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Guo, Chengchen, Gregory P. Holland, and Jeffery L. Yarger. "Lysine-Capped Silica Nanoparticles: A Solid-State NMR Spectroscopy Study." MRS Advances 1, no. 31 (2016): 2261–66. http://dx.doi.org/10.1557/adv.2016.365.
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ABSTRACTTo achieve the goal of biocompatibility in nano-based materials we must first obtain a fundamental understanding of the physical and chemical behavior of biomolecules at the interfaces of nanomaterials. A first step towards understanding protein interactions with nanomaterials is to understand how individual amino acids interact at the interfaces. In this paper, we investigated the lysine adsorption behavior on fumed silica nanoparticles by solid-state NMR spectroscopy. We use 1H, 13C and 15N solid-state magic angle spinning (MAS) NMR techniques to elucidate how lysine is adsorbed on silica nanoparticles surfaces via strong hydrogen-bonding interaction between the protonated side-chain amine group and silanol group on silica nanoparticles surfaces.*
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Vogel, Hans J. "Calmodulin: a versatile calcium mediator protein." Biochemistry and Cell Biology 72, no. 9-10 (September 1, 1994): 357–76. http://dx.doi.org/10.1139/o94-049.
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The level of intracellular calcium is strictly regulated in all cells. In a resting cell, the [Ca2+] is ≤ 10−7 M and during activation it rises to approximately 10−6 M. Calmodulin (CaM) is the secondary messenger protein that has to translate this modest rise in intracellular calcium into a physiological response in all eukaryotic cells. CaM can activate almost 30 different target systems, including smooth muscle contraction, protein kinases and phosphatases, nitric oxide synthases, and calcium-extruding pumps. It is an acidic protein of 148 amino acids with four helix–loop–helix calcium-binding domains and it has a characteristic dumbbell shape in the crystal structure. In this review I discuss which features of CaM allow it to be such a universal and versatile calcium regulator. First of all, the positive cooperative calcium binding to all four binding sites of CaM in the presence of a target protein allows the protein to act effectively during a calcium transient. Secondly, the high Met content of two hydrophobic surface patches on the two domains of CaM creates a flexible and pliable, yet sticky, interaction surface that does not place high demands on the specificity of the interaction. Consequently, calcium-CaM can bind effectively to the CaM-binding domains of all its target proteins, despite their lack of amino acid sequence homology; their only common feature is that they are hydrophobic basic peptides that have a propensity to form an α-helix. CaM's capacity to recognize its CaM-binding domains is further enhanced by its third crucial feature, the intrinsic flexibility of the central linker region; this allows the two domains of CaM to slide over the surface of the α-helical bound peptide, to find their most favourable binding orientation. In this review I have also presented selected examples of a variety of experimental techniques that have contributed to our understanding of this unique multitasking protein. These include studies with well-established techniques such as site-directed mutagenesis, chemical modification, limited proteolysis, circular dichroism, and two-dimensional nuclear magnetic resonance (NMR), as well as novel or less common approaches involving the use of unnatural amino acids, metal-ion NMR, lysine pKa determinations, and isotope-edited Fourier transform infrared spectroscopy. In combination with available structural information, these studies have provided considerable detail in our understanding of this versatile calcium regulatory protein.Key words: calmodulin, calcium metabolism, methionine, lysine, NMR spectroscopy.
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ZIMMERMAN, Aukje W., Martin RADEMACHER, Heinz RüTERJANS, Christian LüCKE, and Jacques H. VEERKAMP. "Functional and conformational characterization of new mutants of heart fatty acid-binding protein." Biochemical Journal 344, no. 2 (November 24, 1999): 495–501. http://dx.doi.org/10.1042/bj3440495.
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In this study we investigated the possible involvement of several amino acids (not located in the ligand-binding centre) in fatty acid binding and conformational stability of heart fatty acid-binding protein (H-FABP). We prepared recombinant human H-FABP proteins with mutations in the hydrophobic patch (Phe4, Trp8 and Phe64), portal region (Phe16), hinge region (Leu66, Gly67), second portal region (Glu72) and at the protein surface (Lys21) respectively. Oleic acid-binding affinity and conformational stability of human H-FABP are significantly decreased or completely lost by mutation of Trp8 or Phe16. NMR spectra confirmed that these residues are important for the stability of the protein fold. Substitution of Phe4 or Phe64 resulted in less stability, but oleic acid-binding affinity was not affected. Mutation of Lys21 had no effect on either structural integrity or fatty acid-binding affinity. Replacement of Leu66 or Gly67 did not affect fatty acid binding, but protein stability was reduced. Finally, mutation of Glu72 to Ser caused no change of affinity, but NMR spectra and urea-denaturation curves showed the extremely poor stability of this mutant. In conclusion, no relationship was observed between fatty acid-binding affinity and conformational stability.
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Ambrosi, Emmanuele, Stefano Capaldi, Michele Bovi, Gianmaria Saccomani, Massimiliano Perduca, and Hugo L. Monaco. "Structural changes in the BH3 domain of SOUL protein upon interaction with the anti-apoptotic protein Bcl-xL." Biochemical Journal 438, no. 2 (August 12, 2011): 291–301. http://dx.doi.org/10.1042/bj20110257.
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The SOUL protein is known to induce apoptosis by provoking the mitochondrial permeability transition, and a sequence homologous with the BH3 (Bcl-2 homology 3) domains has recently been identified in the protein, thus making it a potential new member of the BH3-only protein family. In the present study, we provide NMR, SPR (surface plasmon resonance) and crystallographic evidence that a peptide spanning residues 147–172 in SOUL interacts with the anti-apoptotic protein Bcl-xL. We have crystallized SOUL alone and the complex of its BH3 domain peptide with Bcl-xL, and solved their three-dimensional structures. The SOUL monomer is a single domain organized as a distorted β-barrel with eight anti-parallel strands and two α-helices. The BH3 domain extends across 15 residues at the end of the second helix and eight amino acids in the chain following it. There are important structural differences in the BH3 domain in the intact SOUL molecule and the same sequence bound to Bcl-xL.
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Nguyen, Leonard T., Paulus H. S. Kwakman, David I. Chan, Zhihong Liu, Leonie de Boer, Sebastian A. J. Zaat, and Hans J. Vogel. "Exploring Platelet Chemokine Antimicrobial Activity: Nuclear Magnetic Resonance Backbone Dynamics of NAP-2 and TC-1." Antimicrobial Agents and Chemotherapy 55, no. 5 (February 14, 2011): 2074–83. http://dx.doi.org/10.1128/aac.01351-10.
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ABSTRACTThe platelet chemokines neutrophil-activating peptide-2 (NAP-2) and thrombocidin-1 (TC-1) differ by only two amino acids at their carboxy-terminal ends. Nevertheless, they display a significant difference in their direct antimicrobial activities, with the longer NAP-2 being inactive and TC-1 being active. In an attempt to rationalize this difference in activity, we studied the structure and the dynamics of both proteins by nuclear magnetic resonance (NMR) spectroscopy. Using15N isotope-labeled protein, we confirmed that the two monomeric proteins essentially have the same overall structure in aqueous solution. However, NMR relaxation measurements provided evidence that the negatively charged carboxy-terminal residues of NAP-2 experience a restricted motion, whereas the carboxy-terminal end of TC-1 moves in an unrestricted manner. The same behavior was also seen in molecular dynamic simulations of both proteins. Detailed analysis of the protein motions through model-free analysis, as well as a determination of their overall correlation times, provided evidence for the existence of a monomer-dimer equilibrium in solution, which seemed to be more prevalent for TC-1. This finding was supported by diffusion NMR experiments. Dimerization generates a larger cationic surface area that would increase the antimicrobial activities of these chemokines. Moreover, these data also show that the negatively charged carboxy-terminal end of NAP-2 (which is absent in TC-1) folds back over part of the positively charged helical region of the protein and, in doing so, interferes with the direct antimicrobial activity.
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Aubol, Brandon E., Pedro Serrano, Laurent Fattet, Kurt Wüthrich, and Joseph A. Adams. "Molecular interactions connecting the function of the serine-arginine–rich protein SRSF1 to protein phosphatase 1." Journal of Biological Chemistry 293, no. 43 (September 5, 2018): 16751–60. http://dx.doi.org/10.1074/jbc.ra118.004587.
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Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) proteins. Protein phosphatase 1 (PP1) regulates the SR proteins by controlling phosphorylation of a C-terminal arginine-serine–rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR protein. Although PP1 has been shown to dephosphorylate the prototype SR protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR protein.
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Samuel, Dharmaraj, Hong Cheng, Paul W. Riley, Peter N. Walsh, and Heinrich Roder. "NMR Structural Analysis of Factor XI Apple 4 Domain." Blood 104, no. 11 (November 16, 2004): 1735. http://dx.doi.org/10.1182/blood.v104.11.1735.1735.
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Abstract Blood coagulation is achieved by two closely coordinated mechanisms: i) the contact factor, or intrinsic, pathway initiated by assembly of coagulation proteins on negatively charged surfaces, and ii) the extrinsic pathway initiated by exposure of tissue factor at the site of vascular injury. Although factor XI (FXI), a 160 KDa homodimeric plasma coagulation protein, can be activated either by FXIIa (generated by the contact factor pathway) or by thrombin (generated by the extrinsic or tissue factor pathway), recent evidence suggests that its unique dimeric structure is required for FXI-activation by thrombin on the platelet surface, leading to the initiation of the intrinsic pathway that is required for normal hemostasis. Each FXI monomer consists of an N-terminal heavy chain and a C-terminal trypsin-like catalytic light chain. The heavy chain consists of four homologous subunits called apple domains (designated A1 to A4). The A2 and A3 domains of one monomeric subunit bind to FIX, whereas the A3 domain of the other monomeric subunit binds to platelets. The A4 domain, which shares high (25–38%) sequence identity with other Apple domains, facilitates FXI dimer formation through an intermolecular disulfide bond at Cys-321. In the present study the rA4 domain was cloned and purified to determine its three-dimensional structure. Multidimensional heteronuclear NMR experiments were carried out using C13, N15, H2 labeled samples. Chemical shifts of the C13, N15 and H1 resonance of all the residues were assigned. Assignment of NOE cross peaks between inter- and intra-subunit amino acids is in progress. Preliminary results indicate that the monomeric structure of the A4 domain consists of six anti-parallel β-strands and an α-helix, stabilized by three cystine cross links. The orientations of charged residues and hydrophobic patches on different sides of the molecule may play important roles in the dimerization process of FXI.
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Wang, Jianjun, Daisy Sahoo, Brian D. Sykes, and Robert O. Ryan. "NMR evidence for a conformational adaptation of apolipophorin III upon lipid association." Biochemistry and Cell Biology 76, no. 2-3 (May 1, 1998): 276–83. http://dx.doi.org/10.1139/o98-049.
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A characteristic property of amphipathic exchangeable apolipoproteins is an ability to exist alternately in lipid-free and lipid-bound states. In the present study, we have used 1H-15N-heteronuclear single quantum correlation spectroscopy to probe structural changes of apolipophorin III upon lipid association, by monitoring alterations of the chemical shifts of specific amino acids as a function of lipid titration. 15N-valine-, 15N-leucine-, 15N-lysine-, and 15N-glycine-labeled apolipophorin III were used in titration experiments with the micelle-forming lipid dodecylphosphocholine. In the absence of lipid, valine and leucine residues are located in the hydrophobic interior of the apolipophorin III helix bundle and their resonances resist chemical shift changes below the critical micelle concentration of dodecylphosphocholine. At the critical micelle concentration, however, dramatic and abrupt chemical shift changes occur, apparently coincident with formation of a protein-lipid micelle complex, as judged by significant line-width broadening of the crosspeaks. By contrast, apolipophorin III lysine and glycine residues are located on the hydrophilic surfaces of amphipathic alpha-helices or in loop regions, exposed to solvent. Their crosspeaks display either a chemical shift change similar to that seen for hydrophobic residues or a more gradual chemical shift change, beginning at very low dodecylphosphocholine concentrations. These results indicate that an interaction occurs between specific solvent-exposed lysine residues and dodecylphosphocholine below the critical micelle concentration of this lipid, whereas valine and leucine residues are not accessible to monomeric dodecylphosphocholine. At the critical micelle concentration, however, the availability of a newly formed lipid surface induces apolipophorin III binding, concomitant with conformational opening of the helix bundle, exposing its hydrophobic surfaces for binding to the dodecylphosphocholine micellar surface. Subsequently, hydrophobic residues undergo characteristic spectral changes. Subtle differences in behavior of specific hydrophobic residues, in terms of their response to dodecylphosphocholine titration and relative locations in the helix-bundle conformation, suggest that one end of the molecule may initiate contact with the lipid surface, followed by helix bundle opening.Key words: apolipophorin III, apolipoprotein.
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Genest, Stephanie C., Myrna J. Simpson, André J. Simpson, Ronald Soong, and David J. McNally. "Analysis of soil organic matter at the solid–water interface by nuclear magnetic resonance spectroscopy." Environmental Chemistry 11, no. 4 (2014): 472. http://dx.doi.org/10.1071/en14060.
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Environmental context Structural and conformational information on organic matter–clay complexes and whole soils was obtained using different NMR methods. The results show that organic matter interactions with clay mineral surfaces determine the accessibility of specific organic matter components at the soil–water interface. This physical conformation may also play a role in soil biogeochemical processes and binding to pollutants in terrestrial environments. Abstract Organic matter (OM)–mineral interactions play an important role in OM preservation, global carbon cycling and contaminant transport. Studies have indicated that preferential sorption of OM is dependent on mineral type and solution conditions. In this study, 1H high resolution–magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy was employed to examine OM chemistry in organo-clay complexes. Dissolved OM from a forest soil, Leonardite humic acid and Peat humic acid were sorbed to Ca2+ enriched kaolinite and montmorillonite. As observed using 1H HR-MAS NMR spectroscopy, kaolinite sorbed mainly long-chain aliphatic compounds such as those from plant cuticles whereas montmorillonite sorbed a mixture of aliphatic components and proteins. These results show the preferential sorption of specific dissolved OM components on clay surfaces. This was tested further using solid-state 13C and 1H HR-MAS NMR analysis of whole soils containing kaolinite and montmorillonite as well as a Peat soil for contrast. The species present at the soil–water interface were mainly aliphatic components, carbohydrates and amino acids. Aromatic constituents were present in the soils (observed by solid-state 13C NMR and by 1H HR-MAS NMR spectroscopy when a more penetrating solvent was used) which signifies that these compounds likely exist in more hydrophobic domains that are buried and surface inaccessible. This study highlights the important role of OM interactions with clay minerals in the preservation of OM in soils and suggests that OM–OM associations may also play a role in the protection of specific OM components in soil.
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HE, Qing-Yu, Anne B. MASON, Beatrice M. TAM, Ross T. A. MACGILLIVRAY, and Robert C. WOODWORTH. "[13C]Methionine NMR and metal-binding studies of recombinant human transferrin N-lobe and five methionine mutants: conformational changes and increased sensitivity to chloride." Biochemical Journal 344, no. 3 (December 8, 1999): 881–87. http://dx.doi.org/10.1042/bj3440881.
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The N-lobe of human serum transferrin (hTF/2N) and single point mutants in which each of the five methionine residues was individually mutated have been produced in a mammalian tissue-culture expression system. Since the five methionine residues are well distributed in the transferrin N-lobe, 13C NMR of the [ϵ-13C]methionine-labelled proteins has been used to monitor conformational changes of the protein during metal binding. All five methionine residues have been assigned [Beatty, Cox, Frenkiel, Tam, Mason, MacGillivray, Sadler and Woodworth (1996) Biochemistry 35, 7635-7642]. The tentative two-dimensional NMR assignment for two of the five methionine residues, namely Met26 and Met109, has been corrected. A series of NMR spectra for the complexes of 13C-Met-labelled hTF/2N with six different metal ions, Fe(III), Cu(II), Cr(III), Co(III), Ga(III) and In(III), demonstrate that the conformational change of the protein upon metal binding can be observed by means of the changes in the NMR chemical shifts associated with certain methionine residues, regardless of whether diamagnetic or paramagnetic metals are used. Changing any of the methionine residues should have minimal effects on transferrin function, since structural analysis shows that none of these residues contacts functional amino acids or has any obvious role in iron uptake or release. In fact, UV-visible spectra show little perturbation of the electronic spectra of any of the mutants. Nevertheless, the M109L mutant (Met109 → Leu) releases iron at half the rate of the wild-type N-lobe, and chloride shows a significantly greater retarding effect on the rate of iron release from all five mutants. All the methionine mutants (especially in the apo form) show a poor solubility in Hepes buffer lacking anions such as bicarbonate. These findings imply a more general effect of anion binding to surface residues than previously realized.
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Lin, Yi-Chien, Yan-Hwa Wu Lee, and Jing-Jer Lin. "Genetic analysis reveals essential and non-essential amino acids within the telomeric DNA-binding interface of Cdc13p." Biochemical Journal 403, no. 2 (March 26, 2007): 289–95. http://dx.doi.org/10.1042/bj20061698.
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Cdc13p is a specific single-stranded telomeric DNA-binding protein of Saccharomyces cerevisiae. It is involved in protecting telomeres and regulating telomere length. The telomere-binding domain of Cdc13p is located between residues 497 and 693, and its structure has been resolved by NMR spectroscopy. A series of aromatic, hydrophobic and basic residues located at the DNA-binding surface of Cdc13p are involved in binding to telomeres. Here we applied a genetic approach to analyse the involvements of these residues in telomere binding. A series of mutants within the telomere-binding domain of Cdc13p were identified that failed to complement cdc13 mutants in vivo. Among the amino acids that were isolated, the Tyr522, Arg635, and Ile633 residues were shown to locate at the DNA-binding surface. We further demonstrated that Y522C and R635A mutants failed to bind telomeric DNA in vitro, indicating that these residues are indeed required for telomere binding. We did not, however, isolate other mutant residues located at the DNA-binding surface of Cdc13p beyond these three residues. Instead, a mutant on Lys568 was isolated that did not affect the essential function of Cdc13p. The Lys568 is also located on the DNA-binding surface of Cdc13p. Thus these results suggested that other DNA-binding residues are not essential for telomere binding. In the present study, we have established a genetic test that enabled the identification of telomere-binding residues of Cdc13p in vivo. This type of analysis provides information on those residues that indeed contribute to telomere binding in vivo.
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Miller, Michelle C., Irina V. Nesmelova, Vladimir A. Daragan, Hans Ippel, Malwina Michalak, Aurelio Dregni, Herbert Kaltner, Jürgen Kopitz, Hans-Joachim Gabius, and Kevin H. Mayo. "Pro4 prolyl peptide bond isomerization in human galectin-7 modulates the monomer-dimer equilibrum to affect function." Biochemical Journal 477, no. 17 (September 4, 2020): 3147–65. http://dx.doi.org/10.1042/bcj20200499.
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Human galectin-7 (Gal-7; also termed p53-induced gene 1 product) is a multifunctional effector by productive pairing with distinct glycoconjugates and protein counter-receptors in the cytoplasm and nucleus, as well as on the cell surface. Its structural analysis by NMR spectroscopy detected doubling of a set of particular resonances, an indicator of Gal-7 existing in two conformational states in slow exchange on the chemical shift time scale. Structural positioning of this set of amino acids around the P4 residue and loss of this phenomenon in the bioactive P4L mutant indicated cis–trans isomerization at this site. Respective resonance assignments confirmed our proposal of two Gal-7 conformers. Mapping hydrogen bonds and considering van der Waals interactions in molecular dynamics simulations revealed a structural difference for the N-terminal peptide, with the trans-state being more exposed to solvent and more mobile than the cis-state. Affinity for lactose or glycan-inhibitable neuroblastoma cell surface contact formation was not affected, because both conformers associated with an overall increase in order parameters (S2). At low µM concentrations, homodimer dissociation is more favored for the cis-state of the protein than its trans-state. These findings give direction to mapping binding sites for protein counter-receptors of Gal-7, such as Bcl-2, JNK1, p53 or Smad3, and to run functional assays at low concentration to test the hypothesis that this isomerization process provides a (patho)physiologically important molecular switch for Gal-7.
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Zarrine-Afsar, Arash, Sung Lun Lin, and Philipp Neudecker. "Mutational investigation of protein folding transition states by Φ-value analysis and beyond: lessons from SH3 domain foldingThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process." Biochemistry and Cell Biology 88, no. 2 (April 2010): 231–38. http://dx.doi.org/10.1139/o09-153.
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Understanding how proteins adopt their unique native structures requires a complete structural characterization of the rate-limiting transition state(s) along the folding pathway. By definition, transition states are not significantly populated and are only accessible via folding kinetics studies. In this respect, interpreting the kinetic effects of amino acid substitutions (especially to Ala) via Φ-value analysis is the most common method to probe the structure of these transient, yet important states. A critical review of the key assumptions required for rigorous interpretation of Φ values reveals that a multiple substitution strategy in which a position of interest is mutated to a variety of amino acids, and not exclusively to Ala, provides the best means to characterize folding transition states. This approach has proven useful in revealing non-native interactions and (or) conformations in folding transition states. Moreover, by simultaneously examining the folding kinetics of multiple substitutions made at a single surface-exposed position using the Brønsted analysis the backbone conformation in a folding transition state can be investigated. For folding equilibria with exchange rates on the order of milliseconds, the kinetic parameters for Φ-value analysis can be obtained from NMR relaxation dispersion experiments, under fully native conditions, along with a wealth of high-resolution structural information about the states in exchange (native, denatured, and intermediate states that populate the pathway). This additional structural information, which is not readily obtained through stopped-flow based methods, can significantly facilitate the interpretation of Φ values because it often reports on the validity of the assumptions required for a rigorous interpretation of Φ values.
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MURRAY, Ian, Jörg KÖHL, and Katherine CIANFLONE. "Acylation-stimulating protein (ASP): structure–function determinants of cell surface binding and triacylglycerol synthetic activity." Biochemical Journal 342, no. 1 (August 10, 1999): 41–48. http://dx.doi.org/10.1042/bj3420041.
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Acylation-stimulating protein (ASP or C3adesArg) is a potent lipogenic factor in human and murine adipocytes and fibroblasts. The arginated form of ASP, i.e. complement C3a (C3a), stimulates immunological responses in human granulocytes, mast cells, guinea pig platelets and guinea pig macrophages; however, ASP is inactive in stimulating these responses. Thus both ASP and C3a are bioactive across species but are not functionally interchangeable. Tertiary structure of both proteins by X-ray crystallography and NMR spectroscopy predicts a tightly linked core region consisting of three α-helices linked via three disulphide bonds, with one of the α-helices extending out from the core and terminating in a flexible conformationally irregular carboxy-tail region. The present studies were undertaken in order to define the functionally active domains of ASP, distinctive from those of C3a, using chemical modifications, enzymic cleavage and synthetic peptide fragments. The results indicate that: (i) the N-terminal region (< 10 amino acids) plays little role in ASP receptor binding and triacylglycerol synthesis stimulation; (ii) the native C-terminal region had no activity, but modifications which increased hydrophobicity increased receptor binding, and led to some activation of triacylglycerol synthesis stimulation; (iii) an intact disulphide-linked core region is essential for triacylglycerol synthesis stimulation activity but not for receptor interaction. Finally, basic charges in the carboxy region (His) are essential for ASP triacylglycerol synthesis stimulation but not for receptor binding, whereas both functions are eliminated by the modification of Lys in the disulphide-linked core region. The present results suggest that there are two functional domains in ASP, one that is responsible for the initial binding to the cell surface receptor, and a second domain that activates and increases triacylglycerol synthesis stimulation. This contrasts markedly with the structure-function studies of C3a where both binding competency and function were dependent on the C-terminal Arg. Thus ASP demonstrates distinct bioactivity.
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Kumar, Vasantha, D. Ganavi, B. Sukesh Kumar, Rajesh P. Shastry, A. H. Udaya Kumar, S. Madan Kumar, Mohammed Al-Ghorbani, et al. "Synthesis, Crystal Structure, Hirshfeld, DFT, Quorum Sensing Inhibition and Molecular Docking Studies of N'-{(E)-[3-(3,5-Difluorophenyl)1H-pyrazol-4-yl]methylidene}-4-methoxybenzohydrazide." Asian Journal of Chemistry 33, no. 8 (2021): 1796–804. http://dx.doi.org/10.14233/ajchem.2021.23254.
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In present study, the synthesis, structural, quorum sensing inhibition and computational studies of a new N’-{(E)-[3-(3,5-difluorophenyl)-1H-pyrazol-4-yl]methylidene}-4-methoxybenzohydrazide are reported. The structure of the synthesized compound was confirmed by IR, 1H & 13C NMR and mass spectral data. The single crystals of the compound was obtained using ethanol as a crystallization solvent. The synthesized compound (C40H40N8O6F4) crystallizes in the monoclinic crystal system, C2/c space group. Various intermolecular interactions were quantified using a 3D molecular Hirshfeld surface computational method. The 2D fingerprint plots revealed that the H···H (35.9%) interactions have a major contribution to the total molecular surface. DFT studies was performed to establish the molecular crystal structure of the compound and to study the HOMO-LUMO energies of the compound. The anti-quorum sensing study of the target compound on Chromobacterium violaceum (wild type) biosensor revealed that quorum quenching activity at 300 μg/mL. Interestingly, quantitative assay exhibited more than 80% of quorum sensing inhibition without interfering cell growth (p < 0.05). Molecular docking studies of the compound on CviR protein (PDB: 3QP8) confirmed the observed activity through strong hydrogen bonding interaction with the amino acids in the binding pocket.
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Zou, Jing, Le Tian Lee, Qing Yin Wang, Xuping Xie, Siyan Lu, Yin Hoe Yau, Zhiming Yuan, et al. "Mapping the Interactions between the NS4B and NS3 Proteins of Dengue Virus." Journal of Virology 89, no. 7 (January 14, 2015): 3471–83. http://dx.doi.org/10.1128/jvi.03454-14.
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ABSTRACTFlavivirus RNA synthesis is mediated by a multiprotein complex associated with the endoplasmic reticulum membrane, named the replication complex (RC). Within the flavivirus RC, NS4B, an integral membrane protein with a role in virulence and regulation of the innate immune response, binds to the NS3 protease-helicase. NS4B modulates the RNA helicase activity of NS3, but the molecular details of their interaction remain elusive. Here, we used dengue virus (DENV) to map the determinants for the NS3-NS4B interaction. Coimmunoprecipitation and anin situproximity ligation assay confirmed that NS3 colocalizes with NS4B in both DENV-infected cells and cells coexpressing both proteins. Surface plasmon resonance demonstrated that subdomains 2 and 3 of the NS3 helicase region and the cytoplasmic loop of NS4B are required for binding. Using nuclear magnetic resonance (NMR), we found that the isolated cytoplasmic loop of NS4B is flexible, with a tendency to form a three-turn α-helix and two short β-strands. Upon binding to the NS3 helicase, 12 amino acids within the cytoplasmic loop of NS4B exhibited line broadening, suggesting a participation in the interaction. Sequence alignment showed that 4 of these 12 residues are strictly conserved across different flaviviruses. Mutagenesis analysis showed that three (Q134, G140, and N144) of the four evolutionarily conserved NS4B residues are essential for DENV replication. The mapping of the NS3/NS4B-interacting regions described here can assist the design of inhibitors that disrupt their interface for antiviral therapy.IMPORTANCENS3 and NS4B are essential components of the flavivirus RC. Using DENV as a model, we mapped the interaction between the viral NS3 and NS4B proteins. The subdomains 2 and 3 of NS3 helicase as well as the cytoplasmic loop of NS4B are critical for the interaction. Functional analysis delineated residues within the NS4B cytoplasmic loop that are crucial for DENV replication. Our findings reveal molecular details of how flavivirus NS3 protein cooperates with NS4B within the RC. In addition, this study has established the rationale and assays to search for inhibitors disrupting the NS3-NS4B interaction for antiviral drug discovery.
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Tawk, Caroline S., Ingrid R. Ghattas, and Colin A. Smith. "HK022 Nun Requires Arginine-Rich Motif Residues Distinct from λ N." Journal of Bacteriology 197, no. 22 (September 8, 2015): 3573–82. http://dx.doi.org/10.1128/jb.00466-15.
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ABSTRACTBacteriophage λ N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early λ transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to λ N, the Nun protein of HK022 binds the boxBs of coinfecting λ transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress λ replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude λ coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in λ N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and λ N antitermination complexes, the same Nun libraries were fused to the activation domain of λ N and screened for clones able to complement N-deficient λ. Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions.IMPORTANCEλ N protein interacts with host factors to allow λ nut-containing transcripts to elongate past termination signals. A competing bacteriophage, HK022, expresses Nun protein, which causes termination of λ nut transcripts. λ N and HK022 Nun use similar arginine-rich motifs (ARMs) to bind the same boxB RNAs in nut transcripts. Screening libraries of Nun ARM mutants, both in HK022 Nun and in a λ N fusion, revealed amino acids essential to Nun that could bind one or more host factors. Docking suggests that NusG, which is present in both Nun termination and N antitermination, is a plausible partner. These findings could help understand how transcription elongation is regulated and illustrate how subtle differences allow ARMs to evolve new regulatory functions.
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Cornell, Caitlin E., Roy A. Black, Mengjun Xue, Helen E. Litz, Andrew Ramsay, Moshe Gordon, Alexander Mileant, et al. "Prebiotic amino acids bind to and stabilize prebiotic fatty acid membranes." Proceedings of the National Academy of Sciences 116, no. 35 (August 12, 2019): 17239–44. http://dx.doi.org/10.1073/pnas.1900275116.
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The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+. Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.
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Tauber, Maria, Sarah Kreuz, Alexander Lemak, Papita Mandal, Zhadyra Yerkesh, Alaguraj Veluchamy, Bothayna Al-Gashgari, et al. "Alternative splicing and allosteric regulation modulate the chromatin binding of UHRF1." Nucleic Acids Research 48, no. 14 (July 1, 2020): 7728–47. http://dx.doi.org/10.1093/nar/gkaa520.
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Abstract UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.
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Gerothanassis, Ioannis P., Roger N. Hunston, and JÜRgen Lauterwein. "17O NMR chemical shifts of the twenty protein amino acids in aqueous solution." Magnetic Resonance in Chemistry 23, no. 8 (August 1985): 659–65. http://dx.doi.org/10.1002/mrc.1260230812.
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Zou, Jing, Xuping Xie, Qing-Yin Wang, Hongping Dong, Michelle Yueqi Lee, Congbao Kang, Zhiming Yuan, and Pei-Yong Shi. "Characterization of Dengue Virus NS4A and NS4B Protein Interaction." Journal of Virology 89, no. 7 (January 7, 2015): 3455–70. http://dx.doi.org/10.1128/jvi.03453-14.
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ABSTRACTFlavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimatedKd(dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach.IMPORTANCEFlavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, orin vitrowith recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.
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Fairlie, David P. "Small Molecules that Mimic Components of Bioactive Protein Surfaces." Australian Journal of Chemistry 57, no. 9 (2004): 855. http://dx.doi.org/10.1071/ch04074.
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Small molecules designed to mimic specific structural components of a protein (peptide strands, sheets, turns, helices, or amino acids) can be expected to display agonist or antagonist biological responses by virtue of interacting with the same receptors that recognize the protein. Here we describe some minimalist approaches to structural mimetics of amino acids and of strand, turn, or helix segments of proteins. The designed molecules show potent and selective inhibition of protease, transferase, and phospholipase enzymes, or antagonism of G-protein coupled or transcriptional receptors, and have potent anti-tumour, anti-inflammatory, or antiviral activity.
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Tan, Xin, Na Liu, Min Yang, Mojie Duan, and Jun Zeng. "Design of peptide inhibitors of human papillomavirus 16 (HPV16) transcriptional regulator E1–E2 formation." Journal of Theoretical and Computational Chemistry 16, no. 03 (April 4, 2017): 1750026. http://dx.doi.org/10.1142/s0219633617500262.
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Here, we have proposed a new scheme of the computational combinatorial design approach to identify potential inhibitor peptides. It consists of four steps: (i) using “multiple copy simultaneous search” (MCSS) procedure to locate specific functional groups on the protein surface; (ii) the peptide main chain is constructed based on the location of favored N-methylacetamide (NMA) groups; (iii) molecular dynamics simulations of the complex formed between the constructed peptides with the target protein in explicit water molecules are carried to select the peptides with strong binding to the protein and (iv) the sequences of the stable peptides selected from (iii) are aligned and the frequencies of the amino acids at each position of peptide are calculated. Sequence patterns of potential inhibitors are determined based on the frequency of amino acids at each position. It was applied to design peptide inhibitors that bind to the E2 protein of HPV16 so as to disrupt its transcriptional regulator of E1–E2 complex formation. The sequence pattern of these potential inhibitors is in agreement with known inhibitors obtained from phage display, and the MCSS calculations indicate that a hydrophobic pocket on HPV16 E2 plays a significant role in E1–E2 formation and inhibitor-E2 binding.
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Múdra, Marcela, Martin Breza, Lucia Lintnerová, Juraj Filo, and Jacob Bauer. "The design and NMR structure determination of yttrium-oligopeptide tags for recombinant proteins and antibodies." Acta Chimica Slovaca 11, no. 2 (October 1, 2018): 120–33. http://dx.doi.org/10.2478/acs-2018-0018.
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Abstract A strategy for the design of new yttrium(III) tags consisting of sequences of naturally occurring amino acids is described. These tags are 4–6 amino acids in length and consist of aspartic and glutamic acids. The use of natural amino acids would allow these oligopeptides to be incorporated into recombinant proteins at the DNA level, enabling the protein to be Y(III)-labelled after protein isolation. This allows a radionuclide or heavy atom to be associated with the protein without the necessity of further synthetic modification. Suitable peptides able to chelate Y(III) in stable complexes were designed based on quantum-chemical calculations. The stability of complexes formed by these peptides was tested by isothermal titration calorimetry, giving dissociation constants in the micromolar range. The likely structure of the most tightly bound complex was inferred from a combination of NMR experiments and quantum-chemical calculations. This structure will serve as the basis for future optimizations.
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Shi, Qing, Yanlei Su, Wenjuan Chen, Jinming Peng, Laiyin Nie, Lei Zhang, and Zhongyi Jiang. "Grafting short-chain amino acids onto membrane surfaces to resist protein fouling." Journal of Membrane Science 366, no. 1-2 (January 2011): 398–404. http://dx.doi.org/10.1016/j.memsci.2010.10.032.
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Qiao, Baofu, Felipe Jiménez-Ángeles, Trung Dac Nguyen, and Monica Olvera de la Cruz. "Water follows polar and nonpolar protein surface domains." Proceedings of the National Academy of Sciences 116, no. 39 (September 9, 2019): 19274–81. http://dx.doi.org/10.1073/pnas.1910225116.
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The conformation of water around proteins is of paramount importance, as it determines protein interactions. Although the average water properties around the surface of proteins have been provided experimentally and computationally, protein surfaces are highly heterogeneous. Therefore, it is crucial to determine the correlations of water to the local distributions of polar and nonpolar protein surface domains to understand functions such as aggregation, mutations, and delivery. By using atomistic simulations, we investigate the orientation and dynamics of water molecules next to 4 types of protein surface domains: negatively charged, positively charged, and charge-neutral polar and nonpolar amino acids. The negatively charged amino acids orient around 98% of the neighboring water dipoles toward the protein surface, and such correlation persists up to around 16 Å from the protein surface. The positively charged amino acids orient around 94% of the nearest water dipoles against the protein surface, and the correlation persists up to around 12 Å. The charge-neutral polar and nonpolar amino acids are also orienting the water neighbors in a quantitatively weaker manner. A similar trend was observed in the residence time of the nearest water neighbors. These findings hold true for 3 technically important enzymes (PETase, cytochrome P450, and organophosphorus hydrolase). Our results demonstrate that the water−amino acid degree of correlation follows the same trend as the amino acid contribution in proteins solubility, namely, the negatively charged amino acids are the most beneficial for protein solubility, then the positively charged amino acids, and finally the charge-neutral amino acids.
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Brodin, Peter, Torbjörn Drakenberg, Eva Thulin, Sture Forsén, and Thomas Grundström. "Selective proton labelling of amino acids in deuterated bovine calbindin D9K. A way to simplify 1H-NMR spectra." "Protein Engineering, Design and Selection" 2, no. 5 (1989): 353–57. http://dx.doi.org/10.1093/protein/2.5.353.
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Chasapis, Christos T., and Alexios Vlamis-Gardikas. "Probing Conformational Dynamics by Protein Contact Networks: Comparison with NMR Relaxation Studies and Molecular Dynamics Simulations." Biophysica 1, no. 2 (April 8, 2021): 157–67. http://dx.doi.org/10.3390/biophysica1020012.
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Protein contact networks (PCNs) have been used for the study of protein structure and function for the past decade. In PCNs, each amino acid is considered as a node while the contacts among amino acids are the links/edges. We examined the possible correlation between the closeness centrality measure of amino acids within PCNs and their mobility as known from NMR spin relaxation experiments and molecular dynamic (MD) simulations. The pivotal observation was that plasticity within a protein stretch correlated inversely to closeness centrality. Effects on protein conformational plasticity caused by the formation of disulfide bonds or protein–protein interactions were also identified by the PCN analysis measure closeness centrality and the hereby introduced percentage of closeness centrality perturbation (% CCP). All the comparisons between PCN measures, NMR data, and MDs were performed in a set of proteins of different biological functions and structures: the core protease domain of anthrax lethal factor, the N-terminal RING domain of E3 Ub ligase Arkadia, the reduced and oxidized forms of human thioredoxin 1, and the ubiquitin molecules (Ub) of the catalytic Ub–RING–E3–E2–Ub complex of E3 ligase Ark2.The graph theory analysis of PCNs could thus provide a general method for assessing the conformational dynamics of free proteins and putative plasticity changes between different protein forms (apo/complexed or reduced/oxidized).
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Ben Shir, Ira, Shifi Kababya, and Asher Schmidt. "Binding Specificity of Amino Acids to Amorphous Silica Surfaces: Solid-State NMR of Glycine on SBA-15." Journal of Physical Chemistry C 116, no. 17 (April 19, 2012): 9691–702. http://dx.doi.org/10.1021/jp302431t.
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Asandei, Alina, Aldo E. Rossini, Mauro Chinappi, Yoonkyung Park, and Tudor Luchian. "Protein Nanopore-Based Discrimination between Selected Neutral Amino Acids from Polypeptides." Langmuir 33, no. 50 (December 11, 2017): 14451–59. http://dx.doi.org/10.1021/acs.langmuir.7b03163.
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Špačková, Nad'a, Zuzana Trošanová, Filip Šebesta, Séverine Jansen, Jaroslav V. Burda, Pavel Srb, Milan Zachrdla, Lukáš Žídek, and Jiří Kozelka. "Protein environment affects the water–tryptophan binding mode. MD, QM/MM, and NMR studies of engrailed homeodomain mutants." Physical Chemistry Chemical Physics 20, no. 18 (2018): 12664–77. http://dx.doi.org/10.1039/c7cp08623g.
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Water molecules can interact with the π-face of tryptophan either forming an O–H⋯π hydrogen bond or by a lone-pair⋯π interaction. Surrounding amino acids can favor the one or the other interaction type.
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Xue, Mengjun, Janani Sampath, Rachel N. Gebhart, Havard J. Haugen, S. Petter Lyngstadaas, Jim Pfaendtner, and Gary Drobny. "Studies of Dynamic Binding of Amino Acids to TiO2 Nanoparticle Surfaces by Solution NMR and Molecular Dynamics Simulations." Langmuir 36, no. 35 (July 22, 2020): 10341–50. http://dx.doi.org/10.1021/acs.langmuir.0c01256.
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Tay, Hui Min, Aditya Rawal, and Carol Hua. "S-Mg2(dobpdc): a metal–organic framework for determining chirality in amino acids." Chemical Communications 56, no. 94 (2020): 14829–32. http://dx.doi.org/10.1039/d0cc05539e.
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van der Spoel, David. "The solution conformations of amino acids from molecular dynamics simulations of Gly-X-Gly peptides: comparison with NMR parameters." Biochemistry and Cell Biology 76, no. 2-3 (May 1, 1998): 164–70. http://dx.doi.org/10.1139/o98-025.
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The conformations that amino acids can adopt in the random coil state are of fundamental interest in the context of protein folding research and studies of proteinpeptide interactions. To date, no detailed quantitative data from experimental studies have been reported; only nuclear magnetic resonance parameters such as chemical shifts and J coupling constants have been reported. These experimental nuclear magnetic resonance data represent averages over multiple conformations, and hence they do not provide unique structural information. I have performed relatively long (2.5 ns) molecular dynamics simulations of Gly-X-Gly tripeptides, surrounded by explicit water molecules, where X represents eight different amino acids with long side chains. From the trajectories one can calculate time averaged backbone chemical shifts and 3JNHα coupling constants and compare these with experimental data. These calculated quantities are quite close to the experimental values for most amino acids, suggesting that these simulations are a good model for the random coil state of the tripeptides. On the basis of my simulations I predict 3Jαβ coupling constants and present dihedral distributions for the Φ, Ψ, as well as χ1 and χ2 angles. Finally, I present correlation plots for these dihedral angles.Key words: molecular dynamics (MD), nuclear magnetic resonance (NMR) spectroscopy, J-coupling, chemical shift, dihedral probability distribution.
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Cortés, Gualberto Asencio, and Jesús A. Aguilar-Ruiz. "Predicting protein distance maps according to physicochemical properties." Journal of Integrative Bioinformatics 8, no. 3 (December 1, 2011): 158–75. http://dx.doi.org/10.1515/jib-2011-181.
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SummaryThe prediction of protein structures is a current issue of great significance in structural bioinformatics. More specifically, the prediction of the tertiary structure of a protein con- sists in determining its three-dimensional conformation based solely on its amino acid sequence. This study proposes a method in which protein fragments are assembled according to their physicochemical similarities, using information extracted from known protein structures. Many approaches cited in the literature use the physicochemical properties of amino acids, generally hydrophobicity, polarity and charge, to predict structure. In our method, implemented with parallel multithreading, we used a set of 30 physicochemical amino acid properties selected from the AAindex database. Several protein tertiary structure prediction methods produce a contact map. Our proposed method produces a distance map, which provides more information about the structure of a protein than a contact map. We performed several preliminary analysis of the protein physicochemical data distributions using 3D surfaces. Three main pattern types were found in 3D surfaces, thus it is possible to extract rules in order to predict distances between amino acids according to their physicochemical properties. We performed an experimental validation of our method using five non-homologous protein sets and we showed the generality of this method and its prediction quality using the amino acid properties considered. Finally, we included a study of the algorithm efficiency according to the number of most similar fragments considered and we notably improved the precision with the studied proteins sets.
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Shelly and Minakshi Sharma. "Nitrate Reductase Nanoparticles: Synthesis and Characterization." International Journal of Research in Pharmaceutical Sciences 11, no. 3 (August 7, 2020): 4583–89. http://dx.doi.org/10.26452/ijrps.v11i3.2740.
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Nanoparticles of enzyme Nitrate reductase (NaR) a soluble homodimer enzyme of ∼100 kDa polypeptide with cofactors – FAD, heme-molybdopterin (Mo-MPT) and electron donor NAD(P)H, catalyses the reduction of nitrate to nitrite has been synthesised. Nanoparticles of Nitrate reductase enzyme have been prepared by chemical desolvation method including glutaraldehyde cross-linking to form the nanoaggregate. Characterisation of NaR nanoparticles has been made by Transmission Electron Microscopy (TEM), UV-Visible Spectroscopy and by electrochemical Impedance Spectroscopic Study (EIS). TEM study revealed the size of globular aggregated was in the range of 20–30 nm. UV Visible spectroscopic studies depicted that the absorption of NaR NPS is much higher at 560 nm than that of the free enzyme, which showed maximum absorption at 540 nm. NaR NPs aggregates formed were more active, highly stable, have a higher shelf life and can be reused repeatedly. Enzyme nanoparticles with 10-100 nm dimensions and exhibit unique physical, chemical and catalytic properties due to increased surface area. Nitrate reductase nanoparticles can be used as a biochemical tool to increase protein production and grain yield by promoting amino acids production in plants. The synthesised NaR NPs are used in the fabrication of enzyme-based nanosensor in the detection of nitrates in drinking water and serum samples.
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Lee, Chia Min, Xuping Xie, Jing Zou, Shi-Hua Li, Michelle Yue Qi Lee, Hongping Dong, Cheng-Feng Qin, Congbao Kang, and Pei-Yong Shi. "Determinants of Dengue Virus NS4A Protein Oligomerization." Journal of Virology 89, no. 12 (April 1, 2015): 6171–83. http://dx.doi.org/10.1128/jvi.00546-15.
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ABSTRACTFlavivirus NS4A protein induces host membrane rearrangement and functions as a replication complex component. The molecular details of how flavivirus NS4A exerts these functions remain elusive. Here, we used dengue virus (DENV) as a model to characterize and demonstrate the biological relevance of flavivirus NS4A oligomerization. DENV type 2 (DENV-2) NS4A protein forms oligomers in infected cells or when expressed alone. Deletion mutagenesis mapped amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]) of NS4A as the major determinant for oligomerization, while the N-terminal 50 residues contribute only slightly to the oligomerization. Nuclear magnetic resonance (NMR) analysis of NS4A amino acids 17 to 80 suggests that residues L31, L52, E53, G66, and G67 could participate in oligomerization. Ala substitution for 15 flavivirus conserved NS4A residues revealed that these amino acids are important for viral replication. Among the 15 mutated NS4A residues, 2 amino acids (E50A and G67A) are located within TMD1. Both E50A and G67A attenuated viral replication, decreased NS4A oligomerization, and reduced NS4A protein stability. In contrast, NS4A oligomerization was not affected by the replication-defective mutations (R12A, P49A, and K80A) located outside TMD1.transcomplementation experiments showed that expression of wild-type NS4A alone was not sufficient to rescue the replication-lethal NS4A mutants. However, the presence of DENV-2 replicons could partially restore the replication defect of some lethal NS4A mutants (L26A and K80A), but not others (L60A and E122A), suggesting an unidentified mechanism governing the outcome of complementation in a mutant-dependent manner. Collectively, the results have demonstrated the importance of TMD1-mediated NS4A oligomerization in flavivirus replication.IMPORTANCEWe report that DENV NS4A forms oligomers. Such NS4A oligomerization is mediated mainly through amino acids 50 to 76 (spanning the first transmembrane domain [TMD1]). The biological importance of NS4A oligomerization is demonstrated by results showing that mutations of flavivirus conserved residues (E50A and G67A located within TMD1) reduced the oligomerization and stability of the NS4A protein, leading to attenuated viral replication. A systematic mutagenesis analysis demonstrated that flavivirus conserved NS4A residues are important for DENV replication. A successfultranscomplementation of replication-lethal NS4A mutant virus requires wild-type NS4A in the context of the viral replication complex. The wild-type NS4A protein alone is not sufficient to rescue the replication defect of NS4A mutants. Intriguingly, distinct NS4A mutants yielded different complementation outcomes in the replicon-containing cells. Overall, the study has enhanced our understanding of flavivirus NS4A at the molecular level. The results also suggest that inhibitor blocking of NS4A oligomerization could be explored for antiviral drug discovery.
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Slupsky, Carolyn M., Lisa N. Gentile, and Lawrence P. McIntosh. "Assigning the NMR spectra of aromatic amino acids in proteins: analysis of two Ets pointed domains." Biochemistry and Cell Biology 76, no. 2-3 (May 1, 1998): 379–90. http://dx.doi.org/10.1139/o98-017.
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The measurement of interproton nuclear Overhauser enhancements (NOEs) and dihedral angle restraints of aromatic amino acids is a critical step towards determining the structure of a protein. The complete assignment of the resonances from aromatic rings and the subsequent resolution and identification of their associated NOEs, however, can be a difficult task. Shown here is a strategy for assigning the 1H, 13C, and 15N signals from the aromatic side chains of histidine, tryptophan, tyrosine, and phenylalanine using a suite of homo- and hetero-nuclear scalar and NOE correlation experiments, as well as selective deuterium isotope labelling. In addition, a comparison of NOE information obtained from homonuclear NOE spectroscopy (NOESY) and 13C-edited NOESY - heteronuclear single quantum correlation experiments indicates that high-resolution homonuclear two-dimensional NOESY spectra of selectively deuterated proteins are invaluable for obtaining distance restraints to the aromatic residues.Key words: NMR assignment, aromatic residue, transcription factor, NOE, dihedral angle.
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Serrano, Luis. "Comparison between the φ Distribution of the Amino Acids in the Protein Database and NMR Data Indicates that Amino Acids have Various φ Propensities in the Random Coil Conformation." Journal of Molecular Biology 254, no. 2 (November 1995): 322–33. http://dx.doi.org/10.1006/jmbi.1995.0619.
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Landrieu, Isabelle, Arnaud Leroy, Caroline Smet-Nocca, Isabelle Huvent, Laziza Amniai, Malika Hamdane, Nathalie Sibille, Luc Buée, Jean-Michel Wieruszeski, and Guy Lippens. "NMR spectroscopy of the neuronal tau protein: normal function and implication in Alzheimer's disease." Biochemical Society Transactions 38, no. 4 (July 26, 2010): 1006–11. http://dx.doi.org/10.1042/bst0381006.
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NMR spectroscopy was used to explore the different aspects of the normal and pathological functions of tau, but proved challenging because the protein contains 441 amino acids and has poor signal dispersion. We have set out to dissect the phosphorylation patterns of tau in order to understand better its role in the aggregation process and microtubule-binding regulation. Our current knowledge on the functional consequences of specific phosphorylations is still limited, mainly because producing and assessing quantitatively phosphorylated tau samples is far from straightforward, even in vitro. We use NMR spectroscopy as a proteomics tool to characterize the phosphorylation patterns of tau, after in vitro phosphorylation by recombinant kinases. The phosphorylated tau can next be use for functional assays or interaction assays with phospho-dependent protein partners, such as the prolyl cis–trans isomerase Pin1.
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Thøgersen, Rebekka, Hanne Christine Bertram, Mathias T. Vangsoe, and Mette Hansen. "Krill Protein Hydrolysate Provides High Absorption Rate for All Essential Amino Acids—A Randomized Control Cross-Over Trial." Nutrients 13, no. 9 (September 14, 2021): 3187. http://dx.doi.org/10.3390/nu13093187.
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Background: adequate protein intake is essential to humans and, since the global demand for protein-containing foods is increasing, identifying new high-quality protein sources is needed. In this study, we investigated the acute postprandial bioavailability of amino acids (AAs) from a krill protein hydrolysate compared to a soy and a whey protein isolate. Methods: the study was a randomized, placebo-controlled crossover trial including ten healthy young males. On four non-consecutive days, volunteers consumed water or one of three protein-matched supplements: whey protein isolate, soy protein isolate or krill protein hydrolysate. Blood samples were collected prior to and until 180 min after consumption. Serum postprandial AA concentrations were determined using 1H NMR spectroscopy. Hunger and satiety were assessed using visual analogue scales (VAS). Results: whey and krill resulted in significantly higher AA concentrations compared to soy between 20–60 min and 20–40 min after consumption, respectively. Area under the curve (AUC) analyses revealed that whey resulted in the highest postprandial serum concentrations of essential AAs (EAAs) and branched chain AAs (BCAAs), followed by krill and soy, respectively. Conclusions: krill protein hydrolysate increases postprandial serum EAA and BCAA concentrations in a superior manner to soy protein isolate and thus might represent a promising future protein source in human nutrition.
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Czaja, Kornelia, Jacek Kujawski, Elżbieta Jodłowska-Siewert, Paulina Szulc, Tomasz Ratajczak, Dominika Krygier, Marcin K. Chmielewski, and Marek K. Bernard. "On the Interactions of Fused Pyrazole Derivative with Selected Amino Acids: DFT Calculations." Journal of Chemistry 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/8124323.
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Due to the increasing prevalence of neoplasms, there is a permanent need for new selective cytostatic compounds. Anticancer drugs can act in different ways, affecting protein expression and synthesis, including disruption of signaling pathways within cells. Continuing our previous research aiming at elucidating the mechanism of pyrazole’s anticancer activity, we carried out in silico studies on the interactions of fused pyrazole derivative with alanine, lysine, glutamic acid, and methionine. The objective of the study is to improve our understanding of the possible interactions of pyrazole derivatives with the above-mentioned amino acids. For this purpose, we apply the DFT formalism (optimization using the B3LYP, CAM-B3LYP, PBE0, and M06L functionals) and interaction energy calculations (counterpoise corrected method based on the basis set superposition error, BSSE) together with QTAIM approach and estimation of the 1H NMR chemical shifts of analyzed pyrazole derivative using different basis sets and DFT functionals in CPCM solvation model (and water used as a solvent).
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Ottestad, Inger, Stine M. Ulven, Linn K. L. Øyri, Kristin S. Sandvei, Gyrd O. Gjevestad, Asta Bye, Navida A. Sheikh, Anne S. Biong, Lene F. Andersen, and Kirsten B. Holven. "Reduced plasma concentration of branched-chain amino acids in sarcopenic older subjects: a cross-sectional study." British Journal of Nutrition 120, no. 4 (June 18, 2018): 445–53. http://dx.doi.org/10.1017/s0007114518001307.
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AbstractBranched-chain amino acids (BCAA) are essential amino acids that are necessary for muscle mass maintenance. Little is known about the plasma concentrations of BCAA and the protein intake in relation to sarcopenia. We aimed to compare the non-fasting plasma concentrations of the BCAA and the dietary protein intake between sarcopenic and non-sarcopenic older adults. Norwegian older home-dwelling adults (≥70 years) were invited to a cross-sectional study with no other exclusion criteria than age. Sarcopenic subjects were defined by the diagnostic criteria by the European Working Group on Sarcopenia in Older People. Non-fasting plasma concentrations of eight amino acids were quantified using NMR spectroscopy. Protein intake was assessed using 2×24-h dietary recalls. In this study, ninety out of 417 subjects (22 %) were sarcopenic, and more women (32 %) than men (11 %) were sarcopenic (P<0·0001). Sex-adjusted non-fasting plasma concentrations of leucine and isoleucine, and the absolute intake of protein (g/d), were significantly lower among the sarcopenic subjects, when compared with non-sarcopenic subjects (P=0·003, P=0·026 and P=0·003, respectively). A similar protein intake was observed in the two groups when adjusted for body weight (BW) and sex (1·1 g protein/kg BW per d; P=0·50). We show that sarcopenia is associated with reduced non-fasting plasma concentration of the BCAA leucine and isoleucine, and lower absolute intake of protein. More studies are needed to clarify the clinical relevance of these findings, related to maintenance of muscle mass and prevention of sarcopenia.
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López, Claudia S., R. Sean Peacock, Jorge H. Crosa, and Hans J. Vogel. "Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum." Biochemical Journal 418, no. 1 (January 28, 2009): 49–59. http://dx.doi.org/10.1042/bj20081462.
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In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.
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Bellstedt, Peter, Thomas Seiboth, Sabine Häfner, Henriette Kutscha, Ramadurai Ramachandran, and Matthias Görlach. "Resonance assignment for a particularly challenging protein based on systematic unlabeling of amino acids to complement incomplete NMR data sets." Journal of Biomolecular NMR 57, no. 1 (August 14, 2013): 65–72. http://dx.doi.org/10.1007/s10858-013-9768-0.
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Neochoritis, Constantinos G., Maryam Kazemi Miraki, Eman M. M. Abdelraheem, Ewa Surmiak, Tryfon Zarganes-Tzitzikas, Beata Łabuzek, Tad A. Holak, and Alexander Dömling. "Design of indole- and MCR-based macrocycles as p53-MDM2 antagonists." Beilstein Journal of Organic Chemistry 15 (February 20, 2019): 513–20. http://dx.doi.org/10.3762/bjoc.15.45.
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Macrocycles were designed to antagonize the protein–protein interaction p53-MDM2 based on the three-finger pharmacophore F19W23L25. The synthesis was accomplished by a rapid, one-pot synthesis of indole-based macrocycles based on Ugi macrocyclization. The reaction of 12 different α,ω-amino acids and different indole-3-carboxaldehyde derivatives afforded a unique library of macrocycles otherwise difficult to access. Screening of the library for p53-MDM2 inhibition by fluorescence polarization and 1H,15N HSQC NMR measurements confirm MDM2 binding.
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Zhu, Chongqin, Yurui Gao, Hui Li, Sheng Meng, Lei Li, Joseph S. Francisco, and Xiao Cheng Zeng. "Characterizing hydrophobicity of amino acid side chains in a protein environment via measuring contact angle of a water nanodroplet on planar peptide network." Proceedings of the National Academy of Sciences 113, no. 46 (November 1, 2016): 12946–51. http://dx.doi.org/10.1073/pnas.1616138113.
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Hydrophobicity of macroscopic planar surface is conventionally characterized by the contact angle of water droplets. However, this engineering measurement cannot be directly extended to surfaces of proteins, due to the nanometer scale of amino acids and inherent nonplanar structures. To measure the hydrophobicity of side chains of proteins quantitatively, numerous parameters were developed to characterize behavior of hydrophobic solvation. However, consistency among these parameters is not always apparent. Herein, we demonstrate an alternative way of characterizing hydrophobicity of amino acid side chains in a protein environment by constructing a monolayer of amino acids (i.e., artificial planar peptide network) according to the primary and the β-sheet secondary structures of protein so that the conventional engineering measurement of the contact angle of a water droplet can be brought to bear. Using molecular dynamics simulations, contact angles θ of a water nanodroplet on the planar peptide network, together with excess chemical potentials of purely repulsive methane-sized Weeks−Chandler−Andersen solute, are computed. All of the 20 types of amino acids and the corresponding planar peptide networks are studied. Expectedly, all of the planar peptide networks with nonpolar amino acids are hydrophobic due to θ> 90°, whereas all of the planar peptide networks of the polar and charged amino acids are hydrophilic due to θ< 90°. Planar peptide networks of the charged amino acids exhibit complete-wetting behavior due to θ= 0°. This computational approach for characterization of hydrophobicity can be extended to artificial planar networks of other soft matter.
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Wen, Xinian, Lei Liu, Zhen Geng, and Lifeng He. "Application of Taxol Nanomicelles with Lyp-1 Target in Targeted Therapy of Colon Cancer." Journal of Nanoscience and Nanotechnology 21, no. 2 (February 1, 2021): 805–13. http://dx.doi.org/10.1166/jnn.2021.18676.
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As a new type of non-viral gene drug carrier, paclitaxel with Lyp-1 target has unique transmembrane ability due to its unique structure. In this paper, amino acids and surfactants are used to disperse SWCNTs in water, and non-covalent interactions are used to adsorb paclitaxel to the surface of SWCNTs. DSPE-PEG-Maleimide is then connected to NGR to achieve active targeting. To investigate the effect of NGR-SWCNTs-Paclitaxel on isolated cells, and to observe the antitumor effect of NGR-SWCNTs-Paclitaxel on S180 colon cancer mice in vivo, we provide theoretical and experimental basis for targeted cancer treatment. The luciferase activity test results showed that mi R-218 mimics had no significant effect on the intensity of the blank reporter plasmid group and p MIR-REPORT/UTR mutant luciferase activity, but in mi R-218 mimics and p MIR-REPORT/UTR Luciferase activity decreased after co-transfection of wild-type plasmids into cells. The validation results of the luciferase activity analysis system showed that mi R-218 was able to bind to Sp13’UTR. Overexpression of mi R-218 can significantly reduce the expression level of Sp1 protein but has no significant effect on Sp1 m RNA level, indicating that mi R-218 can target the regulation of Sp1 expression at the translation level.
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Stigers, Dannon J., Zachary I. Watts, James E. Hennessy, Hye-Kyung Kim, Romeo Martini, Matthew C. Taylor, Kiyoshi Ozawa, Jeffrey W. Keillor, Nicholas E. Dixon, and Christopher J. Easton. "Incorporation of chlorinated analogues of aliphatic amino acids during cell-free protein synthesis." Chem. Commun. 47, no. 6 (2011): 1839–41. http://dx.doi.org/10.1039/c0cc02879g.
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Zhai, Luhan, Masayuki Nara, Yuko Otani, and Tomohiko Ohwada. "Unexpectedly rigid short peptide foldamers in which NH–π and CH–π interactions are preserved in solution." Chemical Communications 57, no. 67 (2021): 8344–47. http://dx.doi.org/10.1039/d1cc02998c.
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We found that a group of short peptides with only three amino acids containing a phenylalanine formed highly stable folded structures in solution, wherein CH–π and NH–π main chain–side chain interactions can be clearly observed by NMR and ATR-IR.