Category:Team Trypto-pham

Figure 4-

After determining if M. smegmatis is an appropriate target host cell and it's mycolyl-arabinogalactan-peptidoglycan membrane is hydrolyzed, testing the proposed function of a Serine esterase was tested by Chromatography to determine the release of Mycolic acids. Each time period is noted in figure A. Figure A and B are time and concentration dependent of the release of lipids. This is designed to mock the mycolic acid release known from treating the membrane with tetrabutylammonium. Figure C is classifying the concentration of the specific lipids released in figure A and B. Figure D is the most critical, by measuring the mutant that had the serine 82 residue removed, an important catalyst, along with test of other chemicals and controls, it is evident that Lyse B intact with the Serine and other catalysts intact are essential to releasing the free mycolic acids in the cell membrane.

Of the 253 residue for D29 LysB, 170 are typical for the homologic match to the alpha/beta hydrolase. this structure is composed of a beta sheet in between 2 alpha sheets.

The remaining 81- residues form a C-terminal alpha helix that has a backside attachment to the Beta sheet. the structure and polarization of the end of the C-terminal yields a structure where Gly117-ASN118-PRO119 allowing a 180 degree turn underneath ASP 166 with 117 and 119 forming hydrogen bonds with met120, Arg121, and Asp160 allowing rotation that helps facilitate the catalytic state of the release of mycolic acid from the host cell membrane.

The composition of the residues leads a 21% identity with amino acids. However, it yields a high Z-score of 20.3 for comparison in Dali. This lack of identity is possibly due to the 55 residues that are high in glycine residues that lead to the structure in between the second and third helices.

Fig.3 shows high similarity between ply118 and ply500 protein sequence. wither it's a identical residue or a conservative substitution, the two enzymes share the acceptable shared identity

In fig.8, an optical density assay was performed on Listeria monocytogenes WSLC 1001. the assay shows a decrees in the optical density of L.monocytogenes cell wall after being exposed to PL118 (closed circles).

PL118 and PL500 have acceptable sequence similarity to assume they have the same lytic function. the control(open circles) shows no effect on the cell wall without the addition of PL118.

Figure 1a is a sequence alignment between R^21 Lyz^P1. Lyz^P1 is a previously researched protein that has lysozyme activity. The orange boxes have been identified as SAR domains, and in supplementary figure 2a are assayed to show the SAR domains are necessary for lysozyme activity. The yellow boxes are amino acid sequences that are inserted to produce chimeras for other tests. Alignment is inferred based on the E-8aa-(D/C)-5aa-T catalytic triad (blue and asterisks).

Supplementary Figure 2. is a graph of results of an assay to determine lysozyme activity between purified full-length R^21 and truncated R^21. The truncated version has had the N-terminal SAR (signal anchor release) domain cleaved. Lyz^P1, a protein from phage P1 that had been previously determined to have lyzosyme activity, was included for comparison. The results found that the truncated version of R^21 did not produce any lysozyme activity, while the full-length version produced activity similar to Lyz^P1. The assay was done with the EnzChek® Lysozyme Assay which measures lysozyme activity on fluorescently-labeled Micrococcus lysodeikticus cell walls. When the fluorescently-labeled Micrococcus lysodeikticus cell walls are hydrolyzed, the fluorescein was released from the cell wall resulting in an increase in fluorescence. Trials with different concentrations of egg white lysozyme (EWL) (a standard for the EnzChek test) were run. The different shapes in the graph indicate EWL concentrations. Different line colors indicate which protein was being tested. In each trial, the full-length version of R^21 produced similar activity to Lyz^P1, while the truncated version of R^21 experienced almost total loss of function. These results support the idea that the SAR domain is necessary for and contributes to lysozyme activity in R^21.

in figure 5(A), activity assay shows how the wild type of RtcB ligates a linear RNA to circular RNA. RtcB assayed and found to ligates the 5'-hydroxyl and 2',3'-cyclic phosphate of a 21-nt RNA. the figure also compare the wild type to three other mutant of RtcB. the three mutants did not form a circular RNA, conforming the wild type function.

T7 replication yielding HN-S inhibition and production. Inhibition of phage growth in cell.

Assists in fusion and plating

DNA compaction and supercoiling. Specific binding to H-NS inhibits and effects dna plaque, also binds to A rexAB

system