Stem Mutants in the N-terminal Domain of the Phage P22 Tailspike Protein

Palmer, Chris; Williams, Jeremie; Dean, Dexter N.; Johnson, Sam; Wu, Hongzhuan; Robertson, Boakai K.; Jackson, Doba; Villafañe, Robert

doi:10.12691/ajmr-2-1-1

Cited by 3 publications

(8 citation statements)

References 27 publications

(40 reference statements)

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“…The biological, recombinant and expression protocols have been described [16] [18]. The expression plasmid, pET11a-TSP containing a wild type copy of the P22 tailspike gene, was from Dr Peter Prevelige (University of Alabama at Birmingham, UAB).…”

Section: Biological and Recombinant Protocolsmentioning

confidence: 99%

“…The expression plasmid, pET11a-TSP containing a wild type copy of the P22 tailspike gene, was from Dr Peter Prevelige (University of Alabama at Birmingham, UAB). The protein function of the expressed cloned tail gene or gene variant was determined by the Berget Assay [16]. The Berget Assay is a high-expression variant of the functional petri plate assay devised by Schwarz and Berget [17].…”

Section: Biological and Recombinant Protocolsmentioning

confidence: 99%

“…The Berget Assay is a high-expression variant of the functional petri plate assay devised by Schwarz and Berget [17]. It has been described in detail recently [16].…”

Section: Biological and Recombinant Protocolsmentioning

confidence: 99%

“…The functional nature of hmH4-1 (pET11a-TSP-G29R), hmH3 (pET11a-TSP-G71D) and the wild type tail gene (pET11a-TSP) was determined by the Berget Assay [16]. This test measures but does not distinguish between all three tail activities: binding to the viral particle (NTD function), binding LPS and LPS hydrolysis by tail endorhamnosidase activity (both functions of the β-helix domain).…”

Section: Assay Of Cloned Tail Protein Functionmentioning

confidence: 99%

“…The P22 TSP NTD binds to the crevice formed between the GP4 and GP10 viral structural proteins while a crevice formed by two sets of peptide ridges, located in the β-helix domain is responsible for host cell LPS binding and hydrolysis [14] [15]. Recent studies on the structure and function of the P22 TSP NTD have found amino acids, located within the interior of the NTD dome, are involved in the stability of the domain structure [16]. This Short Communication presents preliminary studies strongly suggesting two amino acids, which are not localized in the stem region, and may be important determinants in the overall stability of the P22 TSP NTD.…”

Section: Introductionmentioning

confidence: 99%

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Non-Stem Amino Acids Are Involved in the Phage P22 TSP NTD Stability

Venkatesan¹,

Williams²,

Villafañe³

2014

AiM

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The P22 phage system is an intensely studied model system. Studies have ranged from biochemical analysis of basic life processes to the use of this phage for phage therapy. The phage tailspike protein (TSP) has itself been the subject of intensive studies over the past fifty years. The P22 TSP is essential for initiation of the infection process and instrumental as the last protein assembled onto the phage particle structure to complete its assembly. It has also been the subject for many structural studies including cryoelectron microscopic analysis and photophysical studies. It has been a model for in vivo and in vitro protein folding including analysis using P22 TSP temperaturesensitive for folding mutations (tsf). Recently the structure and function of the N-terminal domain (NTD), including some aspects of the structural stability of the P22 TSP NTD (aa1-aa108), are being genetically dissected. This report strongly supports the notion that two amino acids, not localized to the internal NTD dome stem, are important in the structural stability of the P22 TSP NTD.

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Section: Biological and Recombinant Protocolsmentioning

confidence: 99%

Section: Biological and Recombinant Protocolsmentioning

confidence: 99%

“…The Berget Assay is a high-expression variant of the functional petri plate assay devised by Schwarz and Berget [17]. It has been described in detail recently [16].…”

Section: Biological and Recombinant Protocolsmentioning

confidence: 99%

Section: Assay Of Cloned Tail Protein Functionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Non-Stem Amino Acids Are Involved in the Phage P22 TSP NTD Stability

Venkatesan¹,

Williams²,

Villafañe³

2014

AiM

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A genetic analysis of an important hydrophobic interaction at the P22 tailspike protein N-terminal domain

et al. 2018

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P22 bacteriophage has been studied extensively and has served as a model for many important processes such as in vivo protein folding, protein aggregation and protein-protein interactions. The trimeric tailspike protein (TSP) serves as the receptor-binding protein for the P22 bacteriophage to the bacterial host. The homotrimeric P22 tail consists of three chains of 666aa in which the first 108aa form a trimeric dome-like structure which is called the N-terminal domain (NTD) and is responsible for attachment of the tailspike protein to the rest of the phage particle structure in the phage assembly pathway. Knowledge of this interaction requires information on what amino acids are interacting in the interface and how the NTD structure is maintained. The first 23aa form the "stem peptide" which originates at the dome top and terminates at the dome bottom. It contains a hydrophobic valine patch (V8-V9-V10) located within the dome structure. It is hypothesized that the interaction between the hydrophobic valine patch located on stem peptide and the adjacent polypeptide is critical for the interchain interaction which should be important for the stability of the P22 TSP NTD itself. To test this hypothesis, each amino acid in the valine residues is substituted by an acid, a basic, and a hydrophobic amino acid. The results of such substitutions are presented as well as associated studies. The data strongly suggest that the valine patch is of critical importance in the hydrophobic interaction between stem peptide valine patch and an adjacent chain.

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Single Amino Acid Change Mutation in the Hydrophobic Core of the N-terminal Domain of P22 TSP affects the Proteins Stability

Ayariga

Villafañe

2021

Preprint

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The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly shifted the attention of researchers to critically investigate most viruses to understand specific characteristics that impart their virulence. For instance, the SARS-CoV-2 has undergone several mutations, with some variants classified as variants of concern, e.g., the Omicron and Delta variant of SARS-CoV-2 are known for their rapid transmission and antigenicity due to mutation in the Spike protein. P22 bacteriophage is a bacterial virus that has a tailspike protein (TSP) that performs similar functions as the Spike protein of SARS-COV-2. We previously carried out a site-directed mutagenesis of the P22 TSP to bear disruptive mutations in the hydrophobic core of the N-terminal Domain (NTD), then partially characterized the properties of the mutant TSPs. In this process, the valine patch (triple valine residues that formed a hydrophobic core) was replaced with charged amino acids (Asp or lysine) or hydrophobic amino acids (Leucine or isoleucine). Some of the mutant TSPs characterized showed significant differences in migration in both native and SDS-PAGE. Mutants with such disruptive mutation are known to show non-native properties, and as expected, most of these mutants obtained showed significantly different properties from the WT P22 TSP. In this work, we further characterized these mutant species by computational and in vitro assays to demonstrate the validity of our previous inference that the valine patch is a critical player in the stability of the N-terminal domain of the P22 TSP.

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Stem Mutants in the N-terminal Domain of the Phage P22 Tailspike Protein

Cited by 3 publications

References 27 publications

Non-Stem Amino Acids Are Involved in the Phage P22 TSP NTD Stability

Non-Stem Amino Acids Are Involved in the Phage P22 TSP NTD Stability

A genetic analysis of an important hydrophobic interaction at the P22 tailspike protein N-terminal domain

Single Amino Acid Change Mutation in the Hydrophobic Core of the N-terminal Domain of P22 TSP affects the Proteins Stability

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