, leading to disruption of AR expression. Our data suggests that this Mediterranean dietary phytochemical possess strong anticancer activity and should be evaluated further as potential chemopreventive and/or chemotherapeutic agent for controlling prostate cancer. Citation Format: Sakina M. Petiwala, Saba Berhe, Gongbo Li, Angela G. Puthenveetil, Larisa Nonn, Jeremy J. Johnson. The Mediterranean herb rosemary modulates ER stress proteins to promote androgen receptor degradation in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1255. doi:10.1158/1538-7445.AM2014-1255

Epithelial sheet folding induces lumen formation by Madin-Darby canine kidney cells in a collagen gel.

Lumen formation is important for morphogenesis; however, an unanswered question is whether it involves the collective migration of epithelial cells. Here, using a collagen gel overlay culture method, we show that Madin-Darby canine kidney cells migrated collectively and formed a luminal structure in a collagen gel. Immediately after the collagen gel overlay, an epithelial sheet folded from the periphery, migrated inwardly, and formed a luminal structure. The inhibition of integrin-β1 or Rac1 activity decreased the migration rate of the peripheral cells after the sheets folded. Moreover, lumen formation was perturbed by disruption of apical-basolateral polarity induced by transforming growth factor-β1. These results indicate that cell migration and cell polarity play an important role in folding. To further explore epithelial sheet folding, we developed a computer-simulated mechanical model based on the rigidity of the extracellular matrix. It indicated a soft substrate is required for the folding movement.

Looping and reconfiguration dynamics of a flexible chain with internal friction

In recent past, experiments and simulations have suggested that apart from the solvent friction, friction arising from the protein itself plays an important role in protein folding by affecting the intra-chain loop formation dynamics. This friction is termed as internal friction in the literature. Using a flexible Gaussian chain with internal friction we analyze the intra-chain reconfiguration and loop formation times for all three topology classes namely end-to-end, end-to-interior and interior-to-interior. In a nutshell, bypassing expensive simulations we show how simple models like that of Rouse and Zimm can support the single molecule experiment and computer simulation results on intra-chain diffusion coefficients, looping time and even can predict the effects of tail length on the looping time.

Dietary soy isoflavones increased hepatic protein disulfide isomerase content and suppressed its enzymatic activity in rats

Protein disulfide isomerase (PDI) is a multifunctional protein and plays important roles in protein folding, triglyceride transfer, insulin degradation, and thyroid hormone transportation. This study examined the modulation of PDI expression by soy consumption using rat as a model. Sprague-Dawley male and female rats at 50 days (d) of age were fed diets containing either 20% casein or alcohol-washed soy protein isolate (SPI, containing 50 mg isoflavones (ISFs)/kg diet) or SPI plus ISF (250 mg/kg diet) and mated at age of 120 d. The offspring (F1) were fed the same diets as their parents. Addition of ISF to SPI diet markedly increased PDI protein content in the liver and testis of the adult rats compared with the casein or SPI diet. PDI mRNA abundance in the liver and protein content in the brain, thyroid, heart, and uterus were unchanged by the diets. Two-dimensional Western blot showed that the rats fed diets containing SPI had a diminished hepatic PDI protein with an isoelectric point (pI) of 6.12, a dephosphorylated form, compared with the rats fed diets containing either casein or SPI with supplemental ISF. Soy ISF added into SPI diet remarkably suppressed hepatic PDI activity of the rats compared with the casein diet. Moreover, soy ISF dose-dependently increased PDI and thyroid hormone receptor (TR) β protein content, whereas reduced TR DNA binding ability in human hepatocytes. Overall, this study shows that soy ISF increased hepatic PDI protein content, but addition of ISF into SPI diet inhibited its enzymatic activities and this effect may be mediated through a post-transcriptional mechanism.

Time-Resolved Temperature-Jump Ir-Measurements on Beta-Hairpin Peptides with Alternate Cross-Strand Interactions

The formation of β-sheets plays an important role in protein folding. We studied the folding dynamics of tryptophan zipper (Trpzip) peptides being ideal simple model systems of β-hairpins stabilized by the hydrophobic interactions of two cross-strand Trp-Trp pairs. Rapid heating of the solvent is induced by a Raman-shifted Nd:YAG pulse (ΔT ∼ 10°C in 10 ns) and ns-to-µs peptide dynamics were monitored at single wavelengths using a quantum cascade laser tunable in the amide I region. Our isotope-edited kinetics support a multistate dynamic behavior being consistent with a hydrophobic collapse hypothesis for the folding process of Trpzip2 β-hairpin variants. In order to analyze hydrophobic interactions, the tryptophans in the Trpzip2 sequence (SWTWENGKWTWK-NH2) were replaced pairwise by either valines or tyrosines. Equilibrium CD and FTIR measurements show lower transition temperatures for all mutants. While the Trp-Trp stabilized peptides have different relaxation rates for the loss of sheet and gain of disordered structure, our mutant studies indicate that the aromatic substitution of Trp→Tyr results in kinetics being faster than for Trpzip2, but essentially the same for the sheet and disordered components, with similar activation energies. However with Val substitutions, different relaxation kinetics were found. Our time-resolved temperature-jump measurements reveal the impact of cross-strand interactions on the folding mechanism of peptides, in particular illustrate different effects of hydrophobic versus aromatic interactions on stability and dynamics of structure formation.

Identification of Key Hinge Residues Important for Nucleotide-Dependent Allostery in E. coli Hsp70/DnaK

DnaK is a molecular chaperone that has important roles in protein folding. The hydrolysis of ATP is essential to this activity, and the effects of nucleotides on the structure and function of DnaK have been extensively studied. However, the key residues that govern the conformational motions that define the apo, ATP-bound, and ADP-bound states are not entirely clear. Here, we used molecular dynamics simulations, mutagenesis, and enzymatic assays to explore the molecular basis of this process. Simulations of DnaK's nucleotide-binding domain (NBD) in the apo, ATP-bound, and ADP/Pi-bound states suggested that each state has a distinct conformation, consistent with available biochemical and structural information. The simulations further suggested that large shearing motions between subdomains I-A and II-A dominated the conversion between these conformations. We found that several evolutionally conserved residues, especially G228 and G229, appeared to function as a hinge for these motions, because they predominantly populated two distinct states depending on whether ATP or ADP/Pi was bound. Consistent with the importance of these “hinge” residues, alanine point mutations caused DnaK to have reduced chaperone activities in vitro and in vivo. Together, these results clarify how sub-domain motions communicate allostery in DnaK.

Dinosolve: a protein disulfide bonding prediction server using context-based features to enhance prediction accuracy

BackgroundDisulfide bonds play an important role in protein folding and structure stability. Accurately predicting disulfide bonds from protein sequences is important for modeling the structural and functional characteristics of many proteins.MethodsIn this work, we introduce an approach of enhancing disulfide bonding prediction accuracy by taking advantage of context-based features. We firstly derive the first-order and second-order mean-force potentials according to the amino acid environment around the cysteine residues from large number of cysteine samples. The mean-force potentials are integrated as context-based scores to estimate the favorability of a cysteine residue in disulfide bonding state as well as a cysteine pair in disulfide bond connectivity. These context-based scores are then incorporated as features together with other sequence and evolutionary information to train neural networks for disulfide bonding state prediction and connectivity prediction.ResultsThe 10-fold cross validated accuracy is 90.8% at residue-level and 85.6% at protein-level in classifying an individual cysteine residue as bonded or free, which is around 2% accuracy improvement. The average accuracy for disulfide bonding connectivity prediction is also improved, which yields overall sensitivity of 73.42% and specificity of 91.61%.ConclusionsOur computational results have shown that the context-based scores are effective features to enhance the prediction accuracies of both disulfide bonding state prediction and connectivity prediction. Our disulfide prediction algorithm is implemented on a web server named "Dinosolve" available at: http://hpcr.cs.odu.edu/dinosolve.

Energetics of β-turn formation in a mutant peptide YPGDV from influenza hemagglutinin: an MD simulation study

Reverse turns play an important role in protein folding, molecular recognition and in eliciting immune response. While sequence determinants of reverse turns are known, not much is known about their energetics. In this paper we have investigated the thermodynamics of a reverse turn sequence YPGDV, an experimentally well characterized turn sequence, using molecular dynamics simulations performed over a range of temperatures from 280-360 K using GROMACS 4.0.4 software and all atom OPLS-AA/L force field. The change in folding free energy (ΔAfolding) for the β-turn formation in YPGDV peptide shows a linear relationship with temperature. We find that the entropy change (ΔSfolding) for the β-turn formation is close to zero and the internal energy change (ΔUfolding) is a modest -3.8 kJ mol(-1). These thermodynamic quantities are interpreted in terms of intra-molecular (intra-peptide) and inter-molecular (peptide-solvent) hydrogen bonding interactions. Implications for protein folding and peptide immunogenicity are discussed.

The Influence of Synonymous Codon Bias on Protein Folding Rates

Based on the present small data of protein folding rates and related works, a bigger protein folding rates data were constructed, and the information of mRNA of each protein was added in the new data. For each protein, the codon usage bias of its corresponding mRNA sequence was calculated, then, for each kind of proteins, the correlation between protein coding rates and the codon usage bias of their corresponding mRNA sequence were analyzed. The results showed that, for some codons, protein coding rates significantly correlate with the codon usage bias of their corresponding mRNA sequence. It means that synonymous codon usage bias may play an important role in protein folding.

Robustness in Protein Folding Revealed by Thermodynamics Calculations

Long-range intraprotein interactions play important roles in protein folding. In the present study, we use two variants of the B domain of protein A (BdpA F14W/G30A and BdpA_ds) and two variants of the Trp cage (TC5b_P1 and TC5b_P2) as models to investigate how long-range hydrophobic interactions affect protein tertiary and secondary structures. The mutation of the selected residues (BdpA F14W/G30A) or the change in the sequence order of Helix1 and Helix2 (BdpA_ds) changes detailed hydrophobic interactions. However, this change does not alter the global three-helix-bundle structure of BdpA and the overall shape of the folding free-energy landscape. It does affect the formation and stability of individual secondary structures. Similarly, the addition of an extra segment to the C-terminus of Trp-cage increases the number of long-range hydrophobic interactions without making any significant change of the native structure of Trp-cage. These results show the robustness of the overall protein folding, where rather large sequence changes exert significant influences on secondary but not tertiary structures.

Protein Dynamics Governed by Interfaces of High Polarity and Low Packing Density

The folding pathway, three-dimensional structure and intrinsic dynamics of proteins are governed by their amino acid sequences. Internal protein surfaces with physicochemical properties appropriate to modulate conformational fluctuations could play important roles in folding and dynamics. We show here that proteins contain buried interfaces of high polarity and low packing density, coined as LIPs: Light Interfaces of high Polarity, whose physicochemical properties make them unstable. The structures of well-characterized equilibrium and kinetic folding intermediates indicate that the LIPs of the corresponding native proteins fold late and are involved in local unfolding events. Importantly, LIPs can be identified using very fast and uncomplicated computational analysis of protein three-dimensional structures, which provides an easy way to delineate the protein segments involved in dynamics. Since LIPs can be retained while the sequences of the interacting segments diverge significantly, proteins could in principle evolve new functional features reusing pre-existing encoded dynamics. Large-scale identification of LIPS may contribute to understanding evolutionary constraints of proteins and the way protein intrinsic dynamics are encoded.

Japanese flounder (Paralichthys olivaceus) Hsp70: Adjuvant effect and its dependence on the intrinsic ATPase activity

Heat shock protein (Hsp) 70 is a molecular chaperone that plays an important role in protein folding and transport. In addition, Hsp70 is also involved in regulation of innate and adaptive immune response. In this study, we examined the biological activity and the immunomodulatory property of an Hsp70 homologue, PoHsp70, from Japanese flounder (Paralichthys olivaceus). Recombinant PoHsp70 purified from Escherichia coli exhibits apparent ATPase activity; however, a mutant PoHsp70, PoHsp70M, that bears mutation of the ATPase-associated domain, was completely abolished in activity. Expression of PoHsp70 was upregulated in a time-dependent manner by vaccination of flounder with a DNA vaccine, pSia10, that expresses a Streptococcus iniae antigen, Sia10. To examine whether PoHsp70 possessed any adjuvant potential, the DNA vaccine plasmids pSia10Hsp70 and pSia10Hsp70M were constructed. pSia10Hsp70 co-expresses Sia10 and PoHsp70, while pSia10Hsp70M co-expresses Sia10 and PoHsp70M. Following vaccination of flounder, production of Sia10 plus PoHsp70 and Sia10 plus PoHsp70M was detected in pSia10Hsp70- and pSia10Hsp70M-vaccinated fish respectively. At one month post-vaccination, comparable levels of serum antibodies were detected in fish vaccinated with pSia10Hsp70, pSia10Hsp70M, and pSia10. Subsequent protection analysis showed that, following S. iniae challenge, pSia10Hsp70 induced a survival rate that was significantly higher than that induced by pSia10, while pSia10Hsp70M induced a survival rate similar to that induced by pSia10. These results indicate that PoHsp70 is an effective adjuvant and that the adjuvanticity of PoHsp70 requires the intrinsic ATPase activity.

Identification of HSP90 gene from the Chinese oak silkworm, Antheraea pernyi

The heat shock proteins (Hsp) play an important role in protein folding and protection of cells from stress. To investigate the role of Hsp90 in silk-producing insect Antheraea pernyi (Lepidoptera: Saturniidae), a full-length cDNA encoding Hsp90 from A. pernyi was cloned, sequenced and characterized. The complete cDNA (2,482 bp) contained a 2,154 bp open reading frame encoding 717 amino acid residues and had 94.5% identity with Antheraea yamamai Hsp90. The relative expression levels of Hsp90 in five different tissues at normal and high temperatures were evaluated with real-time fluorescence quantitative RT-PCR. The expression of Hsp90 was obviously changed in the examined tissues except for fat bodies after induced by high temperature. SDS-PAGE of purified protein demonstrated that an 86 KD recombinant protein was successfully expressed in transformed Escherichia coli cells. These results shed light on studying the mechanism of tolerance in A. pernyi. Key words: Antheraea pernyi, HSP90, sequence analysis, expression.

Intrinsic energy landscapes of amino acid side-chains.

Amino acid side-chain conformational properties influence the overall structural and dynamic properties of proteins and, therefore, their biological functions. In this study, quantum mechanical (QM) potential energy surfaces for the rotation of side-chain χ(1) and χ(2) torsions in dipeptides in the alphaR, beta, and alphaL backbone conformations were calculated. The QM energy surfaces provide a broad view of the intrinsic conformational properties of each amino acid side-chain. The extent to which intrinsic energetics dictates side-chain orientation was studied through comparisons of the QM energy surfaces with χ(1) and χ(2) free energy surfaces from probability distributions obtained from a survey of high resolution crystal structures. In general, the survey probability maxima are centered in minima of the QM surfaces as expected for sp(3) (or sp(2) for χ(2) of Asn, Phe, Trp, and Tyr) atom centers with strong variations between amino acids occurring in the energies of the minima indicating intrinsic differences in rotamer preferences. High correlations between the QM and survey data were found for hydrophobic side-chains except Met, suggesting minimal influence of the protein and solution environments on their conformational distributions. Conversely, low correlations for polar or charged side-chains indicate a dominant role of the environment in stabilizing conformations that are not intrinsically favored. Data also link the presence of off-rotamers in His and Trp to favorable interactions with the backbone. Results also suggest that the intrinsic energetics of the side-chains of Phe and Tyr may play important roles in protein folding and stability. Analyses on whether intrinsic side-chain energetics can influence backbone preference identified a strong correlation for residues in the alphaL backbone conformation. It is suggested that this correlation reflects the intrinsic instability of the alphaL backbone such that assumption of this backbone conformation is facilitated by intrinsically favorable side-chain conformations. Together our results offer a broad overview of the conformational properties of amino acid side-chains and the QM data may be used as target data for force field optimization.

Functional Evolution of Duplicated Odorant-Binding Protein Genes, Obp57d and Obp57e, in Drosophila

Odorant-binding proteins (OBPs) are extracellular proteins found in insect chemosensilla, where they participate in the sensing of odors, tastes, and pheromones. Although a large number of OBP genes have been identified in insect genomes, their molecular functions and biological roles have been clarified in limited cases. Two OBP genes, Obp57d and Obp57e, were involved in the evolution of host-plant preference in Drosophila sechellia. Comparative analyses of the Obp57d/e genomic sequences from 27 closely related species suggested that the two genes arose by tandem gene duplication and functionally diverged from each other. In this study, the functional evolution of Obp57d and Obp57e was examined by in vitro binding assays using recombinant proteins synthesized in a bacterial system. Compared to the ancestral Dpse\\OBP57de, Dmel\\OBP57d was more specialized to tridecanoic acid while Dmel\\OBP57e was generalized regarding their binding affinity, suggesting that the two OBP genes underwent subfunctionalization and neofunctionalization. A behavioral analysis using knockout flies supported that the biological role is different between OBP57d and OBP57e in vivo. Site-directed mutagenesis of the evolutionarily conserved amino acids revealed that these residues play an important role in protein folding. These findings provide a clue to understanding how the repertoire of OBP genes is maintained in a genome under natural selection.

Computing Entropies for Binding and Refinement of Protein Structures

Entropy plays an important role in protein folding and binding, but is difficult to evaluate; consequently its contribution to free energy is often neglected or locally approximated. We propose a novel method for calculating the change in free energy upon binding using coarse-grained models of proteins by combining multi-body potentials with entropies calculated from elastic network models (ENMs). Multi-body energy potentials have been developed to account for the cooperativity in globular protein structures by an improved accounting of three dimensional interactions and residue packing.

Cloning and Characterization of Bombyx mori Cyclophilin A

Cyclophilins are originally identified as cytosolic binding protein of the immunosuppressive drug cyclosporine A. They have an activity of peptidyl prolyl cis/trans-isomerases (PPIase), which may play important roles in protein folding, trafficking, assembly and cell signaling. In this study, we report the cloning and characterization of a Bombyx mori cyclophilin A (bCypA) cDNA. The full-length cDNA of bCypA consist of 947 nucleotides with a polyadenylation signal sequence AATAAA and contain an open reading frame of 498 nucleotides encoding a polypeptide of 166 amino acids. The deduced amino acid sequence of bCypA shares a central peptidyl prolyl cis/trans-isomerase and a cyclosporin-A-binding domain with other cyclophilin sequences. Relative quantification real-time (RT) PCR analysis shows that mRNA transcripts of bCypA are detected in all the investigated tissues and highest expression level in the skin of 3-day-old 5 instar larva. Also, bCypA had PPIase activity on the proline-containing peptides. Accordingly, we suggest that bCypA is a new member of the cyclophilin A (CyPA) family and will be useful for quality control of bioactivity recombinant proteins with proline-containing peptides.