Toward the Structure of the C-terminal Domain of EcoR124I Restriction Enzyme by Nicholas Leudtke
Dublin Core
Title
Toward the Structure of the C-terminal Domain of EcoR124I Restriction Enzyme by Nicholas Leudtke
Subject
Chemistry
Description
Using the type I restriction-modification (R-M) system of plasmid EcoR124I, E. coli cells systematically distinguish their own DNA from foreign DNA. Self DNA is protected by
methylation within a specific recognition sequence, while foreign DNA, which lacks methylation, promotes DNA translocation through the stationary R-M enzyme and cleavage at unspecific sites. The R-M system consists of three subunits: HsdS (specificity), HsdM (modification) and HsdR (restriction).
The published structure of the HsdR subunit of EcoR124I1 contained four functionally integrated domains. The last 150 amino acids in the C-terminal domain were unresolved in the crystal structure. A single point mutation led to a new crystal structure indicating that the last 150 residues form a 5th domain perpendicular to the other four domains.
To facilitate expression and crystallization, the C-terminal part of HsdR was appended after a fluorescent protein domain and a hexahistidine tag. Three constructs that include HsdR residues 705-1038, 867-1038, or 887-1038 were developed through PCR mediated deletions. Following expression, each protein was purified by nickel-NTA affinity and DEAE-Sepharose anion exchange chromatography. Crystals of the construct containing residues 887-1038 diffract x-rays to 8 Å and a model structure has been predicted for the C-terminal domain.
Molecular dynamics simulations with GROMACS software is being used to simulate the restriction subunit. The aim is to estimate if the proposed C-terminal structure maintains its secondary and tertiary structures during molecular dynamics simulations in solvent. Simulations of the protein are being examined for features that can evaluate the acceptability of the current model of EcoR124I.
This project was completed with all of the creators listed below, as well as the support from the Czech Science Foundation (P207/12/2323) and the United States National Science Foundation REU program (award 1358737).
methylation within a specific recognition sequence, while foreign DNA, which lacks methylation, promotes DNA translocation through the stationary R-M enzyme and cleavage at unspecific sites. The R-M system consists of three subunits: HsdS (specificity), HsdM (modification) and HsdR (restriction).
The published structure of the HsdR subunit of EcoR124I1 contained four functionally integrated domains. The last 150 amino acids in the C-terminal domain were unresolved in the crystal structure. A single point mutation led to a new crystal structure indicating that the last 150 residues form a 5th domain perpendicular to the other four domains.
To facilitate expression and crystallization, the C-terminal part of HsdR was appended after a fluorescent protein domain and a hexahistidine tag. Three constructs that include HsdR residues 705-1038, 867-1038, or 887-1038 were developed through PCR mediated deletions. Following expression, each protein was purified by nickel-NTA affinity and DEAE-Sepharose anion exchange chromatography. Crystals of the construct containing residues 887-1038 diffract x-rays to 8 Å and a model structure has been predicted for the C-terminal domain.
Molecular dynamics simulations with GROMACS software is being used to simulate the restriction subunit. The aim is to estimate if the proposed C-terminal structure maintains its secondary and tertiary structures during molecular dynamics simulations in solvent. Simulations of the protein are being examined for features that can evaluate the acceptability of the current model of EcoR124I.
This project was completed with all of the creators listed below, as well as the support from the Czech Science Foundation (P207/12/2323) and the United States National Science Foundation REU program (award 1358737).
Creator
Luedtke, Nicholas
Grinkevich, Pavel
McIntosh, Bennett
Baikova, Tatsiana
Lapkouski, Mikalai
Ettrich, Rüdiger
Carey, Jannette
Source
Senior Showcase Poster presentation
Publisher
Ripon College
Date
April 19, 2016
Rights
The author reserves all rights.
Identifier
Major: Chemistry
Appleton, WI
Files
Collection
Citation
Luedtke, Nicholas et al., “Toward the Structure of the C-terminal Domain of EcoR124I Restriction Enzyme by Nicholas Leudtke,” Senior Showcase Digital Collection, accessed April 16, 2024, https://rcseniorshowcase.omeka.net/items/show/52.