scholarly journals Acidic residues and a predicted, highly conserved α‐helix are critical for the endonuclease/strand separation functions of bacteriophage λ’s TerL

2019 ◽  
Vol 112 (5) ◽  
pp. 1483-1498
Author(s):  
Jean Sippy Arens ◽  
Carol Duffy ◽  
Michael Feiss
Keyword(s):  
Strand Separation ◽  
Acidic Residues ◽  
Α Helix ◽  
Separation Functions

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

2019 ◽  
Vol 26 (7) ◽  
pp. 532-541 ◽  
Author(s):  
Cadena-Cadena Francisco ◽  
Cárdenas-López José Luis ◽  
Ezquerra-Brauer Josafat Marina ◽  
Cinco-Moroyoqui Francisco Javier ◽  
López-Zavala Alonso Alexis ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Cathepsin D ◽  
Thermal Denaturation ◽  
Protein Denaturation ◽  
Marine Organisms ◽  
Effect Of Temperature ◽  
Jumbo Squid ◽  
Α Helix ◽  
Circular Dichroïsm

Background: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. Objective: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. Methods: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. Results: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. Conclusion: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

The Inhibition of Polysialyltranseferase ST8SiaIV Through Heparin Binding to Polysialyltransferase Domain (PSTD)

2019 ◽  
Vol 15 (5) ◽  
pp. 486-495 ◽  
Author(s):  
Li-Xin Peng ◽  
Xue-Hui Liu ◽  
Bo Lu ◽  
Si-Ming Liao ◽  
Feng Zhou ◽  
...  
Keyword(s):  
Fluorescence Quenching ◽  
Polysialic Acid ◽  
Neuronal Cell ◽  
Cd Spectroscopy ◽  
Migration And Invasion ◽  
Heparin Binding ◽  
Clinical Prognosis ◽  
Quenching Analysis ◽  
Different Types ◽  
Α Helix

Background:The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies.Objective:To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes?Methods:Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4.Results:The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4.Conclusion:The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

Scorpion toxin-potassium channel interaction law and its applications.

2020 ◽  
Vol 01 ◽  
Author(s):  
Zheng Zuo ◽  
Zongyun Chen ◽  
Zhijian Cao ◽  
Wenxin Li ◽  
Yingliang Wu
Keyword(s):  
Potassium Channel ◽  
Scorpion Toxin ◽  
Scorpion Toxins ◽  
Toxin Binding ◽  
Channel Interaction ◽  
Binding Interface ◽  
Α Helix ◽  
Interaction Law ◽  
Binding Interfaces ◽  
Β Sheet

: The scorpion toxins are the largest potassium channel-blocking peptide family. The understanding of toxin binding interfaces is usually restricted by two classical binding interfaces: one is the toxin α-helix motif, the other is the antiparallel β-sheet motif. In this review, such traditional knowledge was updated by another two different binding interfaces: one is BmKTX toxin using the turn motif between the α-helix and antiparallel β-sheet domains as the binding interface, the other is Ts toxin using turn motif between the β-sheet in the N-terminal and α-helix domains as the binding interface. Their interaction analysis indicated that the scarce negatively charged residues in the scorpion toxins played a critical role in orientating the toxin binding interface. In view of the toxin negatively charged amino acids as “binding interface regulator”, the law of scorpion toxin-potassium channel interaction was proposed, that is, the polymorphism of negatively charged residue distribution determines the diversity of toxin binding interfaces. Such law was used to develop scorpion toxin-potassium channel recognition control technique. According to this technique, three Kv1.3 channel-targeted peptides, using BmKTX as the template, were designed with the distinct binding interfaces from that of BmKTX through modulating the distribution of toxin negatively charged residues. In view of the potassium channel as the common targets of different animal toxins, the proposed law was also shown to helpfully orientate the binding interfaces of other animal toxins. Clearly, the toxin-potassium channel interaction law would strongly accelerate the research and development of different potassium channelblocking animal toxins in the future.

scholarly journals Peptide-based inhibitors of protein–protein interactions: biophysical, structural and cellular consequences of introducing a constraint

2021 ◽  
Author(s):  
Hongshuang Wang ◽  
Robert S. Dawber ◽  
Peiyu Zhang ◽  
Martin Walko ◽  
Andrew J. Wilson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Α Helix ◽  
Cellular Behaviour

This review summarizes the influence of inserting constraints on biophysical, conformational, structural and cellular behaviour for peptides targeting α-helix mediated protein–protein interactions.

scholarly journals The structure of ORC–Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiang Feng ◽  
Yasunori Noguchi ◽  
Marta Barbon ◽  
Bruce Stillman ◽  
Christian Speck ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Origin Recognition Complex ◽  
Molecular Level ◽  
Dna Recognition ◽  
Winged Helix ◽  
Replicative Helicase ◽  
The Core ◽  
Α Helix ◽  
Replication Initiator ◽  
Winged Helix Domain

AbstractThe Origin Recognition Complex (ORC) binds to sites in chromosomes to specify the location of origins of DNA replication. The S. cerevisiae ORC binds to specific DNA sequences throughout the cell cycle but becomes active only when it binds to the replication initiator Cdc6. It has been unclear at the molecular level how Cdc6 activates ORC, converting it to an active recruiter of the Mcm2-7 hexamer, the core of the replicative helicase. Here we report the cryo-EM structure at 3.3 Å resolution of the yeast ORC–Cdc6 bound to an 85-bp ARS1 origin DNA. The structure reveals that Cdc6 contributes to origin DNA recognition via its winged helix domain (WHD) and its initiator-specific motif. Cdc6 binding rearranges a short α-helix in the Orc1 AAA+ domain and the Orc2 WHD, leading to the activation of the Cdc6 ATPase and the formation of the three sites for the recruitment of Mcm2-7, none of which are present in ORC alone. The results illuminate the molecular mechanism of a critical biochemical step in the licensing of eukaryotic replication origins.

scholarly journals A mixed chirality α-helix in a stapled bicyclic and a linear antimicrobial peptide revealed by X-ray crystallography

2021 ◽  
Author(s):  
Stéphane Baeriswyl ◽  
Hippolyte Personne ◽  
Ivan Di Bonaventura ◽  
Thilo Köhler ◽  
Christian van Delden ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Antimicrobial Peptide ◽  
Crystal Structures ◽  
X Ray ◽  
X Ray Crystallography ◽  
Α Helix

We report the first X-ray crystal structures of mixed chirality α-helices comprising only natural residues as the example of bicyclic and linear membrane disruptive amphiphilic antimicrobial peptides containing seven l- and four d-residues.

scholarly journals Nanostructure of bioactive glass affects bone cell attachment via protein restructuring upon adsorption

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ukrit Thamma ◽  
Tia J. Kowal ◽  
Matthias M. Falk ◽  
Himanshu Jain
Keyword(s):  
Bioactive Glass ◽  
Cell Response ◽  
Interfacial Layer ◽  
Cell Attachment ◽  
Bone Cell ◽  
Nano Structure ◽  
Rgd Sequence ◽  
Engineered Tissue ◽  
Α Helix ◽  
Β Sheet

AbstractThe nanostructure of engineered bioscaffolds has a profound impact on cell response, yet its understanding remains incomplete as cells interact with a highly complex interfacial layer rather than the material itself. For bioactive glass scaffolds, this layer comprises of silica gel, hydroxyapatite (HA)/carbonated hydroxyapatite (CHA), and absorbed proteins—all in varying micro/nano structure, composition, and concentration. Here, we examined the response of MC3T3-E1 pre-osteoblast cells to 30 mol% CaO–70 mol% SiO2 porous bioactive glass monoliths that differed only in nanopore size (6–44 nm) yet resulted in the formation of HA/CHA layers with significantly different microstructures. We report that cell response, as quantified by cell attachment and morphology, does not correlate with nanopore size, nor HA/CHO layer micro/nano morphology, or absorbed protein amount (bovine serum albumin, BSA), but with BSA’s secondary conformation as indicated by its β-sheet/α-helix ratio. Our results suggest that the β-sheet structure in BSA interacts electrostatically with the HA/CHA interfacial layer and activates the RGD sequence of absorbed adhesion proteins, such as fibronectin and vitronectin, thus significantly enhancing the attachment of cells. These findings provide new insight into the interaction of cells with the scaffolds’ interfacial layer, which is vital for the continued development of engineered tissue scaffolds.

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