Orthorhombic lysozyme crystallization at acidic pH values driven by phosphate binding

The structure of orthorhombic lysozyme has been obtained at 298 K and pH 4.5 using sodium chloride as the precipitant and in the presence of sodium phosphate at a concentration as low as 5 mM. Crystals belonging to space groupP212121(unit-cell parametersa= 30,b= 56,c= 73 Å, α = β = γ = 90.00°) diffracted to a resolution higher than 1 Å, and the high quality of these crystals permitted the identification of a phosphate ion bound to Arg14 and His15. The binding of this ion produces long-range conformational changes affecting the loop containing Ser60–Asn74. The negatively charged phosphate ion shields the electrostatic repulsion of the positively charged arginine and histidine residues, resulting in higher stability of the phosphate-bound lysozyme. Additionally, a low-humidity orthorhombic variant was obtained at pH 4.5, and comparison with those previously obtained at pH 6.5 and 9.5 shows a 1.5 Å displacement of the fifth α-helix towards the active-site cavity, which might be relevant to protein function. Since lysozyme is broadly used as a model protein in studies related to protein crystallization and amyloid formation, these results indicate that the interaction of some anions must be considered when analysing experiments performed at acidic pH values.

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Structure of grouper iridovirus purine nucleoside phosphorylase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444909048276 ◽

2010 ◽

Vol 66 (2) ◽

pp. 155-162

Author(s):

You-Na Kang ◽

Yang Zhang ◽

Paula W. Allan ◽

William B. Parker ◽

Jing-Wen Ting ◽

...

Keyword(s):

Amino Acid ◽

Active Site ◽

Binding Sites ◽

Purine Nucleoside Phosphorylase ◽

Purine Nucleoside ◽

Phosphate Binding ◽

Nucleoside Phosphorylase ◽

Cell Parameters ◽

Phosphate Ion ◽

Enzymatic Activity Assay

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides to the corresponding free bases and ribose 1-phosphate. The crystal structure of grouper iridovirus PNP (givPNP), corresponding to the first PNP gene to be found in a virus, was determined at 2.4 Å resolution. The crystals belonged to space groupR3, with unit-cell parametersa= 193.0,c= 105.6 Å, and contained four protomers per asymmetric unit. The overall structure of givPNP shows high similarity to mammalian PNPs, having an α/β structure with a nine-stranded mixed β-barrel flanked by a total of nine α-helices. The predicted phosphate-binding and ribose-binding sites are occupied by a phosphate ion and a Tris molecule, respectively. The geometrical arrangement and hydrogen-bonding patterns of the phosphate-binding site are similar to those found in the human and bovine PNP structures. The enzymatic activity assay of givPNP on various substrates revealed that givPNP can only accept 6-oxopurine nucleosides as substrates, which is also suggested by its amino-acid composition and active-site architecture. All these results suggest that givPNP is a homologue of mammalian PNPs in terms of amino-acid sequence, molecular mass, substrate specificity and overall structure, as well as in the composition of the active site.

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Do carbon nanotubes inhibit or promote amyloid fibrils formation?

Biophysical Bulletin ◽

10.26565/2075-3810-2019-42-04 ◽

2019 ◽

Keyword(s):

Carbon Nanotubes ◽

Conformational Analysis ◽

Conformational Changes ◽

Room Temperature ◽

Molecular Mechanisms ◽

Amyloid Fibrils ◽

Ph Values ◽

Egg White Lysozyme ◽

Characteristic Features ◽

Α Helix

Objectives: The purpose of the work was to study the effect of carbon nanotubes on the formation of fibril structures in lysozyme at room temperature under different pH values. Materials and methods: For the preparation of the samples, hen egg-white lysozyme protein (HEWL, Fluka), as well as single-walled (SWCNT, Sigma-Aldrich) and multi-walled (MWCNT, OOO TM “Spetsmash”, Kyiv, Ukraine) carbon nanotubes were used. Used techniques: IR-Fourier Absorption Spectroscopy; confocal microscopy. Results: In this paper, the study of molecular mechanisms of interaction of lysozyme with carbon nanotubes by vibrational spectroscopy was carried out and a conformational analysis of the formed complexes was performed. It is shown that carbon nanotubes can affect the structure of lysozyme even at room temperature and normal pH values, as evidenced by conformational changes in lysozyme due to interaction with carbon nanotubes. Complexes which are formed as a result of such interaction, have characteristic features of amyloid fibrillar structures. It reveals one of possible mechanisms of carbon nanotubes cytotoxicity. On the other hand, such a technique can be introduced to obtain model amyloid fibrils for further study. Conclusion: The method of vibtarional spectroscopy has shown that carbon nanotubes can influence the structure of lysozyme, as it is shown by the conformational analysis of the absorption band Amide I. After the interaction of lysozyme with CNT, an increase in the contribution of antiparallel β-conformation in the structure of lysozyme is observed, and the contribution of the α-helix conformation is reduced, which are characteristic features in the formation of fibrillar structures. The possibility of amyloid fibril formation without the use of high temperatures at different pH values with the interaction of lysozyme and carbon nanotubes, which can be applied as a method for obtaining the model amyloid fibrils, is shown.

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Major conformational changes in the structure of lysozyme obtained from a crystal with a very low solvent content

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19013189 ◽

2019 ◽

Vol 75 (11) ◽

pp. 687-696

Author(s):

M. Carmen Salinas-Garcia ◽

Marina Plaza-Garrido ◽

Daniel Alba-Elena ◽

Ana Camara-Artigas

Keyword(s):

Conformational Changes ◽

Crystal Form ◽

Catalytic Site ◽

Side Chain ◽

Hydrophobic Core ◽

Orthorhombic Space Group ◽

Solvent Content ◽

Cell Parameters ◽

Saccharide Binding ◽

Α Helix

A new crystal form of lysozyme with a very low solvent content (26.35%) has been obtained in the orthorhombic space group P212121 (with unit-cell parameters a = 30.04, b = 51.68, c = 61.53 Å). The lysozyme structure obtained from these crystals does not show the typical overall fold. Instead, major conformational changes take place in some elements of the secondary structure and in the hydrophobic core of the protein. At the end of the central α-helix (α2), Glu35 is usually buried in the catalytic site and shows an abnormally high pK a value, which is key to the activity of the enzyme. The high pK a value of this glutamate residue is favoured by the hydrophobic environment, particularly by its neighbour Trp108, which is important for structural stability and saccharide binding. In this new structure, Trp108 shows a 90° rotation of its side chain, which results in the rearrangement of the hydrophobic core. Conformational changes also result in the exposure of Glu35 to the solvent, which impairs the catalytic site by increasing the distance between Glu35 and Asp52 and lowering the pK a value of the glutamate. Altogether, this new lysozyme structure reveals major conformational changes in the hydrophobic core and catalytic site that might play a role in the folding and bactericidal function of the protein.

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Is the Acidic pH of the External Auditory Canal Playing a Significant Role in the Treatment of Acute Otitis Externa?

Revista de Chimie ◽

10.37358/rc.18.8.6521 ◽

2018 ◽

Vol 69 (8) ◽

pp. 2304-2305

Author(s):

Oana Ruxandra Iana ◽

Dragos Cristian Stefanescu ◽

Viorel Zainea ◽

Razvan Hainarosie

Keyword(s):

External Auditory Canal ◽

Otitis Externa ◽

Proton Pumps ◽

Acidic Ph ◽

External Ear ◽

Ph Values ◽

The World ◽

Subcutaneous Tissues ◽

Low Levels ◽

Value Changes

Variable pH values for skin have been reported in the literature, all within the acidic range, varying from 4.0 up to 7. 0. The origin of the acidic pH remains conjectural, and several factors have been incriminated with this role, such as eccrine and sebaceous secretions as well as proton pumps. Keeping low levels of pH prevents microbial dispersal as well as multiplication. The skin in the external auditory canal is also covered with this acidic mantle with antimicrobial value. Changes of pH in the external ear can lead to acute otitis externa. This condition is defined by the inflammation and infection of the cutaneous and subcutaneous tissues of the external auditory canal. 10% of the world�s population may suffer from acute otitis externa at least once in their lifetime. This paper aims to consolidate the relevance of an acidic pH in the healthy external ear and its relation to the pathogenesis and treatment of otitis externa through a prospective and interventional clinical study on 80 patients who presented to the outpatient department at Prof. Dr D. Hociota ENT Institute in Buch

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The X-Ray Crystallographic Structure of the Human Neonatal Fc Receptor at Acidic pH Gives Insights into pH-Dependent Conformational Changes

Protein and Peptide Letters ◽

10.2174/0929866523666160404125850 ◽

2016 ◽

Vol 23 (6) ◽

pp. 525-529 ◽

Cited By ~ 2

Author(s):

Mohammed Taha ◽

Sally E. Ward ◽

Hyun-Joo Nam

Keyword(s):

Conformational Changes ◽

Fc Receptor ◽

Crystallographic Structure ◽

Acidic Ph ◽

Neonatal Fc Receptor ◽

X Ray ◽

Ph Dependent

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The key amino acids of E protein involved in early flavivirus infection: viral entry

Virology Journal ◽

10.1186/s12985-021-01611-2 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Tao Hu ◽

Zhen Wu ◽

Shaoxiong Wu ◽

Shun Chen ◽

Anchun Cheng

Keyword(s):

Amino Acids ◽

Conformational Changes ◽

Viral Entry ◽

Envelope Protein ◽

Cell Attachment ◽

Envelope Proteins ◽

Infection Process ◽

Early Infection ◽

Protein Amino Acids ◽

Α Helix

AbstractFlaviviruses are enveloped viruses that infect multiple hosts. Envelope proteins are the outermost proteins in the structure of flaviviruses and mediate viral infection. Studies indicate that flaviviruses mainly use envelope proteins to bind to cell attachment receptors and endocytic receptors for the entry step. Here, we present current findings regarding key envelope protein amino acids that participate in the flavivirus early infection process. Among these sites, most are located in special positions of the protein structure, such as the α-helix in the stem region and the hinge region between domains I and II, motifs that potentially affect the interaction between different domains. Some of these sites are located in positions involved in conformational changes in envelope proteins. In summary, we summarize and discuss the key envelope protein residues that affect the entry process of flaviviruses, including the process of their discovery and the mechanisms that affect early infection.

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In Vitro Incorporation of Helicobacter pylori into Candida albicans Caused by Acidic pH Stress

Pathogens ◽

10.3390/pathogens9060489 ◽

2020 ◽

Vol 9 (6) ◽

pp. 489 ◽

Cited By ~ 1

Author(s):

Kimberly Sánchez-Alonzo ◽

Cristian Parra-Sepúlveda ◽

Samuel Vega ◽

Humberto Bernasconi ◽

Víctor L. Campos ◽

...

Keyword(s):

Helicobacter Pylori ◽

Candida Albicans ◽

Pcr Amplification ◽

Growth Curves ◽

Acidic Ph ◽

Ph Values ◽

16S Gene ◽

Wide Range ◽

H Pylori

Yeasts can adapt to a wide range of pH fluctuations (2 to 10), while Helicobacter pylori, a facultative intracellular bacterium, can adapt to a range from pH 6 to 8. This work analyzed if H. pylori J99 can protect itself from acidic pH by entering into Candida albicans ATCC 90028. Growth curves were determined for H. pylori and C. albicans at pH 3, 4, and 7. Both microorganisms were co-incubated at the same pH values, and the presence of intra-yeast bacteria was evaluated. Intra-yeast bacteria-like bodies were detected using wet mounting, and intra-yeast binding of anti-H. pylori antibodies was detected using immunofluorescence. The presence of the H. pylori rDNA 16S gene in total DNA from yeasts was demonstrated after PCR amplification. H. pylori showed larger death percentages at pH 3 and 4 than at pH 7. On the contrary, the viability of the yeast was not affected by any of the pHs evaluated. H. pylori entered into C. albicans at all the pH values assayed but to a greater extent at unfavorable pH values (pH 3 or 4, p = 0.014 and p = 0.001, respectively). In conclusion, it is possible to suggest that H. pylori can shelter itself within C. albicans under unfavorable pH conditions.

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