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Chemical Structure| 1337883-32-5 Chemical Structure| 1337883-32-5

Structure of DiZPK
CAS No.: 1337883-32-5

Chemical Structure| 1337883-32-5

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DiZPK is a genetically encoded, highly efficient photocrosslinking amino acid valuable in unveiling the physiological interaction partners of given proteins and their functions.

4.5 *For Research Use Only !

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Luis F. Avila-Cobian ; Stefania De Benedetti ; Hidekazu Hoshino ; Van T. Nguyen ; Amr M. El-Araby ; Safaa Sader , et al.

Abstract: Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the partners of Slt span disparate periplasmic families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 μM) include PBPs (PBP1a, KD = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, KD = 0.09 μM; RlpA, KD = 0.4 μM); a type VI secretion system effector (Tse5, KD = 0.3 μM); and a regulatory for alginate biosynthesis (AlgO, KD < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub within the periplasm.

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Avila-Cobian, Luis F. ; Hoshino, Hidekazu ; Horsman, Mark E. ; Nguyen, Van T. ; Qian, Yuanyuan ; Feltzer, Rhona , et al.

Abstract: The 11 lytic transglycosylases of Pseudomonas aeruginosa have overlapping activities in the turnover of the cell-wall peptidoglycan. Rare lipoprotein A (RlpA) is distinct among the 11 by its use of only peptidoglycan lacking peptide stems. The spatial localization of RlpA and its interactome within P. aeruginosa are unknown. We employed suppression of introduced amber codons at sites in the rlpA gene for the introduction of the unnatural-amino-acids Νζ-[(2-azidoethoxy)carbonyl]-L-lysine (compound 1) and Nζ-[[[3-(3-methyl-3H-diazirin-3-yl)propyl]amino]carbonyl]-L-lysine (compound 2). In live P. aeruginosa, full-length RlpA incorporating compound 1 into its sequence was fluorescently tagged using strained-promoted alkyne-azide cycloaddition and examined by fluorescence microscopy. RlpA is present at low levels along the sidewall length of the bacterium, and at higher levels at the nascent septa of replicating bacteria. In intact P. aeruginosa, UV photolysis of full-length RlpA having compound 2 within its sequence generated a transient reactive carbene, which engaged in photoaffinity capture of neighboring proteins. Thirteen proteins were identified. Three of these proteins-PBP1a, PBP5, and MreB-are members of the bacterial divisome. The use of the complementary methodologies of non-canonical amino-acid incorporation, photoaffinity proximity anal., and fluorescent microscopy confirm a dominant septal location for the RlpA enzyme of P. aeruginosa, as a divisome-associated activity. This accomplishment adds to the emerging recognition of the value of these methodologies for identification of the intracellular localization of bacterial proteins.

Keywords: amber-codon suppression ; cell division ; divisome ; fluorescence microscopy ; lytic transglycosylase ; noncanonical-amino-acid incorporation ; protein photoaffinity ; protein-protein interactions

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Product Details of DiZPK

CAS No. :1337883-32-5
Formula : C12H23N5O3
M.W : 285.34
SMILES Code : N[C@H](C(O)=O)CCCCNC(NCCCC1(N=N1)C)=O
MDL No. :MFCD28900721
InChI Key :YGUIFRMUMYTGAV-VIFPVBQESA-N
Pubchem ID :53314876

Safety of DiZPK

GHS Pictogram:
Signal Word:Danger
Hazard Statements:H315-H319-H228
Precautionary Statements:P240-P210-P241-P264-P280-P302+P352-P370+P378-P337+P313-P305+P351+P338-P362+P364-P332+P313
Class:4.1
UN#:1325
Packing Group:

Isoform Comparison

Protocol

Bio Calculators
Preparing Stock Solutions 1mg 5mg 10mg

1 mM

5 mM

10 mM

3.50mL

0.70mL

0.35mL

17.52mL

3.50mL

1.75mL

35.05mL

7.01mL

3.50mL

Dissolving Methods
The prepared working fluid is recommended to be prepared now and used up as soon as possible in a short period of time. The percentage shown in front of the following solvent refers to the volume ratio of the solvent in the final solution; If precipitation or precipitation occurs in the preparation process, it can be assisted by heating and/or ultrasound.
Protocol 1

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