Journal: PLoS ONE

Article Title: Characterization of a nontypeable Haemophilus influenzae thermonuclease

doi: 10.1371/journal.pone.0197010

Figure Lengend Snippet: Fig 1A shows the amino acid sequence of NT Hi 2019 Nuc (HI_1296). The first 20 amino acids in bold are the signal sequence of the protein. Fig 1B shows the alignment of NT Hi Nuc with staphylococcal thermonuclease, which has 35% conserved amino acids and Expect value (number of chance matches) of 2e-10, indicating a significant match.

Article Snippet: Enzyme activity studies were performed with PCR grade staphylococcal thermonuclease (Sigma-Aldrich) and highly purified recombinant NT Hi Nuc without signal sequence.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Characterization of a nontypeable Haemophilus influenzae thermonuclease

doi: 10.1371/journal.pone.0197010

Figure Lengend Snippet: FRET assay of NT Hi Nuc activity.

Article Snippet: Enzyme activity studies were performed with PCR grade staphylococcal thermonuclease (Sigma-Aldrich) and highly purified recombinant NT Hi Nuc without signal sequence.

Techniques: Activity Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Characterization of a nontypeable Haemophilus influenzae thermonuclease

doi: 10.1371/journal.pone.0197010

Figure Lengend Snippet: NT Hi Nuc with Ca 2+ has a hydrodynamic radius of approximately 2.1 nm when in solution. Within minutes of adding Co 2+ , a known inhibitor for staphylococcal thermonuclease, the size of NT Hi Nuc increased dramatically indicating oligomerization or nonspecific aggregation (solid line in Panel A). The aggregates formed by the addition of Co 2+ to NT Hi Nuc were briefly disrupted by passing the solution through a 0.22 μm membrane filter reduced the hydrodynamic radius to approximately 15 nm (start of dashed line in Panel A). However, NT Hi Nuc continued aggregating and increasing in size due to Co 2+ still present in solution (dashed line). Panel A shows representative curves. Activity of NT Hi Nuc was quenched with addition of Co 2+ when measured by FRET, similar to when EDTA was added (Panel B). Panel A shows representative curves and Panel B was done in triplicates.

Article Snippet: Enzyme activity studies were performed with PCR grade staphylococcal thermonuclease (Sigma-Aldrich) and highly purified recombinant NT Hi Nuc without signal sequence.

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Characterization of a nontypeable Haemophilus influenzae thermonuclease

doi: 10.1371/journal.pone.0197010

Figure Lengend Snippet: Effect of pH on thermal stability.

Article Snippet: Enzyme activity studies were performed with PCR grade staphylococcal thermonuclease (Sigma-Aldrich) and highly purified recombinant NT Hi Nuc without signal sequence.

Techniques:

Journal: PLoS ONE

Article Title: Characterization of a nontypeable Haemophilus influenzae thermonuclease

doi: 10.1371/journal.pone.0197010

Figure Lengend Snippet: Fig 7A shows a homology model of NT Hi Nuc shown as a cartoon, blue = N-terminal and red = C-terminal. Fig 7B shows the ab initio shape of NT Hi Nuc determined from SAXS data at pH 7. Fig 7C shows the surface representation of staphylococcal thermonuclease crystal structure. Fig 7B and 7C show similar overall shape and active site (black arrow).

Article Snippet: Enzyme activity studies were performed with PCR grade staphylococcal thermonuclease (Sigma-Aldrich) and highly purified recombinant NT Hi Nuc without signal sequence.

Techniques:

Journal: PLoS ONE

Article Title: Characterization of a nontypeable Haemophilus influenzae thermonuclease

doi: 10.1371/journal.pone.0197010

Figure Lengend Snippet: Overlay of the homology model of NT Hi Nuc (blue), and the active site of staphylococcal thermonuclease (pdb id 1STN) (red). Green sphere represents calcium and cyan sticks represent TdtP (inhibitor); modeled from pdb id 2SNS. There are three loops in the active site of NT Hi Nuc that are shorter than those of staphylococcal thermonculease. The absence of Ω loop near the active site of NT Hi Nuc compared to the staphylococcal thermonuclease could contribute to the stronger activity of NT Hi Nuc.

Article Snippet: Enzyme activity studies were performed with PCR grade staphylococcal thermonuclease (Sigma-Aldrich) and highly purified recombinant NT Hi Nuc without signal sequence.

Techniques: Activity Assay