scholarly journals Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus

2018 ◽  
Vol 19 (8) ◽  
pp. 2447
Author(s):  
Fanghua Wang ◽  
Ruixia Wei ◽  
Abdelkarim Abousalham ◽  
Wuchong Chen ◽  
Bo Yang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Secondary Structure ◽  
Phospholipase D ◽  
Vibrio Parahaemolyticus ◽  
Terminal Segment ◽  
Amino Acid Sequences ◽  
Loop Structure ◽  
Binding Properties ◽  
Phospholipid Monolayers ◽  
Terminal Amino

The effects of N-terminal (1–34 amino acids) and C-terminal (434–487 amino acids) amino acid sequences on the interfacial binding properties of Phospholipase D from Vibrio parahaemolyticus (VpPLD) were characterized by using monomolecular film technology. Online tools allowed the prediction of the secondary structure of the target N- and C-terminal VpPLD sequences. Various truncated forms of VpPLD with different N- or C-terminal deletions were designed, based on their secondary structure, and their membrane binding properties were examined. The analysis of the maximum insertion pressure (MIP) and synergy factor “a” indicated that the loop structure (1–25 amino acids) in the N-terminal segment of VpPLD had a positive effect on the binding of VpPLD to phospholipid monolayers, especially to 1,2-dimyristoyl-sn-glycero-3-phosphoserine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The deletion affecting the N-terminus loop structure caused a significant decrease of the MIP and synergy factor a of the protein for these phospholipid monolayers. Conversely, the deletion of the helix structure (26–34 amino acids) basically had no influence on the binding of VpPLD to phospholipid monolayers. The deletion of the C-terminal amino acids 434–487 did not significantly change the binding selectivity of VpPLD for the various phospholipid monolayer tested here. However, a significant increase of the MIP value for all the phospholipid monolayers strongly indicated that the three-strand segment (434–469 amino acids) had a great negative effect on the interfacial binding to these phospholipid monolayers. The deletion of this peptide caused a significantly greater insertion of the protein into the phospholipid monolayers examined. The present study provides detailed information on the effect of the N- and C-terminal segments of VpPLD on the interfacial binding properties of the enzyme and improves our understanding of the interactions between this enzyme and cell membranes.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Atypical Structural Tendencies Among Low-Complexity Domains in the Protein Data Bank Proteome

2019 ◽  
Author(s):  
Sean M. Cascarina ◽  
Mikaela R. Elder ◽  
Eric D. Ross
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Secondary Structure ◽  
Physical Properties ◽  
Protein Data Bank ◽  
Data Bank ◽  
Low Complexity ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Intrinsically Disordered

AbstractA variety of studies have suggested that low-complexity domains (LCDs) tend to be intrinsically disordered and are relatively rare within structured proteins in the protein data bank (PDB). Although LCDs are often treated as a single class, we previously found that LCDs enriched in different amino acids can exhibit substantial differences in protein metabolism and function. Therefore, we wondered whether the structural conformations of LCDs are likewise dependent on which specific amino acids are enriched within each LCD. Here, we directly examined relationships between enrichment of individual amino acids and secondary structure preferences across the entire PDB proteome. Secondary structure preferences varied as a function of the identity of the amino acid enriched and its degree of enrichment. Furthermore, divergence in secondary structure profiles often occurred for LCDs enriched in physicochemically similar amino acids (e.g. valine vs. leucine), indicating that LCDs composed of related amino acids can have distinct secondary structure preferences. Comparison of LCD secondary structure preferences with numerous pre-existing secondary structure propensity scales resulted in relatively poor correlations for certain types of LCDs, indicating that these scales may not capture secondary structure preferences as sequence complexity decreases. Collectively, these observations provide a highly resolved view of structural preferences among LCDs parsed by the nature and magnitude of single amino acid enrichment.Author SummaryThe structures that proteins adopt are directly related to their amino acid sequences. Low-complexity domains (LCDs) in protein sequences are unusual regions made up of only a few different types of amino acids. Although this is the key feature that classifies sequences as LCDs, the physical properties of LCDs will differ based on the types of amino acids that are found in each domain. For example, the sequences “AAAAAAAAAA”, “EEEEEEEEEE”, and “EEKRKEEEKE” will have very different properties, even though they would all be classified as LCDs by traditional methods. In a previous study, we developed a new method to further divide LCDs into categories that more closely reflect the differences in their physical properties. In this study, we apply that approach to examine the structures of LCDs when sorted into different categories based on their amino acids. This allowed us to define relationships between the types of amino acids in the LCDs and their corresponding structures. Since protein structure is closely related to protein function, this has important implications for understanding the basic functions and properties of LCDs in a variety of proteins.

scholarly journals The N-terminal amino acid sequences of the heavy and light chains of human cathepsin L Relationship to a cDNA clone for a major cysteine proteinase from a mouse macrophage cell line

1986 ◽  
Vol 240 (2) ◽  
pp. 373-377 ◽  
Author(s):  
R W Mason ◽  
J E Walker ◽  
F D Northrop
Keyword(s):  
Amino Acids ◽  
Cell Line ◽  
Heavy Chain ◽  
Cdna Clone ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Amino Acid Sequences ◽  
Macrophage Cell ◽  
Terminal Amino ◽  
Mouse Macrophage Cell Line

Human liver cathepsin L consists of a heavy chain and a light chain with Mr values of 25,000 and 5000 respectively. The chains have been purified and their N-terminal amino acid sequences have been determined. The 40 amino acids determined from the heavy chain and 42 amino acids sequenced in the light chain are homologous with the N-terminal and C-terminal regions respectively of the superfamily of cysteine proteinases. Therefore it is likely that the two chains of cathepsin L are derived by proteolysis of a single polypeptide precursor. Of the amino acids sequenced, 81% are identical with the homologous portions of a protein sequence for a major cysteine proteinase predicted from a cDNA clone from a mouse macrophage cell line. This is the closest relative amongst the known sequences in the superfamily and strongly indicates that the protein encoded by this mRNA is cathepsin L. The mouse protein is also probably the major excreted protein of a transformed cell line [Gal & Gottesman (1986) Biochem. Biophys. Res. Commun. 139, 156-162]. The heavy chain is identical in only 71% of its residues with the sequence of ox cathepsin S, providing further evidence that this latter enzyme is probably not a species variant of cathepsin L. The relationship with a second unidentified cathepsin cDNA clone from a bovine library is much weaker (41% identity), and so this clone remains unidentified.

Anticoagulant And Calcium-Binding Properties Of High Molecular Weight Derivatives Of Human Fibrinogen, Produced By Plasmin (Fragments X)

1981 ◽  
Author(s):  
W Nieuwenhuizen ◽  
M Gravesen
Keyword(s):  
Calcium Ions ◽  
Calcium Binding ◽  
Degradation Products ◽  
Amino Acid Sequences ◽  
Binding Properties ◽  
Human Fibrinogen ◽  
Molecular Weights ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Carboxy Terminal

Early plasmin degradation products (fragments X) of human fibrinogen were prepared and purified on Sepharose 6B-CL. X-fragments were characterized with respect to amino-terminal amino acid sequences, polypeptide-chain composition, anticlotting properties and calcium-binding. Amino-terminal amino acids were alanine and tyrosine. The molecular weights of the chains were about 26,000, 58,000 and 48,000 for Aα-, Bβ- and γ-chains, respectively. Fragments X were about 6 times as potent in anticlotting behaviour as D-fragments prepared in the presence of calcium ions. Calcium-binding properties were similar to those of fibrinogen: No differences were observed between fragments X prepared in the presence of calcium ions and those prepared in the presence of EGTA. Results indicate that the carboxy-terminal parts of the Aα-chains of fibrinogen are not involved in calcium-binding and that differences in chain-remnants as observed in late plasmic degradation products (which depend on the presence of calcium ions or EGTA in the incubation medium) are introduced beyond the stage of fragment X-formation.

Characterization of Glycoprotein Fraction from Carp Pituitaries Isolated Using Concanavalin A as the Affinity Ligand

1998 ◽  
Vol 63 (3) ◽  
pp. 434-440 ◽  
Author(s):  
Irena Hulová ◽  
Jana Barthová ◽  
Helena Ryšlavá ◽  
Václav Kašička
Keyword(s):  
Concanavalin A ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Gel Chromatography ◽  
Affinity Ligand ◽  
Sds Page ◽  
Terminal Amino ◽  
Α And Β Subunits

Glycoproteins that have affinity to Concanavalin A were isolated from the acetone-dried pituitaries of common carp (Cyprinus carpio L.). Two fractions of glycoproteins were separated using gel chromatography on Superdex 75HR. The fraction with lower molecular weight (30 000) corresponding to the carp gonadotropin cGtH II was composed of two subunits as determined using SDS-PAGE. This protein fraction was further divided into four components using reversed-phase HPLC. Two fractions were pure α and β subunits of cGtH II as follows from immunodetection and from determination of N-terminal amino acid sequences. The other two were a mixture of α and β subunits as was also revealed by N-terminal analysis. Capillary electrophoresis was also used for characterization of isolated glycoproteins.

scholarly journals Locking up the AS1411 Aptamer with a Flanking Duplex: Towards an Improved Nucleolin-Targeting

2021 ◽  
Vol 14 (2) ◽  
pp. 121
Author(s):  
André Miranda ◽  
Tiago Santos ◽  
Eric Largy ◽  
Carla Cruz
Keyword(s):  
Loop Structure ◽  
Binding Properties ◽  
Stem Loop ◽  
Affinity Ligand ◽  
Stem Loop Structure ◽  
Topology Maintenance ◽  
G Quadruplex ◽  
Dimeric Form ◽  
Biophysical Techniques ◽  
Melting Experiments

We have designed AS1411-N6, a derivative of the nucleolin (NCL)-binding aptamer AS1411, by adding six nucleotides to the 5′-end that are complementary to nucleotides at the 3′-end forcing it into a stem-loop structure. We evaluated by several biophysical techniques if AS1411-N6 can adopt one or more conformations, one of which allows NCL binding. We found a decrease of polymorphism of G-quadruplex (G4)-forming sequences comparing to AS1411 and the G4 formation in presence of K+ promotes the duplex folding. We also studied the binding properties of ligands TMPyP4, PhenDC3, PDS, 360A, and BRACO-19 in terms of stability, binding, topology maintenance of AS1411-N6, and NCL recognition. The melting experiments revealed promising stabilizer effects of PhenDC3, 360A, and TMPyP4, and the affinity calculations showed that 360A is the most prominent affinity ligand for AS1411-N6 and AS1411. The affinity determined between AS1411-N6 and NCL denoting a strong interaction and complex formation was assessed by PAGE in which the electrophoretic profile of AS1411-N6 showed bands of the dimeric form in the presence of the ligands and NCL.

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