Role of Cel5H protein surface amino acids in binding with clay minerals and measurements of its forces

AbstractOur previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H (BlCel5H) and Paenibacillus polymyxa Cel5A (PpCel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in BlCel5H but not in PpCel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69 ± 19 pN for wild-type, 58 ± 19 pN for M2, 53 ± 19 pN for M3, and 49 ± 19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.

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Mapping of AF1 transactivation domains in duplicated rainbow trout glucocorticoid receptors

Journal of Molecular Endocrinology ◽

10.1677/jme-09-0152 ◽

2010 ◽

Vol 45 (6) ◽

pp. 391-404 ◽

Cited By ~ 6

Author(s):

Armin Sturm ◽

James E Bron ◽

Darren M Green ◽

Nic R Bury

Keyword(s):

Amino Acids ◽

Rainbow Trout ◽

Glucocorticoid Receptors ◽

Marked Decrease ◽

Binding Activity ◽

Wild Type ◽

Sequence Alignments ◽

The Core ◽

Terminal Domain ◽

Transactivation Domains

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor mediating the genomic effects of glucocorticoids. Two activation functions (AFs) are present in the GR. While the N-terminal AF1 is ligand independent, the C-terminal AF2 overlaps with the ligand-binding domain and is ligand dependent. In this study, we have mapped AF1 in duplicated rainbow trout GRs, called rtGR1 and rtGR2, showing a limited homology (24.5%) in the N-terminal domain. Ablation of this domain from rtGR1 or rtGR2 resulted in a marked decrease (>97%) in maximal hormone-dependent transactivation, but did not affect dexamethasone-binding activity or expression levels. This suggested that, similar to the situation in the human GR (hGR), AF1 is the main AF in the trout GRs. Sequence alignments with hGR suggested a localisation of AF1 to residues 70–230 of rtGR1 and 1–119 of rtGR2. These assignments were generally confirmed in the transactivation experiments with rtGR1- and rtGR2-derived mutants showing partial deletions of their N-terminal domains. In dexamethsone-treated cells (10−7 M, 2 h), the subcellular distribution of rtGR1 and rtGR2 mutants lacking the entire N-terminal domain, as well that of an rtGR1 mutant lacking the most N-terminal 234 amino acids, was similar to that of the corresponding wild-type GRs, suggesting that the disruption of transactivation activity was not caused by impairment of nuclear access of the mutants. Bioinformatic analyses predicted the presence of potential helical segments in the core of AF1 of rtGR1 and rtGR2, and further revealed that AF1 in rtGR1, rtGR2, and hGR shares a motif composed of hydrophobic and acidic amino acids.

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Fatty acid binding and conformational stability of mutants of human muscle fatty acid-binding protein

Biochemical Journal ◽

10.1042/bj3140253 ◽

1996 ◽

Vol 314 (1) ◽

pp. 253-260 ◽

Cited By ~ 22

Author(s):

Clemens F. M. PRINSEN ◽

Jacques H. VEERKAMP

Keyword(s):

Amino Acids ◽

Fatty Acid ◽

Fatty Acid Binding Protein ◽

Binding Protein ◽

Binding Activity ◽

Human Muscle ◽

Wild Type ◽

Fatty Acid Binding ◽

Mutant Proteins ◽

Acid Binding Protein

Human muscle fatty acid-binding protein (M-FABP) is a 15 kDa cytosolic protein which may be involved in fatty acid transfer and modulation of non-esterified fatty acid concentration in heart, skeletal muscle, kidney and many other tissues. Crystallographic studies have suggested the importance of the amino acids Thr-40, Arg-106, Arg-126 and Tyr-128 for the hydrogen bonding network of the fatty acid carboxylate group. Two phenylalanines at 16 and 57 are positioned to interact with the acyl chain of the fatty acid. We prepared 13 mutant proteins by site-directed mutagenesis and tested them for fatty acid binding and stability. Substitution of amino acids Phe-16, Arg-106 or Arg-126 created proteins which showed a large decrease in or complete loss of oleic acid binding. Substitution of Phe-57 by Ser or Val and of Tyr-128 by Phe had no great effect. The stability of the mutant proteins was tested by denaturation studies on the basis of fatty acid binding or tryptophan fluorescence and compared with that of the wild-type M-FABP. There was no direct relationship between fatty acid-binding activity and stability. Less stable mutants (F57S and Y128F) did not show a marked change in fatty acid-binding activity. Substitution of Arg-126 by Gln or Arg-106 by Thr eliminated binding activity, but the former mutant protein showed wild-type stability, in contrast to the latter. The results are in agreement with crystallographic data.

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Orphan designation: 505 amino acid protein, corresponding to amino acids 2-506 of the wild-type human histidyl-tRNA synthetase, Treatment of limb-girdle muscular dystrophy

Case Medical Research ◽

10.31525/cmr-2935ce2 ◽

2020 ◽

Author(s):

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Muscular Dystrophy ◽

Trna Synthetase ◽

Limb Girdle Muscular Dystrophy ◽

Wild Type ◽

Orphan Designation ◽

Acid Protein ◽

Amino Acid Protein

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Response of a wild type and a non-nitrogen-fixing mutant ofAnabaena doliolum towards different amino acids

Zeitschrift für allgemeine Mikrobiologie ◽

10.1002/jobm.3630210502 ◽

1981 ◽

Vol 21 (5) ◽

pp. 353-359 ◽

Cited By ~ 1

Author(s):

Ashok Kumar ◽

H. D. Kumar

Keyword(s):

Amino Acids ◽

Nitrogen Fixing ◽

Wild Type

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Identification of a novel homozygous loss-of-function mutation in FUCA1 gene causing severe fucosidosis: A case report

Journal of International Medical Research ◽

10.1177/03000605211005975 ◽

2021 ◽

Vol 49 (4) ◽

pp. 030006052110059

Author(s):

Xinwen Zhang ◽

Shaozhi Zhao ◽

Hongwei Liu ◽

Xiaoyan Wang ◽

Xiaolei Wang ◽

...

Keyword(s):

Amino Acids ◽

Lysosomal Storage Disorder ◽

Autosomal Recessive ◽

Signs And Symptoms ◽

Lysosomal Storage ◽

Loss Of Function ◽

Wild Type ◽

Single Nucleotide ◽

Whole Exome ◽

Exon 1

Fucosidosis is a rare lysosomal storage disorder characterized by deficiency of α-L-fucosidase with an autosomal recessive mode of inheritance. Here, we describe a 4-year-old Chinese boy with signs and symptoms of fucosidosis but his parents were phenotypically normal. Whole exome sequencing (WES) identified a novel homozygous single nucleotide deletion (c.82delG) in the exon 1 of the FUCA1 gene. This mutation will lead to a frameshift which will result in the formation of a truncated FUCA1 protein (p.Val28Cysfs*105) of 132 amino acids approximately one-third the size of the wild type FUCA1 protein (466 amino acids). Both parents were carrying the mutation in a heterozygous state. This study expands the mutational spectrum of the FUCA1 gene associated with fucosidosis and emphasises the benefits of WES for accurate and timely clinical diagnosis of this rare disease.

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DFT Study of Electronic Structure and Optical Properties of Kaolinite, Muscovite, and Montmorillonite

Crystals ◽

10.3390/cryst11060618 ◽

2021 ◽

Vol 11 (6) ◽

pp. 618

Author(s):

Layla Shafei ◽

Puja Adhikari ◽

Wai-Yim Ching

Keyword(s):

Mechanical Properties ◽

Optical Properties ◽

Electronic Structure ◽

Hydrogen Bonds ◽

Clay Minerals ◽

Clay Mineral ◽

Bond Order ◽

Deep Understanding ◽

Pharmaceutical Applications ◽

Structure Properties

Clay mineral materials have attracted attention due to their many properties and applications. The applications of clay minerals are closely linked to their structure and composition. In this paper, we studied the electronic structure properties of kaolinite, muscovite, and montmorillonite crystals, which are classified as clay minerals, by using DFT-based ab initio packages VASP and the OLCAO. The aim of this work is to have a deep understanding of clay mineral materials, including electronic structure, bond strength, mechanical properties, and optical properties. It is worth mentioning that understanding these properties may help continually result in new and innovative clay products in several applications, such as in pharmaceutical applications using kaolinite for their potential in cancer treatment, muscovite used as insulators in electrical appliances, and engineering applications that use montmorillonite as a sealant. In addition, our results show that the role played by hydrogen bonds in O-H bonds has an impact on the hydration in these crystals. Based on calculated total bond order density, it is concluded that kaolinite is slightly more cohesive than montmorillonite, which is consistent with the calculated mechanical properties.

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Volatile Anesthetics Affect Nutrient Availability in Yeast

Genetics ◽

10.1093/genetics/161.2.563 ◽

2002 ◽

Vol 161 (2) ◽

pp. 563-574

Author(s):

Laura K Palmer ◽

Darren Wolfe ◽

Jessica L Keeley ◽

Ralph L Keil

Keyword(s):

Amino Acids ◽

Nutrient Availability ◽

Wild Type Strain ◽

Volatile Anesthetic ◽

Volatile Anesthetics ◽

Wild Type ◽

Anesthetic Exposure ◽

Sites Of Action ◽

Insight Into ◽

Lines Of Evidence

Abstract Volatile anesthetics affect all cells and tissues tested, but their mechanisms and sites of action remain unknown. To gain insight into the cellular activities of anesthetics, we have isolated genes that, when overexpressed, render Saccharomyces cerevisiae resistant to the volatile anesthetic isoflurane. One of these genes, WAK3/TAT1, encodes a permease that transports amino acids including leucine and tryptophan, for which our wild-type strain is auxotrophic. This suggests that availability of amino acids may play a key role in anesthetic response. Multiple lines of evidence support this proposal: (i) Deletion or overexpression of permeases that transport leucine and/or tryptophan alters anesthetic response; (ii) prototrophic strains are anesthetic resistant; (iii) altered concentrations of leucine and tryptophan in the medium affect anesthetic response; and (iv) uptake of leucine and tryptophan is inhibited during anesthetic exposure. Not all amino acids are critical for this response since we find that overexpression of the lysine permease does not affect anesthetic sensitivity. These findings are consistent with models in which anesthetics have a physiologically important effect on availability of specific amino acids by altering function of their permeases. In addition, we show that there is a relationship between nutrient availability and ubiquitin metabolism in this response.

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Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

Nucleic Acids Research ◽

10.1093/nar/gkab041 ◽

2021 ◽

Author(s):

Amit Ketkar ◽

Lane Smith ◽

Callie Johnson ◽

Alyssa Richey ◽

Makayla Berry ◽

...

Keyword(s):

Amino Acids ◽

Mutation Frequency ◽

Control Sequence ◽

Wild Type ◽

Binding Properties ◽

High Affinity ◽

G4 Dna ◽

G Quadruplex ◽

Forward Mutagenesis

Abstract We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

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Host-Dependent Requirement for the Potato leafroll virus 17-kDa Protein in Virus Movement

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.10.1086 ◽

2002 ◽

Vol 15 (10) ◽

pp. 1086-1094 ◽

Cited By ~ 47

Author(s):

Lawrence Lee ◽

Peter Palukaitis ◽

Stewart M. Gray

Keyword(s):

Amino Acids ◽

Solanum Tuberosum ◽

Nicotiana Benthamiana ◽

Systemic Infection ◽

Potato Leafroll Virus ◽

Virus Movement ◽

Wild Type ◽

Mature Leaves ◽

Leafroll Virus ◽

Virus Distribution

The requirement for the 17-kDa protein (P17) of Potato leafroll virus (PLRV) in virus movement was investigated in four plant species: potato (Solanum tuberosum), Physalis floridana, Nicotiana benthamiana, and N. clevelandii. Two PLRV P17 mutants were characterized, one that does not translate the P17 and another that expresses a P17 missing the first four amino acids. The P17 mutants were able to replicate and accumulate in agroinoculated leaves of potato and P. floridana, but they were unable to move into vascular tissues and initiate a systemic infection in these plants. In contrast, the P17 mutants were able to spread systemically from inoculated leaves in both Nicotiana spp., although the efficiency of infection was reduced relative to wild-type PLRV. Examination of virus distribution in N. benthamiana plants using tissue immunoblotting techniques revealed that the wild-type PLRV and P17 mutants followed a similar movement pathway out of the inoculated leaves. Virus first moved upward to the apical tissues and then downward. The P17 mutants, however, infected fewer phloem-associated cells, were slower than wild-type PLRV in moving out of the inoculated tissue and into apical tissues, and were unable to infect any mature leaves present on the plant at the time of inoculation.

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